**Electrophysiology of Woody Plants**

Luis A. Gurovich

*Universidad Católica de Chile Chile* 

#### **1. Introduction**

A fundamental property of all living organisms is related to the continuous gathering of environmental information and the expression of physiological responses aimed to optimize its performance under new environmental conditions. In order to keep homeostasis, plants need to continuously gather information about its environment and to react physiologically, in order to synchronize its normal biological functions. Plant cells become bio electrochemically excited under the influence of environmental changes and the conduction of these electric potential modifications to distant plant organs have been widely reported. Electrochemical phenomena in plants have attracted researchers since the eighteenth century (Bertholon, 1783; Burdon-Sanderson, 1873; Darwin, 1875; Lemström, 1904; Bose, 1926); however, only in the last decade numerous papers related to plant electrophysiology have been published (for a comprehensive review on the subject see Volkov´s book *"Plant Electrophysiology, Theory and Methods"*, 2006). Detection of electrical potentials in plants indicates that electrical signaling is a major system to transmit information over long distances throughout its organs. The reason why plants have developed pathways for electrical signal transmission is probably related to its need to respond rapidly to environmental stress factors (Fromm & Lautner, 2007). Electrophysiological studies of longdistance signals in plants and animals contribute to our knowledge of the living world by revealing important similarities and crucial differences between plants and animals, in an area that might be directly related to their different capacities to respond to environmental change.

The existence of electrophysiological mechanisms for information perception, transmission and processing between different plant organs and tissues, allowing the expression of fast and accurate physiological reactions to specific biotic or abiotic stimuli, is expressed by means of real-time detectable *action* (APs) and *variation* (VPs) potentials (Datta & Palit, 2004; Gil et al., 2008; Lautner et al., 2005; Oyarce & Gurovich, 2010; Volkov et al., 2009; Wang et al., 2009). An additional type of electric potential in plants has been proposed by Zimmermann et al. (2009), to be called *system* potential. In addition to APs that occur also in animals and lower plants (Trebacz et al., 2005) higher plants feature an additional, unique, hydraulically propagated type of electric signals VPs, called also *slow wave potentials* (Stahlberg et al., 2005).

Several models have been proposed to explain the onset of plant cell electric excitation, resulting from external stimuli (Wayne, 1993; Fromm & Lautner, 2007). All plant cells are

Electrophysiology of Woody Plants 3

Research on the subject of electrochemical phenomena in plants is generically known as *plant electrophysiology* (Volkov, 2006); this knowledge is the basis of a newly developed discipline in the field of plant physiology: *plant neurobiology* (Brenner et al, 2006; Stahlberg, 2006; Baluška & Mancuso, 2008; Barlow, 2008). Plant neurobiology is aimed at establishing the structure of information networks that exist within the plant, which is expressed as responses to environmental stimuli by means of electrochemical signals (Baluška et al., 2004; Trewavas, 2005). These signals seem to complement other plant signals: hydraulic, mechanical, volatile and hormonal, already well documented in plant science (Fromm &

Research on plant electrophysiology specifically focused on woody plants like poplar and willow trees, have been seldom reported (Fromm & Spanswick, 1993; Lautner et al, 2005; Gibert et al., 2006). In fruit bearing deciduous and perennial plant species, electrophysiology studies are very limited as well, although it is in such plants that the need for rapid and efficient signals other than chemical and hydraulic signaling becomes more obvious (Gil et al., 2008; Nadler et al. 2008; Gurovich & Hermosilla, 2009; Oyarce & Gurovich, 2011). These studies have associated the effect of water stress, deficit irrigation, light cycles and mechanical or heat injury with electrical signaling in several fruit bearing tree species. Electrical signaling has been also associated to conditions of differential soil water availability; the use of real-time information on tree electrochemical behavior, as early indicator of biotic or abiotic induced water stress conditions, can provide a strategy to quantitatively relate plant physiological reactions to environmental changes and eventually, for the auto-programmed operation of pressurized irrigation systems, aimed to prevent

Additional applications of electrical signals in plants have been postulated, including its eventual use as environmental biosensors (Davies, 2004; Volkov & Brown, 2006) as well as to correlate sap flow based ET measurements with plant electrical behavior has been proposed (Gibert et al., 2006). Artificially applied electric potential differentials between plant organs under field conditions may enhance water use efficiency in woody plants, through its controlled influence on stomata conductance and plant internal water flux (Gil et

For a long time, plants were thought to be living organisms whose limited ability to move and respond was related to its relative limited abilities of sensing (Trewawas, 2003), with the exception only for plants with rapid and/or purposeful movements such as *Mimosa pudica (*also called *the* sensitive plant*)*, *Drosera* (sundews)*, Dionea muscipula* (flytraps) and tendrils of climbing plants. These sensitive plants attracted the attention of outstanding pioneer researchers such as Burdon-Sanderson (1873, 1899), Pfeffer (1873), Haberlandt (1914), Darwin (1896) and Bose (1926). They found plants not only to be equipped with various mechano-receptors that exceeded the sensitivity of a human finger, but also its ability to

The discovery that common plants had propagating APs just as the "sensitive" plants (Gunar & Sinykhin 1962, 1963; Karmanov et al., 1972) was a scientific breakthrough with important consequences, correcting the long-held belief that normal plants are less sensitive

Lautner, 2007; Gil et al., 2009; Dziubinska et al., 2003).

water stress conditions in irrigated trees (Oyarce and Gurovich, 2010).

al., 2008; Jia & Zhang, 2008; Gil et al., 2009; Gurovich, 2009).

trigger action potentials (APs) that implemented these movements.

**2. History of plant electrophysiology** 

surrounded by a plasma membrane (Murphy et al., 2010), composed of a lipid bilayer, with a variety of molecular structures embedded in it, known generically as *ion channels* and *electrogenic pumps* (Hedrich & Schroeder, 1989). Electrochemical excitation is caused by ionic fluxes through the cell plasma membrane (Knudsen, 2002; Blatt, 2008), creating an electric charge modification in the membrane itself, as well as a differential charge on either side. This trans - membrane potential is the difference in voltage (or electrical potential difference) between the interior and exterior of a cell (*Vinterior* − *Vexterior*). Plant plasma membranes always maintain a potential, the cell interior being more negative than the exterior, arising mainly from the activity of electrogenic pumps. As an example, H+ transporting ATPases (Sze et al., 1999) pump protons out of the cell, thus maintaining a pH gradient across the plasma membrane. This process is involved in the simultaneous symport of carbohydrates and amino acids into the cell, which are produced at different plant tissues as photosynthetic derivatives. Other electrogenic ion pumps described for plant cell plasma membranes are related to ion and solute fluxes, underpinning inorganic mineral nutrient uptake; they trigger rapid changes in secondary messengers such as cytosolic-free Ca+2 concentrations, and also power the osmotic gradients that drive cell expansion (Schroeder & Thuleau 1991; Gelli & Blumwald, 1997; Zimmermann et al., 1997; Bonza et al., 2001; Sanders, 2002; Blatt, 2008; Lautner & Fromm, 2010). The K+1-transporting ATPase, also embedded in the cell plasma membrane, enables the onset of different ion concentrations (and therefore electrical charge) on the intracellular and extracellular sides of the membrane (Maathuis & Sanders, 1997).

Ion channels, when active, partially discharge the plasma membrane potential, while the electrogenic pumps restore and maintain it (Fromm & Spanswick, 1993; Neuhaus & Wagner, 2000). The plasma membrane potential has two basic functions. First, it allows a cell to function as a *battery*, providing power to operate the variety of electrogenic pumps embedded in its lipid bilayer. Second, in electrically excitable cells, it is used for transmitting signals between different parts of a cell or to other plant cells, tissues or organs. Opening or closing of ion channels at one point in the membrane produces a local and transient change in the membrane potential, which causes an electric current to flow rapidly to other points in the membrane and eventually, to the plasma membrane of surrounding cells. In nonexcitable cells, and in excitable cells in their baseline state, the membrane potential is held at a relatively stable value, called the *resting potential*, characterized by its absence of fluctuations; the resting potential varies from −20 mV to −200 mV according to cell type. Opening and closing of ion channels can induce a departure from the resting potential, called a *depolarization* if the interior voltage rises, or a *hyperpolarization* if the interior voltage becomes more negative. In excitable cells, a sufficiently large depolarization can evoke an action potential (AP), in which the membrane potential very rapidly undergoes a significant, measurable change, often briefly reversing its sign; AP are short-lasting, all-or-nothing events.

Change in trans – plasma membrane potential creates a *wave of depolarization*, which affects the adjoining resting plasma membranes, thus generating an *impulse*. Once initiated, these impulses can propagate to adjacent excitable cells. Electrical signals can propagate along the plasma membrane (Van Bel & Ehlers, 2005; Volkov et al., 2011) on short distances through plasmodesmata and on long distances in plant phloematic tissue (Ksenzhek & Volkov, 1998; Volkov, 2000; Volkov, 2006; Volkov et al., 2011).

surrounded by a plasma membrane (Murphy et al., 2010), composed of a lipid bilayer, with a variety of molecular structures embedded in it, known generically as *ion channels* and *electrogenic pumps* (Hedrich & Schroeder, 1989). Electrochemical excitation is caused by ionic fluxes through the cell plasma membrane (Knudsen, 2002; Blatt, 2008), creating an electric charge modification in the membrane itself, as well as a differential charge on either side. This trans - membrane potential is the difference in voltage (or electrical potential difference) between the interior and exterior of a cell (*Vinterior* − *Vexterior*). Plant plasma membranes always maintain a potential, the cell interior being more negative than the exterior, arising mainly from the activity of electrogenic pumps. As an example, H+ transporting ATPases (Sze et al., 1999) pump protons out of the cell, thus maintaining a pH gradient across the plasma membrane. This process is involved in the simultaneous symport of carbohydrates and amino acids into the cell, which are produced at different plant tissues as photosynthetic derivatives. Other electrogenic ion pumps described for plant cell plasma membranes are related to ion and solute fluxes, underpinning inorganic mineral nutrient uptake; they trigger rapid changes in secondary messengers such as cytosolic-free Ca+2 concentrations, and also power the osmotic gradients that drive cell expansion (Schroeder & Thuleau 1991; Gelli & Blumwald, 1997; Zimmermann et al., 1997; Bonza et al., 2001; Sanders, 2002; Blatt, 2008; Lautner & Fromm, 2010). The K+1-transporting ATPase, also embedded in the cell plasma membrane, enables the onset of different ion concentrations (and therefore electrical charge) on the intracellular and extracellular sides of the membrane

Ion channels, when active, partially discharge the plasma membrane potential, while the electrogenic pumps restore and maintain it (Fromm & Spanswick, 1993; Neuhaus & Wagner, 2000). The plasma membrane potential has two basic functions. First, it allows a cell to function as a *battery*, providing power to operate the variety of electrogenic pumps embedded in its lipid bilayer. Second, in electrically excitable cells, it is used for transmitting signals between different parts of a cell or to other plant cells, tissues or organs. Opening or closing of ion channels at one point in the membrane produces a local and transient change in the membrane potential, which causes an electric current to flow rapidly to other points in the membrane and eventually, to the plasma membrane of surrounding cells. In nonexcitable cells, and in excitable cells in their baseline state, the membrane potential is held at a relatively stable value, called the *resting potential*, characterized by its absence of fluctuations; the resting potential varies from −20 mV to −200 mV according to cell type. Opening and closing of ion channels can induce a departure from the resting potential, called a *depolarization* if the interior voltage rises, or a *hyperpolarization* if the interior voltage becomes more negative. In excitable cells, a sufficiently large depolarization can evoke an action potential (AP), in which the membrane potential very rapidly undergoes a significant, measurable change, often briefly reversing its sign; AP are short-lasting, all-or-nothing

Change in trans – plasma membrane potential creates a *wave of depolarization*, which affects the adjoining resting plasma membranes, thus generating an *impulse*. Once initiated, these impulses can propagate to adjacent excitable cells. Electrical signals can propagate along the plasma membrane (Van Bel & Ehlers, 2005; Volkov et al., 2011) on short distances through plasmodesmata and on long distances in plant phloematic tissue (Ksenzhek & Volkov, 1998;

(Maathuis & Sanders, 1997).

events.

Volkov, 2000; Volkov, 2006; Volkov et al., 2011).

Research on the subject of electrochemical phenomena in plants is generically known as *plant electrophysiology* (Volkov, 2006); this knowledge is the basis of a newly developed discipline in the field of plant physiology: *plant neurobiology* (Brenner et al, 2006; Stahlberg, 2006; Baluška & Mancuso, 2008; Barlow, 2008). Plant neurobiology is aimed at establishing the structure of information networks that exist within the plant, which is expressed as responses to environmental stimuli by means of electrochemical signals (Baluška et al., 2004; Trewavas, 2005). These signals seem to complement other plant signals: hydraulic, mechanical, volatile and hormonal, already well documented in plant science (Fromm & Lautner, 2007; Gil et al., 2009; Dziubinska et al., 2003).

Research on plant electrophysiology specifically focused on woody plants like poplar and willow trees, have been seldom reported (Fromm & Spanswick, 1993; Lautner et al, 2005; Gibert et al., 2006). In fruit bearing deciduous and perennial plant species, electrophysiology studies are very limited as well, although it is in such plants that the need for rapid and efficient signals other than chemical and hydraulic signaling becomes more obvious (Gil et al., 2008; Nadler et al. 2008; Gurovich & Hermosilla, 2009; Oyarce & Gurovich, 2011). These studies have associated the effect of water stress, deficit irrigation, light cycles and mechanical or heat injury with electrical signaling in several fruit bearing tree species. Electrical signaling has been also associated to conditions of differential soil water availability; the use of real-time information on tree electrochemical behavior, as early indicator of biotic or abiotic induced water stress conditions, can provide a strategy to quantitatively relate plant physiological reactions to environmental changes and eventually, for the auto-programmed operation of pressurized irrigation systems, aimed to prevent water stress conditions in irrigated trees (Oyarce and Gurovich, 2010).

Additional applications of electrical signals in plants have been postulated, including its eventual use as environmental biosensors (Davies, 2004; Volkov & Brown, 2006) as well as to correlate sap flow based ET measurements with plant electrical behavior has been proposed (Gibert et al., 2006). Artificially applied electric potential differentials between plant organs under field conditions may enhance water use efficiency in woody plants, through its controlled influence on stomata conductance and plant internal water flux (Gil et al., 2008; Jia & Zhang, 2008; Gil et al., 2009; Gurovich, 2009).

#### **2. History of plant electrophysiology**

For a long time, plants were thought to be living organisms whose limited ability to move and respond was related to its relative limited abilities of sensing (Trewawas, 2003), with the exception only for plants with rapid and/or purposeful movements such as *Mimosa pudica (*also called *the* sensitive plant*)*, *Drosera* (sundews)*, Dionea muscipula* (flytraps) and tendrils of climbing plants. These sensitive plants attracted the attention of outstanding pioneer researchers such as Burdon-Sanderson (1873, 1899), Pfeffer (1873), Haberlandt (1914), Darwin (1896) and Bose (1926). They found plants not only to be equipped with various mechano-receptors that exceeded the sensitivity of a human finger, but also its ability to trigger action potentials (APs) that implemented these movements.

The discovery that common plants had propagating APs just as the "sensitive" plants (Gunar & Sinykhin 1962, 1963; Karmanov et al., 1972) was a scientific breakthrough with important consequences, correcting the long-held belief that normal plants are less sensitive

Electrophysiology of Woody Plants 5

Hydraulic pressure signals are propagating changes in water pressure inside plant tissues (Malone, 1996); plant tissues have plenty of hydraulic connections (mainly xylematic vessels) which provide a pathway for long-distance transmission of hydraulic signals. Pressure waves can be relatively quick and fast, as they can diffuse through the plant at the speed of sound (~1500 m s−1 in water), but, to be physiologically important, a hydraulic signal must cause a significant change in turgor pressure inside a cell. As plant cells can be elastic, their turgor will change only when a significant influx (or efflux) of water occurs: the needed flux is strictly linked with the hydraulic capacitance of the cell, a widely variable property related to plant water potential and plant cell wall elasticity. Thus, hydraulic signals must involve massive water mass ow; for example, to increase the turgor pressure in leaf cells by 1 bar, a net water inux equivalent to 1–5% of the total volume of a leaf must occur (Malone 1996). For a detailed review on plant hydraulic signaling, see Mancuso &

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses, by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca+2), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Significant research in chemical signaling in plants has been aimed to understand the ability of plants respond to abscisic acid (ABA), often called the *stress hormone*. This hormone controls many of the adaptive responses that plants have evolved to conserve water when they perceive a reduced supply of this commodity. Stomata closure, reduced canopy area, and increased root biomass are three of the major adaptive processes regulated by ABA that can potentially be manipulated to improve crop water use efficiency (Wilkinson & Hartung, 2009; Jiang & Hartung, 2008). A comprehensive review on chemical signaling under abiotic stress environment in plants has been recently published

**4. Facts and hypothesis about electrical signals in woody plants** 

published by Volkov & Ranatunga, 2006 and Gibert et al., 2006.

Rapid plant and animal responses to environmental changes are associated to electrical excitability and signaling, using the same electrochemical pathways to drive physiological responses, characterized in animals by movement (physical displacement) and in plants by continuous growth. In plants and animals, signal transmission can occur over long and short distances and correspond to intra and intercellular communication mechanisms, which determine the physiological behavior of the organism. Electrical pulses can be monitored in plants as *signals*, which are transmitted through excitable phloematic cell membranes, enabling the propagation of electrical pulses in the form of a depolarization wave or "action potential" AP. (Dziubinska et al., 2001; Fromm & Spanswick, 2007). At the onset of a change in the environmental conditions, plants respond to these stimuli at the site of occurrence and bioelectrical pulses are distributed throughout the entire plant, from roots to shoots and vice versa. A working model (Figure 1) to define plant behavior has been adapted from work

Two different types of electrical signals have been reported in plants: AP (Fromm, 2006), which is a rapid propagating electrical pulse, travelling at a constant velocity and maintaining a constant amplitude, and VP (slow wave or "variation potential"),

Mugnai (2006).

by Tuteja & Sopory (2008).

and responsive as compared to the so-called "sensitive plants." Also, it led to studies aimed to understand the meaning of the widely distributed electrical signals in different plant tissues (Pickard, 1973), which carry important messages with a broader relevance than the established induction of organ movements in "sensitive plants".

The first known recording of a plant AP was done on leaves of the Venus flytrap (*Dionea muscipula* Ellis) in 1873 by Burdon-Sanderson, measuring the voltage difference between adaxial and abaxial surfaces of a *Dionea* leaf half, while stimulating the other half mechanically by touching the hairs (Burdon-Sanderson 1873, 1899). The trap closure in *Dionea* has been considered as a model case, showing comparable roles of APs in plants and nerve–muscle preparations of animals (Simons, 1992). Bose (1926) proposed that vascular bundles act analogous to nerves, by enabling the propagation of an excitation that moved from cell to cell. A comprehensive review of the early development of plant electrophysiology is provided by Stahlberg (2006).

For many years, the application of external electrodes to the surface of plant and animal organs was the only available technique for measuring potentials. The introduction of microelectrodes, like KCl-filled glass micropipettes with a tip diameter small enough to be inserted into living cells (Montenegro et al., 1991), enabled to record intracellular, *i.e.* real, membrane potentials (Vm). This technique was first adopted for giant cells from charophytic algae such as *Chara* and *Nitella*. Later on, it was complemented with precise electronic amplifiers and voltage clamp circuits, monitoring the activity of ion channels by direct measurement of ion currents instead of voltages. Parallel voltage (V) and current (I) measurements allowed I-V-curves, used to differentiate between the action of an ion channel (ohmic or parallel changes in I and V) or ion pump (non-ohmic relation between V and I changes) (Higinbotham, 1973).

As a next step to improve recording possibilities, the *patch clamp* technique was developed; by going from single cells to isolated membrane patches, one can record the current of as small a unit as a single ionic channel. Initially developed for animal cells, this technique was rapidly adopted for plant cell studies (Hedrich & Schroeder 1989). Voltage clamp techniques were introduced to demonstrate the contribution of various ion currents involved in the AP in *Chara* cells (Lunevsky et al. 1983; Wayne 1994). To this day, charophytic algae have served as important research models for higher plant cells electric behavior studies.

Additional studies made considerable progress in linking electrical signals with respiration and photosynthesis (Lautner et al, 2005; Koziolek et al. 2003), phloem transport (Fromm & Eschrich, 1988; Fromm & Bauer, 1994) and the rapid, plant-wide deployment of plant defenses (Wildon et al. 1992; Malone et al. 1994; Herde et al. 1995, 1996; Volkov & Haak 1995; Stankovic & Davies, 1996, 1998; Volkov, 2000). The significant development of plant neurobiology in the last decade is mostly related to electrophysiology based research, as an integrated view of plant signaling and behavior (Brenner et al., 2006; Baluška & Mancuso 2008; Barlow, 2008).

#### **3. Hormonal and hydraulic physiological signals in woody plants**

Hydraulic and hormonal signals in woody plants complement signaling electrophysiology in plants, playing a significant role in the dynamics of information processes integrating the plant responses to the environment.

and responsive as compared to the so-called "sensitive plants." Also, it led to studies aimed to understand the meaning of the widely distributed electrical signals in different plant tissues (Pickard, 1973), which carry important messages with a broader relevance than the

The first known recording of a plant AP was done on leaves of the Venus flytrap (*Dionea muscipula* Ellis) in 1873 by Burdon-Sanderson, measuring the voltage difference between adaxial and abaxial surfaces of a *Dionea* leaf half, while stimulating the other half mechanically by touching the hairs (Burdon-Sanderson 1873, 1899). The trap closure in *Dionea* has been considered as a model case, showing comparable roles of APs in plants and nerve–muscle preparations of animals (Simons, 1992). Bose (1926) proposed that vascular bundles act analogous to nerves, by enabling the propagation of an excitation that moved from cell to cell. A comprehensive review of the early development of plant

For many years, the application of external electrodes to the surface of plant and animal organs was the only available technique for measuring potentials. The introduction of microelectrodes, like KCl-filled glass micropipettes with a tip diameter small enough to be inserted into living cells (Montenegro et al., 1991), enabled to record intracellular, *i.e.* real, membrane potentials (Vm). This technique was first adopted for giant cells from charophytic algae such as *Chara* and *Nitella*. Later on, it was complemented with precise electronic amplifiers and voltage clamp circuits, monitoring the activity of ion channels by direct measurement of ion currents instead of voltages. Parallel voltage (V) and current (I) measurements allowed I-V-curves, used to differentiate between the action of an ion channel (ohmic or parallel changes in I and V) or ion pump (non-ohmic relation between V

As a next step to improve recording possibilities, the *patch clamp* technique was developed; by going from single cells to isolated membrane patches, one can record the current of as small a unit as a single ionic channel. Initially developed for animal cells, this technique was rapidly adopted for plant cell studies (Hedrich & Schroeder 1989). Voltage clamp techniques were introduced to demonstrate the contribution of various ion currents involved in the AP in *Chara* cells (Lunevsky et al. 1983; Wayne 1994). To this day, charophytic algae have served

Additional studies made considerable progress in linking electrical signals with respiration and photosynthesis (Lautner et al, 2005; Koziolek et al. 2003), phloem transport (Fromm & Eschrich, 1988; Fromm & Bauer, 1994) and the rapid, plant-wide deployment of plant defenses (Wildon et al. 1992; Malone et al. 1994; Herde et al. 1995, 1996; Volkov & Haak 1995; Stankovic & Davies, 1996, 1998; Volkov, 2000). The significant development of plant neurobiology in the last decade is mostly related to electrophysiology based research, as an integrated view of plant signaling and behavior (Brenner et al., 2006; Baluška & Mancuso

Hydraulic and hormonal signals in woody plants complement signaling electrophysiology in plants, playing a significant role in the dynamics of information processes integrating the

as important research models for higher plant cells electric behavior studies.

**3. Hormonal and hydraulic physiological signals in woody plants** 

established induction of organ movements in "sensitive plants".

electrophysiology is provided by Stahlberg (2006).

and I changes) (Higinbotham, 1973).

2008; Barlow, 2008).

plant responses to the environment.

Hydraulic pressure signals are propagating changes in water pressure inside plant tissues (Malone, 1996); plant tissues have plenty of hydraulic connections (mainly xylematic vessels) which provide a pathway for long-distance transmission of hydraulic signals. Pressure waves can be relatively quick and fast, as they can diffuse through the plant at the speed of sound (~1500 m s−1 in water), but, to be physiologically important, a hydraulic signal must cause a significant change in turgor pressure inside a cell. As plant cells can be elastic, their turgor will change only when a significant influx (or efflux) of water occurs: the needed flux is strictly linked with the hydraulic capacitance of the cell, a widely variable property related to plant water potential and plant cell wall elasticity. Thus, hydraulic signals must involve massive water mass ow; for example, to increase the turgor pressure in leaf cells by 1 bar, a net water inux equivalent to 1–5% of the total volume of a leaf must occur (Malone 1996). For a detailed review on plant hydraulic signaling, see Mancuso & Mugnai (2006).

Many chemicals are critical for plant growth and development and play an important role in integrating various stress signals and controlling downstream stress responses, by modulating gene expression machinery and regulating various transporters/pumps and biochemical reactions. These chemicals include calcium (Ca+2), cyclic nucleotides, polyphosphoinositides, nitric oxide (NO), sugars, abscisic acid (ABA), jasmonates (JA), salicylic acid (SA) and polyamines. Significant research in chemical signaling in plants has been aimed to understand the ability of plants respond to abscisic acid (ABA), often called the *stress hormone*. This hormone controls many of the adaptive responses that plants have evolved to conserve water when they perceive a reduced supply of this commodity. Stomata closure, reduced canopy area, and increased root biomass are three of the major adaptive processes regulated by ABA that can potentially be manipulated to improve crop water use efficiency (Wilkinson & Hartung, 2009; Jiang & Hartung, 2008). A comprehensive review on chemical signaling under abiotic stress environment in plants has been recently published by Tuteja & Sopory (2008).

#### **4. Facts and hypothesis about electrical signals in woody plants**

Rapid plant and animal responses to environmental changes are associated to electrical excitability and signaling, using the same electrochemical pathways to drive physiological responses, characterized in animals by movement (physical displacement) and in plants by continuous growth. In plants and animals, signal transmission can occur over long and short distances and correspond to intra and intercellular communication mechanisms, which determine the physiological behavior of the organism. Electrical pulses can be monitored in plants as *signals*, which are transmitted through excitable phloematic cell membranes, enabling the propagation of electrical pulses in the form of a depolarization wave or "action potential" AP. (Dziubinska et al., 2001; Fromm & Spanswick, 2007). At the onset of a change in the environmental conditions, plants respond to these stimuli at the site of occurrence and bioelectrical pulses are distributed throughout the entire plant, from roots to shoots and vice versa. A working model (Figure 1) to define plant behavior has been adapted from work published by Volkov & Ranatunga, 2006 and Gibert et al., 2006.

Two different types of electrical signals have been reported in plants: AP (Fromm, 2006), which is a rapid propagating electrical pulse, travelling at a constant velocity and maintaining a constant amplitude, and VP (slow wave or "variation potential"),

Electrophysiology of Woody Plants 7

potential lasts as long as the stimulus is present, being an electrical replica of the initial stimulus. If the stimulus is sufficiently large to cause the membrane potential to depolarize below a certain threshold, this will cause an action potential to be generated. It shows a large transient depolarization which is self perpetuating and therefore allows the rapid

Action potentials can propagate over short distances through plasmodesmata, and after it has reached the sieve element/companion cell (SE/CC) complex (Figure 3), it can travel

Fig. 3. Action and variation potentials in plants. (*After Lautner et al. 2005; Fromm & Lautner,* 

In contrast, a VP is generated at the plasma membrane of parenchyma cells (PAs) adjacent to xylem vessels (VEs) (Figure 3) by a hydraulic wave or a wounding substance. Because VPs were measured in SEs, it is suggested that they also can pass through the plasmodesmal network and can reach the phloem pathway. However, in contrast to APs, their amplitude

over long distances along the SE plasma membrane in both directions.

will be reduced with increasing distance from the site of generation.

Fig. 4. An action potential recorded in Aloe vera spp. (*After Volkov et al., 2007*).

Action potentials (AP) induced in leaves of an Aloe vera spp. plant by thermal shock (flame) are described by Volkov et al., 2007 (Figure 4). Measurements were recorded at 500,000

transmission of information over long distances.

*2007*).

corresponding to a long range of a variation pulse (Stahlberg et al., 2006), which varies with the intensity of the stimulus, and its amplitude and speed decrease with increasing distance from its generation site (Davies, 2004, 2006). AP is an all-or-none depolarization that spreads passively from the excited cellular membrane region to the neighboring non-excited region. Excitation in plant cells depends on Ca+2 depolarization and Cl- and K+ repolarization, that spreads passively from the excited cellular membrane region to the neighboring non-excited region (Brenner et al., 2006). A similitude on electrical signal transmission between animal and plant organs has been postulated by Volkov & Ranatunga (2006), using the model presented in Figure 2.

Fig. 1. Proposed mechanism of electric potential signals in plants *(Adapted from Volkov & Ranatunga, 2006 and Gibert et al., 2006).*

Fig. 2. The Hodgkin-Huxley (HH, 1952) equivalent circuit for an axon (A) and the modified HH circuit for sieve tubes in phloem (B) (*Volkov & Ranatunga*, *2006*).

Electrical conduction rate of most of the plant action potentials studied so far is in the range of 0.01-0.2 m s-1 , i.e. much slower than the conduction velocity of action potentials in animal nerves, which is between 0.4 and 42 m s-1 (van Bel & Ehlers 2005). Usually, the receptor

corresponding to a long range of a variation pulse (Stahlberg et al., 2006), which varies with the intensity of the stimulus, and its amplitude and speed decrease with increasing distance from its generation site (Davies, 2004, 2006). AP is an all-or-none depolarization that spreads passively from the excited cellular membrane region to the neighboring non-excited region. Excitation in plant cells depends on Ca+2 depolarization and Cl- and K+ repolarization, that spreads passively from the excited cellular membrane region to the neighboring non-excited region (Brenner et al., 2006). A similitude on electrical signal transmission between animal and plant organs has been postulated by Volkov & Ranatunga (2006), using the model

Fig. 1. Proposed mechanism of electric potential signals in plants *(Adapted from Volkov &* 

Fig. 2. The Hodgkin-Huxley (HH, 1952) equivalent circuit for an axon (A) and the modified

Electrical conduction rate of most of the plant action potentials studied so far is in the range of 0.01-0.2 m s-1 , i.e. much slower than the conduction velocity of action potentials in animal nerves, which is between 0.4 and 42 m s-1 (van Bel & Ehlers 2005). Usually, the receptor

HH circuit for sieve tubes in phloem (B) (*Volkov & Ranatunga*, *2006*).

presented in Figure 2.

*Ranatunga, 2006 and Gibert et al., 2006).*

potential lasts as long as the stimulus is present, being an electrical replica of the initial stimulus. If the stimulus is sufficiently large to cause the membrane potential to depolarize below a certain threshold, this will cause an action potential to be generated. It shows a large transient depolarization which is self perpetuating and therefore allows the rapid transmission of information over long distances.

Action potentials can propagate over short distances through plasmodesmata, and after it has reached the sieve element/companion cell (SE/CC) complex (Figure 3), it can travel over long distances along the SE plasma membrane in both directions.

Fig. 3. Action and variation potentials in plants. (*After Lautner et al. 2005; Fromm & Lautner, 2007*).

In contrast, a VP is generated at the plasma membrane of parenchyma cells (PAs) adjacent to xylem vessels (VEs) (Figure 3) by a hydraulic wave or a wounding substance. Because VPs were measured in SEs, it is suggested that they also can pass through the plasmodesmal network and can reach the phloem pathway. However, in contrast to APs, their amplitude will be reduced with increasing distance from the site of generation.

Fig. 4. An action potential recorded in Aloe vera spp. (*After Volkov et al., 2007*).

Action potentials (AP) induced in leaves of an Aloe vera spp. plant by thermal shock (flame) are described by Volkov et al., 2007 (Figure 4). Measurements were recorded at 500,000

Electrophysiology of Woody Plants 9

repolarization phases – and b. the mechanisms and pathways of signal propagation. The generation of APs occurs under different environmental and internal influences, like touch, light changes, cold treatment or cell expansion that trigger a voltage-dependent depolarization spike in an all-or-nothing manner. The depolarizations of a VP arise with an increase in turgor pressure cells experience as a result of a hydraulic pressure wave, that spreads through the xylem conduits after rain, embolism, bending, local wounds, organ excision or local burning. While APs and VPs can be triggered in excised organs, VPs depend on the pressure difference between the atmosphere and an intact plant interior.

The ionic mechanism of the VP is thought to involve a transient shutdown of a P-type H+- ATPase in the plasma membrane and differs from the mechanism underlying APs. Another defining characteristic of VPs is the hydraulic mode of propagation, that enables them but not APs — to pass through killed or poisoned areas. Unlike APs they can easily communicate between leaf and stem. VPs can move in both directions of the plant axis, while their amplitudes show a decrement of about 2.5% cm−1 and move with speeds that can be slower than APs in darkness and faster in bright light. The VPs move with a rapid pressure increase, establishing an axial pressure gradient in the xylem. This gradient translates distance (perhaps via changing kinetics in the rise of turgor pressure) into increasing lag phases for the pressure-induced depolarizations in the epidermis cells. VPs are not only ubiquitous among higher plants but represent a unique, defining characteristic

without parallels in lower plants or animals (Stahlberg et al., 2005; Baluska, 2010).

Fig. 7. Apple tree (*Malus domestica* Borkh), cv. Granny Schmidt electric behavior after tipping (A) and basal shoot removal (B). Electrodes are separated by 35 cm. (*After Gurovich, Rivera &* 

Figure 8) are presented below.

*García, 2011, unpublished data*).

Electric signals in different fruit bearing trees and other plants species are evaluated at the present, and the effects of different environmental stimuli on its magnitudes and interpretation is a major subject of research. Also, the large number of experiences, yet to be published and now on the peer review referral process in several scientific journals is indicative of a major breakthrough in our knowledge of plant electrical physiology. As an example, data on the effects of tipping and shoot removal in apple trees (Gurovich, Rivera & García, 2011, Figure 7), and dark – light cycles in olive trees (Gurovich and Cano, 2011,

High humidity and prolonged darkness will also suppress VP signaling.

scans/second and 2,000,000 scans/sample. Channel 1 is located on the leaf treated by thermal shock and channel 2 is located on a different leaf of the same plant. Distance between Ag/AgCl electrodes for each channel was 1 cm.

Stankovic et al. (1998) provide data on APs and VPs measured in *Helianthus annuus* stems by extracellular electrodes (Figure 5). The AP was elicited by electrical stimulation (±), and the VP by wounding (*W*).

Fig. 5. Action potentials (*APs*) and variation potentials (*VPs*) recorded in the stem of *Helianthus annuus* by extracellular electrodes, *E1*–*E4*. *Vertical arrows* indicate the moment of stimulation. *Arrowheads* point to the direction of propagation. (*After Stankovic et al., 1998*).

After a transient change in the membrane potential of plant cells (depolarization and subsequent repolarization), VPs and APs make use of the vascular bundles to achieve a potentially systemic spread through the entire plant. The principal difference used to differentiate VPs from APs is that VPs show longer, delayed repolarizations, as shown in Figure 6.

Fig. 6. APs (a to e) and VP (f to h) in plants (*After Stahlberg et al., 2006*).

VPs repolarizations show a large range of variation that makes a clear distinction to APs difficult; however, VPs and APs do differ more clearly in two aspects: a. the causal factors stimulating their appearance - the ionic mechanisms of their depolarization and

scans/second and 2,000,000 scans/sample. Channel 1 is located on the leaf treated by thermal shock and channel 2 is located on a different leaf of the same plant. Distance

Stankovic et al. (1998) provide data on APs and VPs measured in *Helianthus annuus* stems by extracellular electrodes (Figure 5). The AP was elicited by electrical stimulation (±), and the

Fig. 5. Action potentials (*APs*) and variation potentials (*VPs*) recorded in the stem of *Helianthus annuus* by extracellular electrodes, *E1*–*E4*. *Vertical arrows* indicate the moment of stimulation. *Arrowheads* point to the direction of propagation. (*After Stankovic et al., 1998*).

Fig. 6. APs (a to e) and VP (f to h) in plants (*After Stahlberg et al., 2006*).

After a transient change in the membrane potential of plant cells (depolarization and subsequent repolarization), VPs and APs make use of the vascular bundles to achieve a potentially systemic spread through the entire plant. The principal difference used to differentiate VPs from APs is that VPs show longer, delayed repolarizations, as shown in

VPs repolarizations show a large range of variation that makes a clear distinction to APs difficult; however, VPs and APs do differ more clearly in two aspects: a. the causal factors stimulating their appearance - the ionic mechanisms of their depolarization and

between Ag/AgCl electrodes for each channel was 1 cm.

VP by wounding (*W*).

Figure 6.

repolarization phases – and b. the mechanisms and pathways of signal propagation. The generation of APs occurs under different environmental and internal influences, like touch, light changes, cold treatment or cell expansion that trigger a voltage-dependent depolarization spike in an all-or-nothing manner. The depolarizations of a VP arise with an increase in turgor pressure cells experience as a result of a hydraulic pressure wave, that spreads through the xylem conduits after rain, embolism, bending, local wounds, organ excision or local burning. While APs and VPs can be triggered in excised organs, VPs depend on the pressure difference between the atmosphere and an intact plant interior. High humidity and prolonged darkness will also suppress VP signaling.

The ionic mechanism of the VP is thought to involve a transient shutdown of a P-type H+- ATPase in the plasma membrane and differs from the mechanism underlying APs. Another defining characteristic of VPs is the hydraulic mode of propagation, that enables them but not APs — to pass through killed or poisoned areas. Unlike APs they can easily communicate between leaf and stem. VPs can move in both directions of the plant axis, while their amplitudes show a decrement of about 2.5% cm−1 and move with speeds that can be slower than APs in darkness and faster in bright light. The VPs move with a rapid pressure increase, establishing an axial pressure gradient in the xylem. This gradient translates distance (perhaps via changing kinetics in the rise of turgor pressure) into increasing lag phases for the pressure-induced depolarizations in the epidermis cells. VPs are not only ubiquitous among higher plants but represent a unique, defining characteristic without parallels in lower plants or animals (Stahlberg et al., 2005; Baluska, 2010).

Electric signals in different fruit bearing trees and other plants species are evaluated at the present, and the effects of different environmental stimuli on its magnitudes and interpretation is a major subject of research. Also, the large number of experiences, yet to be published and now on the peer review referral process in several scientific journals is indicative of a major breakthrough in our knowledge of plant electrical physiology. As an example, data on the effects of tipping and shoot removal in apple trees (Gurovich, Rivera & García, 2011, Figure 7), and dark – light cycles in olive trees (Gurovich and Cano, 2011, Figure 8) are presented below.

Fig. 7. Apple tree (*Malus domestica* Borkh), cv. Granny Schmidt electric behavior after tipping (A) and basal shoot removal (B). Electrodes are separated by 35 cm. (*After Gurovich, Rivera & García, 2011, unpublished data*).

Electrophysiology of Woody Plants 11

Several micro-electrodes have been used for electrophysiological studies in plants. In most of our publications, electrical potentials are monitored continuously using own designed nonpolarizable Ag/AgCl microelectrodes inserted into different positions along the trunk; microelectrode characteristics have been reported by Gurovich & Hermosilla (2009), Gil et al. (2009), Oyarce & Gurovich (2011), and consist on a 0.35 mm-diameter silver wire (99.99% Ag), chlorated in a solution of HCl 0.1N for 30 s using a differential voltage of 2.5 V, to obtain an Ag/AgCl coating, which is inserted in a stainless steel hypodermic needle, 0.5 mm in diameter, filled with a KCl 3M solution; both needle ends are heat-sealed with polyethylene. Electrodes were inserted into the trunk using a low velocity electric microdriller, with a barbed microreel, penetrating the phloematic and cambium tissue; needle tip was further inserted into the xylematic tissue, 0.5–0.75 cm, by mechanical pressure. Each Ag/AgCl microelectrode was referenced to an identical microelectrode

In our work on electrophysiology, EP real time measurements are implemented using a multi channel voltmeter (Model 2701, Keithley Instruments, including a 20 channel switch module Keithley, model 7700), measuring DC and AC voltage in the range from 100 mV to 1000 V, in testing intervals from 1 to 100 ms. Signals obtained are analyzed with the software ExceLINX-1, an utility provided by Microsoftc Excel. All EP measurements are made by keeping the trees within a Faraday-type electromagnetic insulation cage, installed

Trees live in a continuously changing environment and although not all parts of the tree are exposed to the same stimuli at the same time, tree organs respond in a coordinated fashion, for example, by fast stomata closing under even mild water stress buildup, demonstrating the existence of communication between various regions of the tree. For years, researchers have concentrated their efforts on the study of chemical (hormonal) signals in trees, and very seldom considering that plants simultaneously show distinct electrical and hydraulic signals, which correlate to water stress conditions and other physiological stimuli as well. Considering the large leaf area of a tree, very large amounts of chemicals would need to be synthesized, transported and be perceived at the canopy, in order to respond to a signal

in the laboratory to control constant light and temperature conditions (Figure 10).

installed in the sand media, within the root system (Figure 9).

Fig. 9. The Ag/AgCl microelectrode construction.

**1 cm** 

coming from the roots.

**6. Research on plant electrophysiology of woody plants** 

In Figure 7A an electrical pulse is transmitted from the tree distal upper tipped point down to the microelectrode located 50 cm in the trunk, within the canopy, with a 3 s delay, and led to a maximal EP reduction of 6.93 ± 1.2 mV in 15 s, with an almost complete EP recovery in 90 s; however, no changes in the EP were measured at the base of the trunk. Elimination of a basal shoot from the rootstock (Figure 7 B) resulted in a EP 15.76 mV reduction, measured with a microelectrode located 5 cm above the rootstock – tree grafting area and a slight increase of 3.88 mV measured at the canopy.

Olive plants kept for 48 hr in total darkness were cyclically illuminated every 5 min for 1000 s periods and EP was measured at the root, rootstock, grafted tree and 2 shoots (Figure 8). A sharp reduction in EP values (on average 50 mV, with a polarity change) take place 3 to 5 s after each illumination cycle, with a slow EP recovery when dark conditions are restored. This behavior is much intense in shoots than in roots, grafted tree and rootstock, and each electric impulse travels throughout the whole plant with similar patterns and velocities.

#### **5. Plant electrophysiology research technology and applications**

Two techniques for the measurement of electrical currents in plant studies have been developed: a. non invasive surface recording and b. measurements using inserted thin metal electrodes (Fromm & Lautner, 2007). At different positions of the plant, from roots to fruits, electrodes are connected by insulated cables to a high – input impedance multichannel electrometer and a reference electrode is inserted in the soil. When all channels are stabilized electrically, the effect of many treatments on plant electric behavior can be evaluated, such as electrical stimulation at different organs in the symplastic continuum, to study its transmission dynamics within the plant, resulting from environmental stimuli like light – darkness sequences, drought - irrigation cycles, heat pulses at a specific leaf, localized chemical product applications, variable wind speed and air relative humidity conditions, or plant organ mechanical wounding, like trunk girdling, pruning, leaf and fruit thinning or root excision by underground tillage.

Fig. 8. Electrical behavior of Olive (*Olea europea*) trees) in alternate dark – light cycles (average values from 10 plants) (*After Gurovich & Cano, 2011, unpublished data*). L = light period at constant 45 watt m-2, at the canopy top).

In Figure 7A an electrical pulse is transmitted from the tree distal upper tipped point down to the microelectrode located 50 cm in the trunk, within the canopy, with a 3 s delay, and led to a maximal EP reduction of 6.93 ± 1.2 mV in 15 s, with an almost complete EP recovery in 90 s; however, no changes in the EP were measured at the base of the trunk. Elimination of a basal shoot from the rootstock (Figure 7 B) resulted in a EP 15.76 mV reduction, measured with a microelectrode located 5 cm above the rootstock – tree grafting area and a

Olive plants kept for 48 hr in total darkness were cyclically illuminated every 5 min for 1000 s periods and EP was measured at the root, rootstock, grafted tree and 2 shoots (Figure 8). A sharp reduction in EP values (on average 50 mV, with a polarity change) take place 3 to 5 s after each illumination cycle, with a slow EP recovery when dark conditions are restored. This behavior is much intense in shoots than in roots, grafted tree and rootstock, and each electric impulse travels throughout the whole plant with similar patterns and velocities.

Two techniques for the measurement of electrical currents in plant studies have been developed: a. non invasive surface recording and b. measurements using inserted thin metal electrodes (Fromm & Lautner, 2007). At different positions of the plant, from roots to fruits, electrodes are connected by insulated cables to a high – input impedance multichannel electrometer and a reference electrode is inserted in the soil. When all channels are stabilized electrically, the effect of many treatments on plant electric behavior can be evaluated, such as electrical stimulation at different organs in the symplastic continuum, to study its transmission dynamics within the plant, resulting from environmental stimuli like light – darkness sequences, drought - irrigation cycles, heat pulses at a specific leaf, localized chemical product applications, variable wind speed and air relative humidity conditions, or plant organ mechanical wounding, like trunk girdling, pruning, leaf and fruit thinning or

Fig. 8. Electrical behavior of Olive (*Olea europea*) trees) in alternate dark – light cycles (average values from 10 plants) (*After Gurovich & Cano, 2011, unpublished data*). L = light

**5. Plant electrophysiology research technology and applications** 

slight increase of 3.88 mV measured at the canopy.

root excision by underground tillage.

period at constant 45 watt m-2, at the canopy top).

Several micro-electrodes have been used for electrophysiological studies in plants. In most of our publications, electrical potentials are monitored continuously using own designed nonpolarizable Ag/AgCl microelectrodes inserted into different positions along the trunk; microelectrode characteristics have been reported by Gurovich & Hermosilla (2009), Gil et al. (2009), Oyarce & Gurovich (2011), and consist on a 0.35 mm-diameter silver wire (99.99% Ag), chlorated in a solution of HCl 0.1N for 30 s using a differential voltage of 2.5 V, to obtain an Ag/AgCl coating, which is inserted in a stainless steel hypodermic needle, 0.5 mm in diameter, filled with a KCl 3M solution; both needle ends are heat-sealed with polyethylene. Electrodes were inserted into the trunk using a low velocity electric microdriller, with a barbed microreel, penetrating the phloematic and cambium tissue; needle tip was further inserted into the xylematic tissue, 0.5–0.75 cm, by mechanical pressure. Each Ag/AgCl microelectrode was referenced to an identical microelectrode installed in the sand media, within the root system (Figure 9).

In our work on electrophysiology, EP real time measurements are implemented using a multi channel voltmeter (Model 2701, Keithley Instruments, including a 20 channel switch module Keithley, model 7700), measuring DC and AC voltage in the range from 100 mV to 1000 V, in testing intervals from 1 to 100 ms. Signals obtained are analyzed with the software ExceLINX-1, an utility provided by Microsoftc Excel. All EP measurements are made by keeping the trees within a Faraday-type electromagnetic insulation cage, installed in the laboratory to control constant light and temperature conditions (Figure 10).

Fig. 9. The Ag/AgCl microelectrode construction.

#### **6. Research on plant electrophysiology of woody plants**

Trees live in a continuously changing environment and although not all parts of the tree are exposed to the same stimuli at the same time, tree organs respond in a coordinated fashion, for example, by fast stomata closing under even mild water stress buildup, demonstrating the existence of communication between various regions of the tree. For years, researchers have concentrated their efforts on the study of chemical (hormonal) signals in trees, and very seldom considering that plants simultaneously show distinct electrical and hydraulic signals, which correlate to water stress conditions and other physiological stimuli as well. Considering the large leaf area of a tree, very large amounts of chemicals would need to be synthesized, transported and be perceived at the canopy, in order to respond to a signal coming from the roots.

Electrophysiology of Woody Plants 13

Recent studies have associated the effect of water stress build-up, irrigation and light with electrical signaling in fruit bearing tree species including avocado (*Persea americana* Mill.), blueberry (*Vaccinium spp*.), lemon (*Citrus limon* (L.) Buró) and olive (*Olea europaea* L.) (Gil et al., 2008; Gurovich & Hermosilla, 2009; Oyarce & Gurovich, 2010, 2011). Some results are included below as examples on this research line, aimed to develop new real – time plant stress sensors based on tree electric behavior, for the automation of irrigation systems

Electric potential (EP) differences have been detected between the base of the stem and leaf petiole and between the base of the stem and the leaf area, located in the upper half of the tree canopy, in response to drought, irrigation and diurnal light and dark cycles (Figure 12). Orders of magnitude of the observed EP variation in those studies were similar to values observed by other authors (Fromm, 2006; Davies, 2006). Electric potential variations observed in avocado trees in response to decreased soil water content have been associated with a decrease in stomata conductance (gs) (Gil et al., 2009), indicating that stomata closure might be induced or at least associated with an electrical signal that travels through the phloem at a speed of 2.4 cm min-1. Larger changes in electric potential behavior have been detected in response to drought compared to watering. Thus, an extra-cellular electrical signal appears to be involved in root to leaf communication, initiating stomata closure at a very early stage of drought stress. These drought-induced electrical signals were also related

to changes in gs, in concordance to other studies published by Fromm & Fei (1998).

Fig. 12. Electrical potential responses of avocado plants to light and dark and irrigation. (A) EP responses according to the day time. (B) Effect of irrigation on EP behavior *(Adapted from* 

*Gurovich & Hermosilla 2009).* 

operation, optimizing water and energy efficiency in fruit production.

Fig. 10. Schematic diagram of the digital acquisition system for recording voltage differences between the base of the trunk and the canopy. (*After Gurovich and Hermosilla, 2009*).

Limited reaearch has been reported on signaling in woody trees (*Tilia* and *Prunus*, Boari & Malone 1993; *Salix*, Fromm & Spanswick 1993; Grindl et al., 1999; Oak, Morat et al., 1994; Koppan et al., 2000, 2002; *Vitis*, Mancuso,1999; *Poplar*, Gibert et al., 2006) although it is in such plants that the need for rapid and efficient signals other than chemicals becomes more obvious.

Gibert et al., 2006 present relevant information on the electric long term (2 year) behavior of a single poplar tree, focused on the spatial and temporal variations of the electric potential distribution (Figure 11), with its correlation to air temperature, concluding that seasonal fluctuations of EP trends may be correlated to sap flow patterns, largely influenced by seasonal sap constituents and concentrations.

Fig. 11. Top: potential signals for the December 2003–April 2004 period, expressed as relative potential values *(see Gibert et al., 2006, Fig. 1 for electrode location).* Bottom: outdoor temperature measured near the tree. Tick marks fall at midday.

Fig. 10. Schematic diagram of the digital acquisition system for recording voltage differences

Limited reaearch has been reported on signaling in woody trees (*Tilia* and *Prunus*, Boari & Malone 1993; *Salix*, Fromm & Spanswick 1993; Grindl et al., 1999; Oak, Morat et al., 1994; Koppan et al., 2000, 2002; *Vitis*, Mancuso,1999; *Poplar*, Gibert et al., 2006) although it is in such plants that the need for rapid and efficient signals other than chemicals becomes more

Gibert et al., 2006 present relevant information on the electric long term (2 year) behavior of a single poplar tree, focused on the spatial and temporal variations of the electric potential distribution (Figure 11), with its correlation to air temperature, concluding that seasonal fluctuations of EP trends may be correlated to sap flow patterns, largely influenced by

Fig. 11. Top: potential signals for the December 2003–April 2004 period, expressed as relative potential values *(see Gibert et al., 2006, Fig. 1 for electrode location).* Bottom: outdoor

temperature measured near the tree. Tick marks fall at midday.

between the base of the trunk and the canopy. (*After Gurovich and Hermosilla, 2009*).

obvious.

seasonal sap constituents and concentrations.

Recent studies have associated the effect of water stress build-up, irrigation and light with electrical signaling in fruit bearing tree species including avocado (*Persea americana* Mill.), blueberry (*Vaccinium spp*.), lemon (*Citrus limon* (L.) Buró) and olive (*Olea europaea* L.) (Gil et al., 2008; Gurovich & Hermosilla, 2009; Oyarce & Gurovich, 2010, 2011). Some results are included below as examples on this research line, aimed to develop new real – time plant stress sensors based on tree electric behavior, for the automation of irrigation systems operation, optimizing water and energy efficiency in fruit production.

Electric potential (EP) differences have been detected between the base of the stem and leaf petiole and between the base of the stem and the leaf area, located in the upper half of the tree canopy, in response to drought, irrigation and diurnal light and dark cycles (Figure 12). Orders of magnitude of the observed EP variation in those studies were similar to values observed by other authors (Fromm, 2006; Davies, 2006). Electric potential variations observed in avocado trees in response to decreased soil water content have been associated with a decrease in stomata conductance (gs) (Gil et al., 2009), indicating that stomata closure might be induced or at least associated with an electrical signal that travels through the phloem at a speed of 2.4 cm min-1. Larger changes in electric potential behavior have been detected in response to drought compared to watering. Thus, an extra-cellular electrical signal appears to be involved in root to leaf communication, initiating stomata closure at a very early stage of drought stress. These drought-induced electrical signals were also related to changes in gs, in concordance to other studies published by Fromm & Fei (1998).

Fig. 12. Electrical potential responses of avocado plants to light and dark and irrigation. (A) EP responses according to the day time. (B) Effect of irrigation on EP behavior *(Adapted from Gurovich & Hermosilla 2009).* 

Electrophysiology of Woody Plants 15

Fig. 13. Electric potentials (EP) in avocado trees during 4 irrigated days. (Average values for 7 trees). Micro electrodes inserted at 25 (A) and 85 (B) cm above the soil surface. (*Adapted* 

behavior have been proposed by Trewavas & Malho (1997), Zimmermann et al. (1997), Stankovic et al. (1998), Volkov & Brown (2006), Volkov et al. (2008), Baluska et al. (2004); Brenner et al. (2006). All these authors agree with the idea that a certain stimuli receptor must be present at the cell membrane, and that a transient polarization, induced by specific

Results presented in these papers indicate a clear and rapid mechanism of electrical signal generation and transmission in woody plants, positively correlated to the intensity and duration of stimuli, such as light intensity, water availability and mechanical injury. The electrical signal is generated in a specific organ or tissue and is transmitted rapidly in the form of AP or VP to other tissues or organs of the plant. The measurement of electrical potentials can be used as a tool for real-time measurement of plant physiological responses, opening the possibility of using this technology as a tool for early detection of stress and for

Sedimenting amyloplasts act as statoliths in root and shoot cells specialized for gravisensing; also different auxins are involved in the gravi - stimulated differential growth known a *gravitropism*. However, no comprehensive explanation is available related to gravity signal perception and its transduction pathways in plants from the sedimenting

ion fluxes through this membrane, is the ultimate agent of the EP signal generation.

the operation of automatic high frequency irrigation systems.

statoliths to the motoric response of organ bending (Baluska et al., 2006).

**7. Electrophysiology of some plant tropisms** 

*from Oyarce & Gurovich, 2010*).

According to Gurovich & Hermosilla (2009) effects of sunset, daybreak and water application are clearly reflected as fast changes in the EP between the base and leaf area electrode locations on the trunk or stem (Figure 12). Electrical potential fluctuations during light and dark periods may be due to differential sap flow velocity at different times of the day as a result of stomata closure during the night. Electrical potential values were reduced during the initial hours after daybreak, and started to increase after midday, as a result of transient water stress conditions; the first dark hours after sunset resulted in rapid increases of voltages and after midnight these increases tended to slow down. Also, a small but consistent increase in voltage was detected about 1–2 hours before daybreak. Explanations for this behavior may also be related to circadian rhythms detected in plants, but need further study to be fully understood (Dodd et al, 2005: Horta et al., 2007).

The effects of irrigation and day – night cycles on the electric behavior of avocado trees has been reported also by Oyarce & Gurovich (2010) under controlled conditions (Figure 13) EP vary in daily cycles throughout the measurement period: during the morning (2:00 to 7:59 AM), the mean 4-day EP average is in the range -89.991 ± 0, 46 mV at 25 cm and -121.53± 0.5 mV at 85 cm above the ground, respectively. During the afternoon (14:00 at 19:59 PM), EP values rise, reaching mean values of -79.71 ± 2.16 mV at 25 cm and -104.05 ± 1.21 mV at 85 cm above the ground, respectively, and maximum values of -76.16 ± 20 mV at 17:10 PM (25 cm) and -101.35 ± 5.05 mV at 18:30 PM (85 cm). These values indicate the existence of significant differences in EP between the periods compared (see Oyarce & Gurovich, 2010, Table 2). The effect of irrigation applied every day at 11:00 AM is clearly expressed by a significant decrease in EP, of the order of 7.10 ± 1.56 mV and 7.53 ± 1.39 mV, for micro electrodes inserted in the tree trunk at 25 and 85 cm above the soil surface respectively, representing specific characteristics of an action potential (AP). The recovery of EP values measured before irrigation requires an average period of 16 minutes. On the fourth day, irrigation applied at 15:35 PM did not induce changes in the electrical potential probably due to a low atmospheric demand at that time.

Oyarce & Gurovich (2011) examined the nature and specific characteristics of the electrical response to wounding in the woody plant Persea americana (avocado) cv. Hass. Under field conditions, wounds can be the result of insect activity, strong winds or handling injury during fruit harvest. Evidence for extracellular EP signaling in avocado trees after mechanical injury is expressed in the form of variation potentials. For tipping and pruning, signal velocities of 8.7 and 20.9 cm/s-1, respectively, are calculated, based on data measured with Ag/AgCl microelectrodes inserted at different positions of the trunk (Figure 14 *a* to *d*). EP signal intensity decreased with increasing distance between the tipping and pruning point and the electrode. Recovery time to pre-tipping or pre-pruning EP values was also affected by the distance and signal intensity from the tipping or pruning point to the specific electrode position.

A significant EP signal, corresponding to a variation potential, is generated as a response of tipping or pruning avocado plants (Figure 14 a to d); the signal was transmitted along the tree trunk at a specific velocity, which is dependent on the distance to the mechanical injury. Mancuso (1999) reported a propagation velocity of the front of the main negative-going signal(VP) of 2.7 mm s−1, while an AP propagated along the shoot with a velocity of about 100 mm s−1. The EP signal intensity also decreases with distance between the mechanical injury sites to the electrode position in the trunk. Several physiological explanations for this

According to Gurovich & Hermosilla (2009) effects of sunset, daybreak and water application are clearly reflected as fast changes in the EP between the base and leaf area electrode locations on the trunk or stem (Figure 12). Electrical potential fluctuations during light and dark periods may be due to differential sap flow velocity at different times of the day as a result of stomata closure during the night. Electrical potential values were reduced during the initial hours after daybreak, and started to increase after midday, as a result of transient water stress conditions; the first dark hours after sunset resulted in rapid increases of voltages and after midnight these increases tended to slow down. Also, a small but consistent increase in voltage was detected about 1–2 hours before daybreak. Explanations for this behavior may also be related to circadian rhythms detected in plants, but need

The effects of irrigation and day – night cycles on the electric behavior of avocado trees has been reported also by Oyarce & Gurovich (2010) under controlled conditions (Figure 13) EP vary in daily cycles throughout the measurement period: during the morning (2:00 to 7:59 AM), the mean 4-day EP average is in the range -89.991 ± 0, 46 mV at 25 cm and -121.53± 0.5 mV at 85 cm above the ground, respectively. During the afternoon (14:00 at 19:59 PM), EP values rise, reaching mean values of -79.71 ± 2.16 mV at 25 cm and -104.05 ± 1.21 mV at 85 cm above the ground, respectively, and maximum values of -76.16 ± 20 mV at 17:10 PM (25 cm) and -101.35 ± 5.05 mV at 18:30 PM (85 cm). These values indicate the existence of significant differences in EP between the periods compared (see Oyarce & Gurovich, 2010, Table 2). The effect of irrigation applied every day at 11:00 AM is clearly expressed by a significant decrease in EP, of the order of 7.10 ± 1.56 mV and 7.53 ± 1.39 mV, for micro electrodes inserted in the tree trunk at 25 and 85 cm above the soil surface respectively, representing specific characteristics of an action potential (AP). The recovery of EP values measured before irrigation requires an average period of 16 minutes. On the fourth day, irrigation applied at 15:35 PM did not induce changes in the electrical potential probably

Oyarce & Gurovich (2011) examined the nature and specific characteristics of the electrical response to wounding in the woody plant Persea americana (avocado) cv. Hass. Under field conditions, wounds can be the result of insect activity, strong winds or handling injury during fruit harvest. Evidence for extracellular EP signaling in avocado trees after mechanical injury is expressed in the form of variation potentials. For tipping and pruning, signal velocities of 8.7 and 20.9 cm/s-1, respectively, are calculated, based on data measured with Ag/AgCl microelectrodes inserted at different positions of the trunk (Figure 14 *a* to *d*). EP signal intensity decreased with increasing distance between the tipping and pruning point and the electrode. Recovery time to pre-tipping or pre-pruning EP values was also affected by the distance and signal intensity from the tipping or pruning point to the specific

A significant EP signal, corresponding to a variation potential, is generated as a response of tipping or pruning avocado plants (Figure 14 a to d); the signal was transmitted along the tree trunk at a specific velocity, which is dependent on the distance to the mechanical injury. Mancuso (1999) reported a propagation velocity of the front of the main negative-going signal(VP) of 2.7 mm s−1, while an AP propagated along the shoot with a velocity of about 100 mm s−1. The EP signal intensity also decreases with distance between the mechanical injury sites to the electrode position in the trunk. Several physiological explanations for this

further study to be fully understood (Dodd et al, 2005: Horta et al., 2007).

due to a low atmospheric demand at that time.

electrode position.

Fig. 13. Electric potentials (EP) in avocado trees during 4 irrigated days. (Average values for 7 trees). Micro electrodes inserted at 25 (A) and 85 (B) cm above the soil surface. (*Adapted from Oyarce & Gurovich, 2010*).

behavior have been proposed by Trewavas & Malho (1997), Zimmermann et al. (1997), Stankovic et al. (1998), Volkov & Brown (2006), Volkov et al. (2008), Baluska et al. (2004); Brenner et al. (2006). All these authors agree with the idea that a certain stimuli receptor must be present at the cell membrane, and that a transient polarization, induced by specific ion fluxes through this membrane, is the ultimate agent of the EP signal generation.

Results presented in these papers indicate a clear and rapid mechanism of electrical signal generation and transmission in woody plants, positively correlated to the intensity and duration of stimuli, such as light intensity, water availability and mechanical injury. The electrical signal is generated in a specific organ or tissue and is transmitted rapidly in the form of AP or VP to other tissues or organs of the plant. The measurement of electrical potentials can be used as a tool for real-time measurement of plant physiological responses, opening the possibility of using this technology as a tool for early detection of stress and for the operation of automatic high frequency irrigation systems.

#### **7. Electrophysiology of some plant tropisms**

Sedimenting amyloplasts act as statoliths in root and shoot cells specialized for gravisensing; also different auxins are involved in the gravi - stimulated differential growth known a *gravitropism*. However, no comprehensive explanation is available related to gravity signal perception and its transduction pathways in plants from the sedimenting statoliths to the motoric response of organ bending (Baluska et al., 2006).

Electrophysiology of Woody Plants 17

**8. Plant electrophysiology modulated by neurotransmitters, neuroregulators** 

Plants produce a wide range of phytochemicals that mediate cell functions and translate environmental cues for survival; many of these molecules are also found as neuro regulatory molecules in animals, including humans. For example, the human neurotransmitter melatonin (N-acetyl-5-methoxytryptamine) is a common molecule associated with timing of circadian rhythms in many organisms, including higher plants. Its major concentrations are located within the phloem conducting vessels and it has been suggested that its action is centered in the electrochemical processes involved in plasmodesmata synaptic – like contacts. Plant synapse has been proposed, since actin cytoskeleton-based adhesive contacts between plant cells resemble the neuronal and immune synapses found in animals (Baluska et al., 2005). A comprehensive review of neurotransmitters in plants is provided by V. V. Roschina in the book *"Neurotransmitters in* 

Whereas glutamate and glycine were shown to gate Ca+2-permeable channels in plants, glutamate was reported to rapidly depolarize the plant cell plasma membrane in a process mediated by glutamate receptors (Baluška, 2010; Felle & Zimmermann, 2007); plant glutamate receptors have all the features of animal neuronal glutamate receptors, inducing plant APs (Stolarz et al., 2010) . These publications strongly suggest that glutamate serves as a neurotransmitter-like in cell-to-cell communication in plants too. Whereas glutamate might represent a plant excitatory transmitter, gamma-aminobutyric acid (GABA) seems to act as an inhibitory transmitter in plants, as it does similarly in animal neurons. For instance, it is well documented that GABA is rapidly produced under diverse stress situations and also that GABA can be transported from cell-to-cell across plant tissues (Bouche et al., 2003). Many fascinating questions in future research will define the role of neurotransmitters, neuroregulators and neurotoxins in the growth and development of plants. As newer technologies emerge, it will become possible to understand more about the role of neurological compounds in the inner workings of plant metabolism, plant environment interactions and plant electrophysiology. However, signaling molecules, by their nature, are short lived, unstable, difficult to detect and quantify, because they are highly reactive, and

**and neurotoxins** 

*plant life"* (2001).

al., (2008).

**10. Conclusions** 

present in small concentrations within plant tissues.

**9. Electrophysiological control of cyclical oscillations in plants.** 

Sanchez et al. (2011) reviewed the interaction between the circadian clock of higher plants to that of metabolic and physiological processes that coordinate growth and performance under a predictable, albeit changing environment. The circadian clock of plants and abioticstress tolerance appear to be firmly interconnected processes, by means of electrophysiological signaling (Volkov et al., 2011). Time oscillations (circadian clocks) in plant membrane transport, including model predictions, experimental validation, and physiological implications has been reported by Mancuso & Shabala (2006) and Shabala et

Plants have evolved sophisticated systems to sense environmental abiotic and biotic stimuli for adaptation and to produce signals to other cells for coordinated actions, synchronizing

Fig. 14. *a*) Average EP speed of transmission along the trunk, as a result of tipping (n = 5 plants), t (s) = time at which the electrode detected the electric signal, ξ (cm) = distance of electrodes from the tipping point. Error bars represents ±1 std. dev. *b*) Relative intensity of EP as a result of tipping (n = 5 plants). ɸ ex = relative intensity of the signal (%), ξ (cm) = distance from the electrode to the tipping point. Error bars represents ±1 std. dev. *c*) Average EP speed of transmission along the trunk after pruning, measured above and below the pruned branch (n = 5 plants), t (s) = time at which the electrode detected the electric signal, ξ (cm) = distance of electrodes from the pruning point. Error bars represents +1 std. dev. *d*) Recovery time of the pre-tipping EP potential (n = 5 plants), τ = recovery time signal, ξ (cm) = distance from the electrode to the tipping point. Error bars represents ±1 std. dev (*after Oyarce & Gurovich, 2011*).

Bioelectrochemical signaling in green plants induced by photosensory systems have been reported by Volkov et al., (2004). The generation of electrophysiological responses induced by blue and red photosensory systems was observed in soybean plants. A phototropic response is a sequence of the following four processes: reception of a directional light signal, signal transduction, transformation of the signal into a physiological response, and the production of a directional growth response. It was found that the irradiation of soybean plants at 450±50, 670, and 730 nm induces APs with duration times and amplitudes of approximately 0.3 ms and 60 mV. Plants respond to light ranging from ultraviolet to far-red using specific photoreceptors and natural radiation simultaneously activates more than one photoreceptor in higher plants; these receptors initiate distinct signaling pathways leading to wavelength-specific light responses. Three types of plant photoreceptors that have been identified at the molecular level are phototropins, cryptochromes, and phytochromes respectively.

(a) (b)

(c) (d)

dev (*after Oyarce & Gurovich, 2011*).

respectively.

Fig. 14. *a*) Average EP speed of transmission along the trunk, as a result of tipping (n = 5 plants), t (s) = time at which the electrode detected the electric signal, ξ (cm) = distance of electrodes from the tipping point. Error bars represents ±1 std. dev. *b*) Relative intensity of EP as a result of tipping (n = 5 plants). ɸ ex = relative intensity of the signal (%), ξ (cm) = distance from the electrode to the tipping point. Error bars represents ±1 std. dev. *c*) Average EP speed of transmission along the trunk after pruning, measured above and below the pruned branch (n = 5 plants), t (s) = time at which the electrode detected the electric signal, ξ (cm) = distance of electrodes from the pruning point. Error bars represents +1 std. dev. *d*) Recovery time of the pre-tipping EP potential (n = 5 plants), τ = recovery time signal, ξ (cm) = distance from the electrode to the tipping point. Error bars represents ±1 std.

Bioelectrochemical signaling in green plants induced by photosensory systems have been reported by Volkov et al., (2004). The generation of electrophysiological responses induced by blue and red photosensory systems was observed in soybean plants. A phototropic response is a sequence of the following four processes: reception of a directional light signal, signal transduction, transformation of the signal into a physiological response, and the production of a directional growth response. It was found that the irradiation of soybean plants at 450±50, 670, and 730 nm induces APs with duration times and amplitudes of approximately 0.3 ms and 60 mV. Plants respond to light ranging from ultraviolet to far-red using specific photoreceptors and natural radiation simultaneously activates more than one photoreceptor in higher plants; these receptors initiate distinct signaling pathways leading to wavelength-specific light responses. Three types of plant photoreceptors that have been identified at the molecular level are phototropins, cryptochromes, and phytochromes

#### **8. Plant electrophysiology modulated by neurotransmitters, neuroregulators and neurotoxins**

Plants produce a wide range of phytochemicals that mediate cell functions and translate environmental cues for survival; many of these molecules are also found as neuro regulatory molecules in animals, including humans. For example, the human neurotransmitter melatonin (N-acetyl-5-methoxytryptamine) is a common molecule associated with timing of circadian rhythms in many organisms, including higher plants. Its major concentrations are located within the phloem conducting vessels and it has been suggested that its action is centered in the electrochemical processes involved in plasmodesmata synaptic – like contacts. Plant synapse has been proposed, since actin cytoskeleton-based adhesive contacts between plant cells resemble the neuronal and immune synapses found in animals (Baluska et al., 2005). A comprehensive review of neurotransmitters in plants is provided by V. V. Roschina in the book *"Neurotransmitters in plant life"* (2001).

Whereas glutamate and glycine were shown to gate Ca+2-permeable channels in plants, glutamate was reported to rapidly depolarize the plant cell plasma membrane in a process mediated by glutamate receptors (Baluška, 2010; Felle & Zimmermann, 2007); plant glutamate receptors have all the features of animal neuronal glutamate receptors, inducing plant APs (Stolarz et al., 2010) . These publications strongly suggest that glutamate serves as a neurotransmitter-like in cell-to-cell communication in plants too. Whereas glutamate might represent a plant excitatory transmitter, gamma-aminobutyric acid (GABA) seems to act as an inhibitory transmitter in plants, as it does similarly in animal neurons. For instance, it is well documented that GABA is rapidly produced under diverse stress situations and also that GABA can be transported from cell-to-cell across plant tissues (Bouche et al., 2003).

Many fascinating questions in future research will define the role of neurotransmitters, neuroregulators and neurotoxins in the growth and development of plants. As newer technologies emerge, it will become possible to understand more about the role of neurological compounds in the inner workings of plant metabolism, plant environment interactions and plant electrophysiology. However, signaling molecules, by their nature, are short lived, unstable, difficult to detect and quantify, because they are highly reactive, and present in small concentrations within plant tissues.

#### **9. Electrophysiological control of cyclical oscillations in plants.**

Sanchez et al. (2011) reviewed the interaction between the circadian clock of higher plants to that of metabolic and physiological processes that coordinate growth and performance under a predictable, albeit changing environment. The circadian clock of plants and abioticstress tolerance appear to be firmly interconnected processes, by means of electrophysiological signaling (Volkov et al., 2011). Time oscillations (circadian clocks) in plant membrane transport, including model predictions, experimental validation, and physiological implications has been reported by Mancuso & Shabala (2006) and Shabala et al., (2008).

#### **10. Conclusions**

Plants have evolved sophisticated systems to sense environmental abiotic and biotic stimuli for adaptation and to produce signals to other cells for coordinated actions, synchronizing

Electrophysiology of Woody Plants 19

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#### **11. References**


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**2** 

*Ferrara Italy* 

**Pacemaker Currents in Dopaminergic Neurones** 

In the olfactory bulb (OB) dopaminergic (DA) neurones constitute a fraction of the cells occupying the most external (glomerular) layer (Halász et al.1977). In this region, populated by three types of interneurons, periglomerular (PG) cells, short-axon cells and external tufted (ET) cells (Halász1990) - often collectively referred to as juxtaglomerular cells - an estimated 10% of the neurones in adulthood are positive for tyrosine hydroxylase (TH) (McLean and Shipley1988; Kratskin and Belluzzi2003), the rate limiting enzyme for dopamine synthesis. Dopaminergic neurones in the glomerular layer include PG cells (Gall et al.1987; Kosaka et al.1985) and a fraction of ET cells (Halász1990). Several studies have focused on the role of dopamine in the olfactory bulb, using immunohistochemical (Baker et al.1983; Guthrie et al.1991), behavioral (Doty and Risser1989), and electrophysiological techniques (Nowycky et al.1983; Ennis et al.2001; Davila et al.2003). The more complete description of the functional properties of DA neurons in the OB is probably the paper of Pignatelli (Pignatelli et al.2005), but it was incomplete, as it did not consider the contribution

A property shared by many DA neurons in the CNS is their capacity to generate rhythmic action potentials even in the absence of synaptic inputs (Grace and Onn1989; Hainsworth et al.1991; Yung et al.1991; Feigenspan et al.1998; Neuhoff et al.2002). In this paper we show for the first time that DA cells in the glomerular layer of the olfactory bulb possess a pacemaker activity, and we provide an explanation for the ionic basis of rhythm generation in these

There is an additional reason to study the functional properties of DA neurones in the OB other than their role in olfaction. The olfactory bulb is one of the rare regions of the mammalian CNS in which new cells, derived from stem cells in the anterior subventricular zone, are also added in adulthood (Gross2000). In the OB, these cells differentiate in interneurones in the granular and glomerular layers. Among these cells there are DA neurones (Betarbet et al.1996; Baker et al.2001), and this has raised a remarkable interest because, for their accessibility, they could provide a convenient source of autologous DA neurons for transplant therapies in neurodegenerative diseases, like Parkinson's disease.

of the inward rectifier currents, a lacuna which is filled in the present work.

**1. Introduction** 

cells.

**of the Mice Olfactory Bulb** 

Angela Pignatelli, Cristina Gambardella,

*Università di Ferrara, Dip. Biologia ed Evoluzione,* 

Mirta Borin, Alex Fogli Iseppe and Ottorino Belluzzi

*Sezione di Fisiologia & Biofisica – Centro di Neuroscienze,* 


## **Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb**

Angela Pignatelli, Cristina Gambardella, Mirta Borin, Alex Fogli Iseppe and Ottorino Belluzzi *Università di Ferrara, Dip. Biologia ed Evoluzione, Sezione di Fisiologia & Biofisica – Centro di Neuroscienze, Ferrara Italy* 

### **1. Introduction**

24 Electrophysiology – From Plants to Heart

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wounding. Plant Physiology 149: 1593–1600.

1885–1891.

In the olfactory bulb (OB) dopaminergic (DA) neurones constitute a fraction of the cells occupying the most external (glomerular) layer (Halász et al.1977). In this region, populated by three types of interneurons, periglomerular (PG) cells, short-axon cells and external tufted (ET) cells (Halász1990) - often collectively referred to as juxtaglomerular cells - an estimated 10% of the neurones in adulthood are positive for tyrosine hydroxylase (TH) (McLean and Shipley1988; Kratskin and Belluzzi2003), the rate limiting enzyme for dopamine synthesis. Dopaminergic neurones in the glomerular layer include PG cells (Gall et al.1987; Kosaka et al.1985) and a fraction of ET cells (Halász1990). Several studies have focused on the role of dopamine in the olfactory bulb, using immunohistochemical (Baker et al.1983; Guthrie et al.1991), behavioral (Doty and Risser1989), and electrophysiological techniques (Nowycky et al.1983; Ennis et al.2001; Davila et al.2003). The more complete description of the functional properties of DA neurons in the OB is probably the paper of Pignatelli (Pignatelli et al.2005), but it was incomplete, as it did not consider the contribution of the inward rectifier currents, a lacuna which is filled in the present work.

A property shared by many DA neurons in the CNS is their capacity to generate rhythmic action potentials even in the absence of synaptic inputs (Grace and Onn1989; Hainsworth et al.1991; Yung et al.1991; Feigenspan et al.1998; Neuhoff et al.2002). In this paper we show for the first time that DA cells in the glomerular layer of the olfactory bulb possess a pacemaker activity, and we provide an explanation for the ionic basis of rhythm generation in these cells.

There is an additional reason to study the functional properties of DA neurones in the OB other than their role in olfaction. The olfactory bulb is one of the rare regions of the mammalian CNS in which new cells, derived from stem cells in the anterior subventricular zone, are also added in adulthood (Gross2000). In the OB, these cells differentiate in interneurones in the granular and glomerular layers. Among these cells there are DA neurones (Betarbet et al.1996; Baker et al.2001), and this has raised a remarkable interest because, for their accessibility, they could provide a convenient source of autologous DA neurons for transplant therapies in neurodegenerative diseases, like Parkinson's disease.

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 27

After disruption, about 60% of the cells continued to fire spontaneous action potentials under current-clamp condition (Fig. 2B) without any significant alteration of the firing frequency (7.84 + 2.44 Hz, n = 14). Interspike intervals were rather constant in most of the cells (Fig. 2C), and irregular in others for the presence of sporadic misses. Occasionally, especially in isolated cells (see below), the firing was structured in bursts. We found no

This spontaneous activity was completely blocked by TTX (0.3 M) or by Cd+ (100 M), but persisted after block of glutamatergic and GABAergic synaptic transmission with kynurenate 1 mM and bicuculline (10 M), suggesting that it was due to intrinsic properties of the cell membrane and was not driven by external synaptic inputs, as it resulted even more obviously by the observation that spontaneous activity was maintained also in

Occasionally we did observe spontaneous synaptic currents, which were completely blocked by a mixture of 1 mM kynurenate and 10 M bicuculline (not shown), and which

Fig. 2. Spontaneous activity in DA neurones in thin slices. A – Action currents in cellattached mode B – Action potentials in whole-cell mode C – Frequency distribution of the inter-event time for the cell shown in panel B. D – Frequency of spontaneous firing in TH-GFP cells under the indicated experimental conditions. CA cell attached, WC whole cell.

currents were elicited both by step and ramp depolarisations.

We studied the dopaminergic neurones under current- and voltage-clamp conditions to characterise the ionic currents underlying spontaneous firing. In voltage-clamped neurones,

Depolarisation activates a complicated pattern of current flow, in which a variety of conductances coexist, the most prominent of which were a fast transient sodium current and a non-inactivating potassium current (Fig. 3A, B). We identified specific ionic currents present in the cells by measurements of their voltage-dependence and kinetics during step

correlation of the firing frequency with cell size.

were not further investigated for the purpose of this study.

dissociated cells (Fig. 2D).

### **2. Results**

#### **2.1 Localisation and general properties of TH-GFP cells**

Generation of transgenic mice (TH-GFP/21-31) was described in previous papers (Sawamoto et al.2001; Matsushita et al.2002). The transgene construct contained the 9.0-kb 5'-flanking region of the rat tyrosine hydroxylase (TH) gene, the second intron of the rabbit -globin gene, cDNA encoding green fluorescent protein (GFP), and polyadenylation signals of the rabbit -globin and simian virus 40 early genes.

Cells expressing the GFP transgene under the TH promoter (TH-GFP) occurred primarily in the glomerular layer of the main olfactory bulb (Fig. 1A,B). The intraglomerular processes of these cells displayed high levels of TH-GFP expression, and their intertwine delimitates the glomeruli, with the soma of GFP+ cells laying around them.

Recordings with the patch-clamp technique in the whole-cell configuration were obtained from 368 DA cells in the glomerular layer following the procedures described in Pignatelli et al., 2005.

Cell dimensions were rather variable, as shown in Fig. 1C. Previous studies have suggested that there are two populations of DA neurones in the adult OB, based on size or location (Halász1990; Baker et al.1983). In fact, the distribution of the mean cell diameter of GFP+ cells could be best fitted with two Gaussian curves, identifying two subpopulations with average sizes of 5.67 + 0.96 μm and 9.48 + 2.39 μm (R2 = 0.991); the same result could be obtained from the analysis of the membrane capacitances, whose frequency distribution could be best fitted by two Gaussians (5.41 ± 1.5 pF and 10.63 ± 3.45 pF, R2 = 0.975, not shown). However, we found no significant differences in the properties of the two populations.

Fig. 1. Morphological properties of TH-GFP cells. A, B - Expression pattern of the TH-GFP transgene in the glomerular layer of the main olfactory bulb in a coronal section. Scale bar 50m. C - Frequency distribution of the soma diameter of the cells used in this study. The distribution could be best fitted by two Gaussian curves, identifying two distinct subpopulations of cells.

About 80% of DA neurones were spontaneously active. In the cell-attached configuration, action currents were recorded across the patch, usually structured in a regular, rhythmic pattern (Fig. 2A) with an average frequency of 7.30 + 1.35 Hz (n = 31).

Generation of transgenic mice (TH-GFP/21-31) was described in previous papers (Sawamoto et al.2001; Matsushita et al.2002). The transgene construct contained the 9.0-kb 5'-flanking region of the rat tyrosine hydroxylase (TH) gene, the second intron of the rabbit -globin gene, cDNA encoding green fluorescent protein (GFP), and polyadenylation signals

Cells expressing the GFP transgene under the TH promoter (TH-GFP) occurred primarily in the glomerular layer of the main olfactory bulb (Fig. 1A,B). The intraglomerular processes of these cells displayed high levels of TH-GFP expression, and their intertwine delimitates the

Recordings with the patch-clamp technique in the whole-cell configuration were obtained from 368 DA cells in the glomerular layer following the procedures described in Pignatelli et

Cell dimensions were rather variable, as shown in Fig. 1C. Previous studies have suggested that there are two populations of DA neurones in the adult OB, based on size or location (Halász1990; Baker et al.1983). In fact, the distribution of the mean cell diameter of GFP+ cells could be best fitted with two Gaussian curves, identifying two subpopulations with average sizes of 5.67 + 0.96 μm and 9.48 + 2.39 μm (R2 = 0.991); the same result could be obtained from the analysis of the membrane capacitances, whose frequency distribution could be best fitted by two Gaussians (5.41 ± 1.5 pF and 10.63 ± 3.45 pF, R2 = 0.975, not shown). However, we found no significant differences in the properties of the two

Fig. 1. Morphological properties of TH-GFP cells. A, B - Expression pattern of the TH-GFP transgene in the glomerular layer of the main olfactory bulb in a coronal section. Scale bar 50m. C - Frequency distribution of the soma diameter of the cells used in this study. The

About 80% of DA neurones were spontaneously active. In the cell-attached configuration, action currents were recorded across the patch, usually structured in a regular, rhythmic

distribution could be best fitted by two Gaussian curves, identifying two distinct

pattern (Fig. 2A) with an average frequency of 7.30 + 1.35 Hz (n = 31).

**2.1 Localisation and general properties of TH-GFP cells** 

of the rabbit -globin and simian virus 40 early genes.

glomeruli, with the soma of GFP+ cells laying around them.

**2. Results** 

al., 2005.

populations.

subpopulations of cells.

After disruption, about 60% of the cells continued to fire spontaneous action potentials under current-clamp condition (Fig. 2B) without any significant alteration of the firing frequency (7.84 + 2.44 Hz, n = 14). Interspike intervals were rather constant in most of the cells (Fig. 2C), and irregular in others for the presence of sporadic misses. Occasionally, especially in isolated cells (see below), the firing was structured in bursts. We found no correlation of the firing frequency with cell size.

This spontaneous activity was completely blocked by TTX (0.3 M) or by Cd+ (100 M), but persisted after block of glutamatergic and GABAergic synaptic transmission with kynurenate 1 mM and bicuculline (10 M), suggesting that it was due to intrinsic properties of the cell membrane and was not driven by external synaptic inputs, as it resulted even more obviously by the observation that spontaneous activity was maintained also in dissociated cells (Fig. 2D).

Occasionally we did observe spontaneous synaptic currents, which were completely blocked by a mixture of 1 mM kynurenate and 10 M bicuculline (not shown), and which were not further investigated for the purpose of this study.

Fig. 2. Spontaneous activity in DA neurones in thin slices. A – Action currents in cellattached mode B – Action potentials in whole-cell mode C – Frequency distribution of the inter-event time for the cell shown in panel B. D – Frequency of spontaneous firing in TH-GFP cells under the indicated experimental conditions. CA cell attached, WC whole cell.

We studied the dopaminergic neurones under current- and voltage-clamp conditions to characterise the ionic currents underlying spontaneous firing. In voltage-clamped neurones, currents were elicited both by step and ramp depolarisations.

Depolarisation activates a complicated pattern of current flow, in which a variety of conductances coexist, the most prominent of which were a fast transient sodium current and a non-inactivating potassium current (Fig. 3A, B). We identified specific ionic currents present in the cells by measurements of their voltage-dependence and kinetics during step

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 29

(and occasionally also in the intracellular solution to complete the blockade). Under these conditions, depolarising voltage steps to potentials positive to –60 mV evoked a large, transient inward current, peaking in 0.4 ms at 0 mV which reached its maximum amplitude for steps near –30 mV (Fig. 4A). Its sensitivity to TTX (0.3 M) at all voltages, and its abolition following removal of sodium ions from the perfusing medium indicate that it is a

Although it was not always possible to exert an accurate control of membrane potential during the transient sodium current in DA cells in slice preparations (presumably because of currents generated at a distance from the soma on the axon or dendrites), an adequate space clamp and series resistance compensation could be achieved in 7 neurones in which we could obtain a complete series of recordings with and without TTX. The kinetic characterisation of the fast transient Na-current showed in Fig. 4 (and on which is based the numerical reconstruction of this current presented below), was carried out in a homogeneous group of 12 dissociated neurones, averaging 4.5 + 0.12 pF, which were electrotonically compact and thus allowed for a more precise space clamp. The results were similar in the two cases, with I/V relationships showing a slightly larger maximum inward current in slices (3784 + 369 pA, n=7) than in dissociated cells (3219 + 223 pA, n=12), but

The peak INa(F) I-V relationship for a group of twelve dissociated neurones over a range of voltage pulses extending from -80 to +40 mV is shown in Fig. 4B. Reversal potentials for INa(F) could not be measured directly in our experiments because of uncertainties regarding leakage correction in the presence of large non-specific outward currents. The Na equilibrium potential, evaluated indirectly from the positive limb of the I-V plot (Fig. 4B), is close to +40 mV, about 20 mV more negative than the value predicted by the Nernst

The activation process is illustrated in Fig. 4 A-C and G. The fast Na-current develops following a third-order exponential; the activation time constant, m, studied in the –60 to +30 mV range, was computed from the least squares fit of a cubic exponential to the rising phase of the Na-current. In some cases the activation time constant was computed using the method proposed by Bonifazzi et al. (Bonifazzi et al.1988), allowing for the determination of m from the time-to-peak (tp) and the decay time constant, and consisting in the solution of the equation tp = m ln(1+n·h/m), where n is the order of the activation kinetics. Nachannels activate rapidly, with time constants extending from 0.66 to 0.14 ms in the –60 to +10 mV range. The continuous function describing the dependence of m upon voltage in the

The open channel current as a function of voltage was obtained in a 12 neurones sample from the extrapolation at the time zero of the decaying phase of the current. From the obtained values, the open-channel Na conductance, gNa(F), was calculated using the equation gNa(F)(V) = INa0(V)/(V- ENa), where V is the membrane potential, ENa the sodium equilibrium

The conductance-voltage relationship, gNa(F)(V), was described by the Boltzmann equation exhibiting a threshold at about -60 mV, with a slope of 4.34 mV, midpoint at –39.9 mV and a maximum conductance gNa(F)max of 101 nS at –20 mV (Fig. 4C). Finally, the voltagedependence of the steady-state activation parameter, m, was computed by extracting the

potential and INa0 is the extrapolation at the zero time of the Na-current.

classical Na-current.

with the same overall voltage dependence and kinetics.

range studied, is indicated in the legend of figure 4.

equation for a pure Na potential.

depolarisations, together with ionic substitution and blocking agents to isolate individual components of the currents. After block of the potassium currents, obtained by adding 20 mM TEA in the perfusing solution and by equimolar substitution of internal K+ ions with Cs+, a persistent inward current was observed after the fast transient inward current had completely subsided (Fig 3C, D). The amplitude of this persistent component, measured as the average of the current amplitude during the last 10 ms of the depolarising step, had a maximum amplitude of 223.3 + 32.2 pA (n=21) at –20 mV, and could be separated in two components, sustained by sodium and calcium ions (see below).

Fig. 3. Responses of DA neurones (PG cells) in thin slices to depolarising voltage steps under different conditions. A, B – Voltage-clamp recordings from the same cell, in normal saline, held at –70 mV (A) and at –50 mV (B), and depolarised to potentials ranging from –50 to +50 mV. C – Inward currents recorded under voltage-clamp conditions in response to depolarising steps ranging from –80 to +50 mV; holding potential was –100 mV. Potassium currents were suppressed by ionic substitution of intracellular K+ ions with Cs+, and addition of 20 mM TEA in the extracellular medium. The inset shows the current-voltage relationship of the persistent inward current, averaged at the times indicated by the box. D – Details of some of the traces shown in panel C, at higher magnification, to show the persistent inward current.

#### **2.2 Fast transient Na current**

The elimination of the concomitant currents was obtained by blocking the Ca2+ current with 100 M Cd+, and by equimolar substitution of intracellular K+ with Cs+ or NMDG; in addition, the K+ channels were blocked by adding 20 mM TEA in the perfusing solution

depolarisations, together with ionic substitution and blocking agents to isolate individual components of the currents. After block of the potassium currents, obtained by adding 20 mM TEA in the perfusing solution and by equimolar substitution of internal K+ ions with Cs+, a persistent inward current was observed after the fast transient inward current had completely subsided (Fig 3C, D). The amplitude of this persistent component, measured as the average of the current amplitude during the last 10 ms of the depolarising step, had a maximum amplitude of 223.3 + 32.2 pA (n=21) at –20 mV, and could be separated in two

Fig. 3. Responses of DA neurones (PG cells) in thin slices to depolarising voltage steps under different conditions. A, B – Voltage-clamp recordings from the same cell, in normal saline, held at –70 mV (A) and at –50 mV (B), and depolarised to potentials ranging from –50 to +50

depolarising steps ranging from –80 to +50 mV; holding potential was –100 mV. Potassium currents were suppressed by ionic substitution of intracellular K+ ions with Cs+, and addition of 20 mM TEA in the extracellular medium. The inset shows the current-voltage relationship of the persistent inward current, averaged at the times indicated by the box. D –

The elimination of the concomitant currents was obtained by blocking the Ca2+ current with 100 M Cd+, and by equimolar substitution of intracellular K+ with Cs+ or NMDG; in addition, the K+ channels were blocked by adding 20 mM TEA in the perfusing solution

mV. C – Inward currents recorded under voltage-clamp conditions in response to

Details of some of the traces shown in panel C, at higher magnification, to show the

persistent inward current.

**2.2 Fast transient Na current** 

components, sustained by sodium and calcium ions (see below).

(and occasionally also in the intracellular solution to complete the blockade). Under these conditions, depolarising voltage steps to potentials positive to –60 mV evoked a large, transient inward current, peaking in 0.4 ms at 0 mV which reached its maximum amplitude for steps near –30 mV (Fig. 4A). Its sensitivity to TTX (0.3 M) at all voltages, and its abolition following removal of sodium ions from the perfusing medium indicate that it is a classical Na-current.

Although it was not always possible to exert an accurate control of membrane potential during the transient sodium current in DA cells in slice preparations (presumably because of currents generated at a distance from the soma on the axon or dendrites), an adequate space clamp and series resistance compensation could be achieved in 7 neurones in which we could obtain a complete series of recordings with and without TTX. The kinetic characterisation of the fast transient Na-current showed in Fig. 4 (and on which is based the numerical reconstruction of this current presented below), was carried out in a homogeneous group of 12 dissociated neurones, averaging 4.5 + 0.12 pF, which were electrotonically compact and thus allowed for a more precise space clamp. The results were similar in the two cases, with I/V relationships showing a slightly larger maximum inward current in slices (3784 + 369 pA, n=7) than in dissociated cells (3219 + 223 pA, n=12), but with the same overall voltage dependence and kinetics.

The peak INa(F) I-V relationship for a group of twelve dissociated neurones over a range of voltage pulses extending from -80 to +40 mV is shown in Fig. 4B. Reversal potentials for INa(F) could not be measured directly in our experiments because of uncertainties regarding leakage correction in the presence of large non-specific outward currents. The Na equilibrium potential, evaluated indirectly from the positive limb of the I-V plot (Fig. 4B), is close to +40 mV, about 20 mV more negative than the value predicted by the Nernst equation for a pure Na potential.

The activation process is illustrated in Fig. 4 A-C and G. The fast Na-current develops following a third-order exponential; the activation time constant, m, studied in the –60 to +30 mV range, was computed from the least squares fit of a cubic exponential to the rising phase of the Na-current. In some cases the activation time constant was computed using the method proposed by Bonifazzi et al. (Bonifazzi et al.1988), allowing for the determination of m from the time-to-peak (tp) and the decay time constant, and consisting in the solution of the equation tp = m ln(1+n·h/m), where n is the order of the activation kinetics. Nachannels activate rapidly, with time constants extending from 0.66 to 0.14 ms in the –60 to +10 mV range. The continuous function describing the dependence of m upon voltage in the range studied, is indicated in the legend of figure 4.

The open channel current as a function of voltage was obtained in a 12 neurones sample from the extrapolation at the time zero of the decaying phase of the current. From the obtained values, the open-channel Na conductance, gNa(F), was calculated using the equation gNa(F)(V) = INa0(V)/(V- ENa), where V is the membrane potential, ENa the sodium equilibrium potential and INa0 is the extrapolation at the zero time of the Na-current.

The conductance-voltage relationship, gNa(F)(V), was described by the Boltzmann equation exhibiting a threshold at about -60 mV, with a slope of 4.34 mV, midpoint at –39.9 mV and a maximum conductance gNa(F)max of 101 nS at –20 mV (Fig. 4C). Finally, the voltagedependence of the steady-state activation parameter, m, was computed by extracting the

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 31

cubic root from the ratio gNa(V) / gNa(F)max (Fig 4H, upward triangles). The steady-state

The steady-state voltage dependence of fast Na inactivation, h∞, was studied by evaluating the non-inactivated fraction of the Na current as a function of membrane potential (Hodgkin and Huxley1952)(Fig. 4D). The protocol used is illustrated in the inset of the same figure. INa(F) was measured in each experiment at a constant test voltage of 0 mV after 200 ms preconditioning pulses to various potentials, and plotted after normalisation to the

The 200 ms pre-pulse was sufficient to allow the inactivation variable to reach its steadystate value (see Fig. 4E). The steady-state inactivation curve h∞(V) thus obtained from a twelve-neurone sample, shows a sigmoidal dependence on voltage which can be fitted by the equation: h∞(V) = (1+exp[(V-Vh)/k])-1, where Vh (midpoint) is -58.7 mV and k (slope) is 4.5 mV (Fig. 4H, downward triangles). It should be noted that the inactivating fraction of INa(F) falls virtually to zero at -30 mV, which is the potential at which INa(P) reaches its

 Fast sodium channels inactivate rapidly. The decay phase of the current, studied in the –60 to +30 mV range, could be adequately fitted with a single exponential. The continuous function describing the voltage-dependence of inactivation time constant, h, illustrated in Fig. 4F for a ten-neurone sample, can be approximated by the equation indicated in the

The rate of recovery from inactivation, was measured using the double-pulse protocol. Nachannel inactivation is removed over a course of a few tens of milliseconds, and the process is markedly voltage-dependent. This is illustrated in Fig. 4E for a typical neurone. The cell was maintained in normal saline at a holding potential of –100 mV, and stepped to –20 mV for 10 ms (h is 0.74 ms at this potential). INa(F) was then allowed to recover by applying a

Recovery was evaluated by measuring INa(F) during a second test pulse to -20 mV. In Fig. 4E, a family of INa(F) currents is shown for the conditioning potential of –80 mV. The current peak as a function of the conditioning pulse duration could be fitted by a simple exponential

A delayed rectifier-type potassium current was present in TH-GFP cells (e.g. Fig. 3B), and it has been kinetically characterised (Fig. 5). The current, isolated by blocking the sodium current with TTX, was calcium-dependent only for a small part (at 0 mV the fraction suppressed by Cd+ 100 M was about 10%), and thus it has been modelled as a single component. The equations describing the time- and voltage-dependence of the potassium

In DA cells, after the fast transient Na-current had completely subsided, a persistent inward

figure, and has values spanning from 4.4 and 0.6 ms in the voltage range considered.

hyperpolarising pre-pulse of variable duration at different potentials.

with a mean value of 42.1 + 8.9 ms (n = 14) at –80 mV.

current are shown in Fig. 5 and detailed in the relative legend.

current showing no sign of inactivation after 200 ms was observed.

**2.3 Delayed rectifier potassium current** 

**2.4 Persistent sodium current** 

activation, m(V), had a midpoint at –47.64 mV and a slope of 5.8 mV.

maximum current evoked with hyperpolarisation.

maximum amplitude (Fig. 4B).

Fig. 4. Properties of fast transient sodium current. A - Family of fast transient sodium current in a TH-GFP cell (PG) in thin slice. Responses to depolarising voltage steps ( –90 to + 50 mV ) from a holding potential of –100 mV. B – I/V relationship for a group of 12 dissociated cells (average values ± SEM). C – Conductance-voltage relationship for the group of cells shown in B. The continuous curve is drawn according to the Boltzmann equation, with the upper asymptote at 101 nS, midpoint at –39.9 mV and slope of 4,34 mV. D – Development of inactivation. Family of tracings obtained in response to the protocol shown in the inset. E – Time course of removal of inactivation at –80 mV. Family of tracings obtained with a double-pulse protocol, consisting in two subsequent steps to –20 mV, the first from a holding potential of –100 mV, the second after a variable time at –80 mV. F – Voltage-dependence of inactivation time constant, measured from the decay of the current. The continuous curve, describing h in the -60/+30 mV range, obeys the equation: h(V) = .58 + .019 \* exp(-V / 11.3). G – Voltage-dependence of activation time constant, calculated as explained in the text in a 12 neurone sample. The continuous curve, describing m in the – 60/+10 mV range, obeys the equation: m(V) = 0.155 + (23.2 / (36.4 \*( /2))) \* exp(- 2\*((V+60.62) /36.4)2) H – Steady-state values of activation and inactivation variables (m and h) of the fast sodium current. The continuous curves obey the equations: m∞(V) = 1/(1+exp((-47.6-V)/5.8)), h∞(V) = 1/(1+exp((V+58.7)/4.5)).

Fig. 4. Properties of fast transient sodium current. A - Family of fast transient sodium current in a TH-GFP cell (PG) in thin slice. Responses to depolarising voltage steps ( –90 to +

50 mV ) from a holding potential of –100 mV. B – I/V relationship for a group of 12 dissociated cells (average values ± SEM). C – Conductance-voltage relationship for the group of cells shown in B. The continuous curve is drawn according to the Boltzmann equation, with the upper asymptote at 101 nS, midpoint at –39.9 mV and slope of 4,34 mV. D – Development of inactivation. Family of tracings obtained in response to the protocol shown in the inset. E – Time course of removal of inactivation at –80 mV. Family of tracings obtained with a double-pulse protocol, consisting in two subsequent steps to –20 mV, the first from a holding potential of –100 mV, the second after a variable time at –80 mV. F – Voltage-dependence of inactivation time constant, measured from the decay of the current. The continuous curve, describing h in the -60/+30 mV range, obeys the equation: h(V) = .58 + .019 \* exp(-V / 11.3). G – Voltage-dependence of activation time constant, calculated as explained in the text in a 12 neurone sample. The continuous curve, describing m in the – 60/+10 mV range, obeys the equation: m(V) = 0.155 + (23.2 / (36.4 \*( /2))) \* exp(-

2\*((V+60.62) /36.4)2) H – Steady-state values of activation and inactivation variables (m and

h) of the fast sodium current. The continuous curves obey the equations: m∞(V) =

1/(1+exp((-47.6-V)/5.8)), h∞(V) = 1/(1+exp((V+58.7)/4.5)).

cubic root from the ratio gNa(V) / gNa(F)max (Fig 4H, upward triangles). The steady-state activation, m(V), had a midpoint at –47.64 mV and a slope of 5.8 mV.

The steady-state voltage dependence of fast Na inactivation, h∞, was studied by evaluating the non-inactivated fraction of the Na current as a function of membrane potential (Hodgkin and Huxley1952)(Fig. 4D). The protocol used is illustrated in the inset of the same figure. INa(F) was measured in each experiment at a constant test voltage of 0 mV after 200 ms preconditioning pulses to various potentials, and plotted after normalisation to the maximum current evoked with hyperpolarisation.

The 200 ms pre-pulse was sufficient to allow the inactivation variable to reach its steadystate value (see Fig. 4E). The steady-state inactivation curve h∞(V) thus obtained from a twelve-neurone sample, shows a sigmoidal dependence on voltage which can be fitted by the equation: h∞(V) = (1+exp[(V-Vh)/k])-1, where Vh (midpoint) is -58.7 mV and k (slope) is 4.5 mV (Fig. 4H, downward triangles). It should be noted that the inactivating fraction of INa(F) falls virtually to zero at -30 mV, which is the potential at which INa(P) reaches its maximum amplitude (Fig. 4B).

 Fast sodium channels inactivate rapidly. The decay phase of the current, studied in the –60 to +30 mV range, could be adequately fitted with a single exponential. The continuous function describing the voltage-dependence of inactivation time constant, h, illustrated in Fig. 4F for a ten-neurone sample, can be approximated by the equation indicated in the figure, and has values spanning from 4.4 and 0.6 ms in the voltage range considered.

The rate of recovery from inactivation, was measured using the double-pulse protocol. Nachannel inactivation is removed over a course of a few tens of milliseconds, and the process is markedly voltage-dependent. This is illustrated in Fig. 4E for a typical neurone. The cell was maintained in normal saline at a holding potential of –100 mV, and stepped to –20 mV for 10 ms (h is 0.74 ms at this potential). INa(F) was then allowed to recover by applying a hyperpolarising pre-pulse of variable duration at different potentials.

Recovery was evaluated by measuring INa(F) during a second test pulse to -20 mV. In Fig. 4E, a family of INa(F) currents is shown for the conditioning potential of –80 mV. The current peak as a function of the conditioning pulse duration could be fitted by a simple exponential with a mean value of 42.1 + 8.9 ms (n = 14) at –80 mV.

#### **2.3 Delayed rectifier potassium current**

A delayed rectifier-type potassium current was present in TH-GFP cells (e.g. Fig. 3B), and it has been kinetically characterised (Fig. 5). The current, isolated by blocking the sodium current with TTX, was calcium-dependent only for a small part (at 0 mV the fraction suppressed by Cd+ 100 M was about 10%), and thus it has been modelled as a single component. The equations describing the time- and voltage-dependence of the potassium current are shown in Fig. 5 and detailed in the relative legend.

#### **2.4 Persistent sodium current**

In DA cells, after the fast transient Na-current had completely subsided, a persistent inward current showing no sign of inactivation after 200 ms was observed.

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 33

The persistent sodium current, INa(P), was activated at potentials more negative than –70 mV, and reached a maximum amplitude of –27.5 + 2.97 pA at –30 mV. The corresponding conductance-voltage relationship, calculated by dividing the current amplitude by the sodium driving force, could be fitted by a Boltzmann equation with a midpoint at –48.8 mV and a slope of 6.51 mV (Fig 6B). The maximum value of gNa(P) conductance was 0.41 nS, about 200 times smaller than that of the fast sodium current (INa(F), see below), but contrary to the latter, this current is activated in the pacemaker range, showing an amplitude of –7.3

After block of the TTX-sensitive component and suppression of the K-current by equimolar substitution of intracellular K+ with Cs+, a persistent inward current could be observed at the end of prolonged depolarising steps (Fig. 7A, upper trace). This residual fraction could be almost completely blocked by Cd2+ or Co2+ ions (Fig. 7A, lower traces), suggesting that this second component was sustained by calcium ions. The very small fraction of current remaining after TTX and Cd2+ block has not been further investigated in the present study. Using classical pharmacological tools, ionic substitutions and voltage-clamp protocols, we

The larger of these components, by its overall kinetics, its voltage-dependence and the absence of inactivation was identified as a possible L-type Ca-current. Its properties were studied in slices, after blockage of the Na-currents with 0.3 – 1.2 M TTX and of the Kcurrents by equimolar substitution with Cs+ in the pipette-filling solution and 20 mM TEA in the perfusing bath. The protocols used were either voltage steps or voltage ramps, giving virtually identical results. The I/V relationship of the Ca-current (Fig 7C), measured in a 10 neurones sample averaging the last 5 ms at the end of a 40 ms depolarising step, had a maximum amplitude of –108.7 11.9 pA at –10 mV. The corresponding conductance-voltage relationship showed a maximum conductance of 2.3 nS, with a midpoint at –25.6 mV.

Equimolar substitution of Ca2+ with Ba2+ increased by a factor of about 3 the amplitude of this current (Fig. 7C), without changing the I/V relationship or the time constant of activation. On this current we tested the effects of two blockers of L-type calcium channels, nifedipine and calcicludine. The fraction of current blocked by the two drugs at different voltages was quantified by subtraction of I-V data acquired before and after treatment.

The effects of 10M nifedipine on peak Ca2+ current amplitude was assessed in 6 PG cells (Fig. 7E and F). On average, the drug blocked 61.1 + 14 % of the current measured at the

A 60 aminoacid peptide isolated from the venom of the green mamba (*Dendroaspis augusticeps*), calcicludine (CaC) has been described to have a powerful effect on all type of high-voltage-activated Ca-channels (L-, N-, and P-type) (Schweitz et al.1994). Since one of the regions of the CNS presenting the highest densities of 125I-labeled CaC binding sites is the glomerular layer of the olfactory bulb (Schweitz et al.1994), we tested the ability of this toxin in suppressing the non-inactivating Ca-current found in the DA neurones. CaC (1 μM) was much more effective than nifedipine, with an inhibitory action averaging 72.7

point of its maximum amplitude (–10 mV).

+ 3.13 %.

could dissect the voltage-dependent Ca currents, Cav, into several components.

pA at –60 mV.

**2.5 Calcium currents** 

Fig. 5. Properties of delayed rectifier potassium current. A – Conductance-voltage relationship. The continuous curve is drawn according to the Boltzmann equation, with the upper asymptote (maximum conductance) at 50.5 nS, midpoint at –4.1 mV and slope of 12.5 mV. B - Steady-state values of activation (*n*) for the potassium current, obtained from the fourth root of the data shown in *A* after normalisation. The continuous curve obey the equations: n∞(V) = 1/(1+exp[(-32.2-V)/14.9]). C - Voltage-dependence of activation time constant (n), obtained by fitting a fourth-order exponential to the rising phase of the current. The continuous curve obeys the equation: n(V) = .42 + .097 \* exp(-V / 21.67). D – Voltage-dependence of de-activation time constant (nd). The continuous curve, describing nd in the -105/-15 mV range, obeys the equation: nd = 6.1 - 5.88/(1+exp((V+57.6)/14.75)). This parameter was calculated from the tail currentsindependently from n.

We first applied TTX (0.3 to 1.2 M), which did suppress a significant fraction of the persistent inward current, indicating it received a contribution from a non-inactivating, TTX-sensitive channels. This current-voltage relationship was virtually coincident with the residual persistent current measured after treatment with Cd2+ 100 M (see below), and therefore the data were pooled together. The current-voltage relationship of the fraction of current abolished by TTX or remaining after Cd2+ treatment is shown in Fig. 6A.

Fig. 6. Properties of persistent sodium current. A – I/V relationship. Pooled data obtained as fraction of non-inactivating current suppressed by TTX (n=12) and residual persistent current after Cd2+ block (n=6). B – Conductance-voltage relationship of persistent sodium current, obtained from the average data shown in A. The continuous curve is the Boltzmann fit, with upper asymptote of 0.41 nS, midpoint at –48.8 mV and slope of 6.51 mV.

The persistent sodium current, INa(P), was activated at potentials more negative than –70 mV, and reached a maximum amplitude of –27.5 + 2.97 pA at –30 mV. The corresponding conductance-voltage relationship, calculated by dividing the current amplitude by the sodium driving force, could be fitted by a Boltzmann equation with a midpoint at –48.8 mV and a slope of 6.51 mV (Fig 6B). The maximum value of gNa(P) conductance was 0.41 nS, about 200 times smaller than that of the fast sodium current (INa(F), see below), but contrary to the latter, this current is activated in the pacemaker range, showing an amplitude of –7.3 pA at –60 mV.

#### **2.5 Calcium currents**

32 Electrophysiology – From Plants to Heart

Fig. 5. Properties of delayed rectifier potassium current. A – Conductance-voltage

This parameter was calculated from the tail currentsindependently from n.

current abolished by TTX or remaining after Cd2+ treatment is shown in Fig. 6A.

relationship. The continuous curve is drawn according to the Boltzmann equation, with the upper asymptote (maximum conductance) at 50.5 nS, midpoint at –4.1 mV and slope of 12.5 mV. B - Steady-state values of activation (*n*) for the potassium current, obtained from the fourth root of the data shown in *A* after normalisation. The continuous curve obey the equations: n∞(V) = 1/(1+exp[(-32.2-V)/14.9]). C - Voltage-dependence of activation time constant (n), obtained by fitting a fourth-order exponential to the rising phase of the current. The continuous curve obeys the equation: n(V) = .42 + .097 \* exp(-V / 21.67). D – Voltage-dependence of de-activation time constant (nd). The continuous curve, describing nd in the -105/-15 mV range, obeys the equation: nd = 6.1 - 5.88/(1+exp((V+57.6)/14.75)).

We first applied TTX (0.3 to 1.2 M), which did suppress a significant fraction of the persistent inward current, indicating it received a contribution from a non-inactivating, TTX-sensitive channels. This current-voltage relationship was virtually coincident with the residual persistent current measured after treatment with Cd2+ 100 M (see below), and therefore the data were pooled together. The current-voltage relationship of the fraction of

Fig. 6. Properties of persistent sodium current. A – I/V relationship. Pooled data obtained as fraction of non-inactivating current suppressed by TTX (n=12) and residual persistent current after Cd2+ block (n=6). B – Conductance-voltage relationship of persistent sodium current, obtained from the average data shown in A. The continuous curve is the Boltzmann

fit, with upper asymptote of 0.41 nS, midpoint at –48.8 mV and slope of 6.51 mV.

After block of the TTX-sensitive component and suppression of the K-current by equimolar substitution of intracellular K+ with Cs+, a persistent inward current could be observed at the end of prolonged depolarising steps (Fig. 7A, upper trace). This residual fraction could be almost completely blocked by Cd2+ or Co2+ ions (Fig. 7A, lower traces), suggesting that this second component was sustained by calcium ions. The very small fraction of current remaining after TTX and Cd2+ block has not been further investigated in the present study.

Using classical pharmacological tools, ionic substitutions and voltage-clamp protocols, we could dissect the voltage-dependent Ca currents, Cav, into several components.

The larger of these components, by its overall kinetics, its voltage-dependence and the absence of inactivation was identified as a possible L-type Ca-current. Its properties were studied in slices, after blockage of the Na-currents with 0.3 – 1.2 M TTX and of the Kcurrents by equimolar substitution with Cs+ in the pipette-filling solution and 20 mM TEA in the perfusing bath. The protocols used were either voltage steps or voltage ramps, giving virtually identical results. The I/V relationship of the Ca-current (Fig 7C), measured in a 10 neurones sample averaging the last 5 ms at the end of a 40 ms depolarising step, had a maximum amplitude of –108.7 11.9 pA at –10 mV. The corresponding conductance-voltage relationship showed a maximum conductance of 2.3 nS, with a midpoint at –25.6 mV.

Equimolar substitution of Ca2+ with Ba2+ increased by a factor of about 3 the amplitude of this current (Fig. 7C), without changing the I/V relationship or the time constant of activation. On this current we tested the effects of two blockers of L-type calcium channels, nifedipine and calcicludine. The fraction of current blocked by the two drugs at different voltages was quantified by subtraction of I-V data acquired before and after treatment.

The effects of 10M nifedipine on peak Ca2+ current amplitude was assessed in 6 PG cells (Fig. 7E and F). On average, the drug blocked 61.1 + 14 % of the current measured at the point of its maximum amplitude (–10 mV).

A 60 aminoacid peptide isolated from the venom of the green mamba (*Dendroaspis augusticeps*), calcicludine (CaC) has been described to have a powerful effect on all type of high-voltage-activated Ca-channels (L-, N-, and P-type) (Schweitz et al.1994). Since one of the regions of the CNS presenting the highest densities of 125I-labeled CaC binding sites is the glomerular layer of the olfactory bulb (Schweitz et al.1994), we tested the ability of this toxin in suppressing the non-inactivating Ca-current found in the DA neurones. CaC (1 μM) was much more effective than nifedipine, with an inhibitory action averaging 72.7 + 3.13 %.

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 35

Since *in situ* hybridisation experiments have localised the expression of two transcripts (1G and 1I) of the T-type calcium channel in the glomerular layer of the olfactory bulb (Talley et al.1999; Klugbauer et al.1999), we checked for the presence of LVA Ca-current. Unfortunately several characteristics of these channels hampered their study in our preparation. First, contrary to the cardiac cells or transfected cells, in TH-GFP interneurones this current is small: we have calculated a maximum conductance of 0.35 nS, corresponding to a peak current of about 20 pA at –35 mV. The problem was further complicated by the fact that on one hand it was difficult to get accurate space clamping in slices, and on the other hand, in isolated cell preparations the current was difficult to resolve, probably because of the preferential localisation of these channels on the dendrites (Perez-Reyes2003). Second, the conductance of these channels cannot be significantly increased by substitution of Ca2+ with Ba2+ ions, as it can be done with HVA channels. Third, there are no effective pharmacological tools for the study of T-type Cachannels, because they are relatively resistant to most organic calcium channel blockers, such as dihydropyridines, that block the L-type, or peptide toxins, such as -conotoxin or

Despite these difficulties, we succeeded in isolating a T-type calcium current in dissociated cells (Fig. 8). The protocol used was a rapid ramp (7 V/s) from –100 to 40 mV, in the presence of TTX (1M) and after substitution of Ca2+ with Sr2+, which in known to have a slightly higher permeability than Ca2+ in T-type Ca-channels (Takahashi et al.1991). Under these conditions, an inward inflection peaking at about –40 mV, distinct from the peak due to the L-type Ca-channels (Fig. 8A), could be seen. Nickel is a nonselective inhibitor of calcium channels, but transient low voltage-activated (LVA) T-type (Perchenet et al.2000; Wolfart and Roeper2002) Ca-channels are particularly sensitive (IC50 < 50 M; (Perez-Reyes et al.1998) whereas other HVA Cav channels (L-, P/Q, and N-type) are less sensitive (IC50 > 90 M; (Zhang et al.1993; Randall1998). In fact Nickel did selectively eliminate the first peak, leaving the second unaltered. The difference, averaged in three cells, is shown in figure 8B, and the conductance, calculated assuming a Ca2+ equilibrium potential of 45 mV, is illustrated in figure 8C. The current activates at potentials positive to –65 mV and peaks at about –35 mV, with a maximum conductance of 0.35 nS. The point of half activation is –45.3

mV, which is in line with the known values for this current (Perez-Reyes2003).

suppressed by ivabradina 10 μM, ZD7288 30 μM and Cs+ 1 mM (Fig. 9D).

A likely candidate for the pacemaking process was the inward rectifier current (Ih). In a previous work (Pignatelli et al.2005), analysing the excitability profile of DA PG cells, we failed to detect any significant component activated by hyperpolarization (Fig. 9A). It was therefore with some disconcert that we observed that a drug blocking the h-current (ivabradine, 10M) did block the spontaneous activity (see below). We then switched to ionic settings known to enhance the amplitude of the h-current, i.e. high [K+]o, using an external saline where [K+]o was 32.5 instead of 2.5 mM. In these conditions, we observed a measurable current activated by hyperpolarization (Fig. 9B), which could be separated in a classical inward rectifier (KIR) and a typical h-current (Fig. 9C). The h-current was


**2.6 h-current** 

Fig. 7. Properties of HVA calcium currents. A – Calcium current recorded in response to depolarising voltage steps to potentials ranging from –70 to +50 mV from a holding potential of –100 mV. Above: tracings recorded in the presence of 1.2M TTX, 20 mM TEA in the extracellular solution, and with Cs+ as a substitute for K+ ions in the intracellular solution. Lower tracings were recorded in the same conditions after addition of 100M Cd2+. B – Barium currents recorded in the same conditions described for A. C – I/V relationship of Ca and Ba currents in a 10 neurones sample from thin slices. D – Activation time constant, measured in a 10 neurones sample by fitting a single exponential to the rising phase of the current. The continuous line is described by the equation: Ca(L) = 1.23 + exp (-V/74.6). E – Effect of 10M nifedipine on calcium current in a group of six TH-GFP PG cells in slices. F - Histogram showing the effect of nifedipine (10M, cells shown in E) and calcicludine (1M, not shown, n=7) measured at –10 mV.

We also tried to define if other types of HVA neuronal Cav channels (P/Q-, N-) were present in TH-GFP cells. Using classical blockers, like -conotoxin GVIA (0.82M) that blocks the N-type, or spider toxin -agatoxin IVA (10 nM) that blocks P/Q-type channels, we observed the suppression of a fraction of HVA Ca-current remaining after nifedipine block (at –10 mV 38% and 42%, respectively, not shown), suggesting the presence of limited amounts of the corresponding HVA Ca-channels.

Since the long-lasting HVA Cav currents were not directly involved in the pacemaker process (see below), and for the dominance of L- over N- and P/Q-type current, for the purpose of the numerical reconstruction of the electrical activity of these cells (see below), they were kinetically modelled as a unique, non inactivating component. The rising phase of the current was fitted by a single exponential, with a time constant of 1.2 + 0.3 ms at 0 mV (n=10, see legend of figure 7D for further details).

Since *in situ* hybridisation experiments have localised the expression of two transcripts (1G and 1I) of the T-type calcium channel in the glomerular layer of the olfactory bulb (Talley et al.1999; Klugbauer et al.1999), we checked for the presence of LVA Ca-current. Unfortunately several characteristics of these channels hampered their study in our preparation. First, contrary to the cardiac cells or transfected cells, in TH-GFP interneurones this current is small: we have calculated a maximum conductance of 0.35 nS, corresponding to a peak current of about 20 pA at –35 mV. The problem was further complicated by the fact that on one hand it was difficult to get accurate space clamping in slices, and on the other hand, in isolated cell preparations the current was difficult to resolve, probably because of the preferential localisation of these channels on the dendrites (Perez-Reyes2003). Second, the conductance of these channels cannot be significantly increased by substitution of Ca2+ with Ba2+ ions, as it can be done with HVA channels. Third, there are no effective pharmacological tools for the study of T-type Cachannels, because they are relatively resistant to most organic calcium channel blockers, such as dihydropyridines, that block the L-type, or peptide toxins, such as -conotoxin or -agatoxin.

Despite these difficulties, we succeeded in isolating a T-type calcium current in dissociated cells (Fig. 8). The protocol used was a rapid ramp (7 V/s) from –100 to 40 mV, in the presence of TTX (1M) and after substitution of Ca2+ with Sr2+, which in known to have a slightly higher permeability than Ca2+ in T-type Ca-channels (Takahashi et al.1991). Under these conditions, an inward inflection peaking at about –40 mV, distinct from the peak due to the L-type Ca-channels (Fig. 8A), could be seen. Nickel is a nonselective inhibitor of calcium channels, but transient low voltage-activated (LVA) T-type (Perchenet et al.2000; Wolfart and Roeper2002) Ca-channels are particularly sensitive (IC50 < 50 M; (Perez-Reyes et al.1998) whereas other HVA Cav channels (L-, P/Q, and N-type) are less sensitive (IC50 > 90 M; (Zhang et al.1993; Randall1998). In fact Nickel did selectively eliminate the first peak, leaving the second unaltered. The difference, averaged in three cells, is shown in figure 8B, and the conductance, calculated assuming a Ca2+ equilibrium potential of 45 mV, is illustrated in figure 8C. The current activates at potentials positive to –65 mV and peaks at about –35 mV, with a maximum conductance of 0.35 nS. The point of half activation is –45.3 mV, which is in line with the known values for this current (Perez-Reyes2003).

#### **2.6 h-current**

34 Electrophysiology – From Plants to Heart

Fig. 7. Properties of HVA calcium currents. A – Calcium current recorded in response to depolarising voltage steps to potentials ranging from –70 to +50 mV from a holding potential of –100 mV. Above: tracings recorded in the presence of 1.2M TTX, 20 mM TEA in the extracellular solution, and with Cs+ as a substitute for K+ ions in the intracellular solution. Lower tracings were recorded in the same conditions after addition of 100M Cd2+. B – Barium currents recorded in the same conditions described for A. C – I/V relationship of Ca and Ba currents in a 10 neurones sample from thin slices. D – Activation time constant, measured in a 10 neurones sample by fitting a single exponential to the rising phase of the current. The continuous line is described by the equation: Ca(L) = 1.23 + exp (-V/74.6). E – Effect of 10M nifedipine on calcium current in a group of six TH-GFP PG cells in slices. F - Histogram showing the effect of nifedipine (10M, cells shown in E) and

We also tried to define if other types of HVA neuronal Cav channels (P/Q-, N-) were present in TH-GFP cells. Using classical blockers, like -conotoxin GVIA (0.82M) that blocks the N-type, or spider toxin -agatoxin IVA (10 nM) that blocks P/Q-type channels, we observed the suppression of a fraction of HVA Ca-current remaining after nifedipine block (at –10 mV 38% and 42%, respectively, not shown), suggesting the presence of limited amounts of the

Since the long-lasting HVA Cav currents were not directly involved in the pacemaker process (see below), and for the dominance of L- over N- and P/Q-type current, for the purpose of the numerical reconstruction of the electrical activity of these cells (see below), they were kinetically modelled as a unique, non inactivating component. The rising phase of the current was fitted by a single exponential, with a time constant of 1.2 + 0.3 ms at 0 mV

calcicludine (1M, not shown, n=7) measured at –10 mV.

(n=10, see legend of figure 7D for further details).

corresponding HVA Ca-channels.

A likely candidate for the pacemaking process was the inward rectifier current (Ih). In a previous work (Pignatelli et al.2005), analysing the excitability profile of DA PG cells, we failed to detect any significant component activated by hyperpolarization (Fig. 9A). It was therefore with some disconcert that we observed that a drug blocking the h-current (ivabradine, 10M) did block the spontaneous activity (see below). We then switched to ionic settings known to enhance the amplitude of the h-current, i.e. high [K+]o, using an external saline where [K+]o was 32.5 instead of 2.5 mM. In these conditions, we observed a measurable current activated by hyperpolarization (Fig. 9B), which could be separated in a classical inward rectifier (KIR) and a typical h-current (Fig. 9C). The h-current was suppressed by ivabradina 10 μM, ZD7288 30 μM and Cs+ 1 mM (Fig. 9D).

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 37

Fig. 9. Properties of the h-current. A,B – Currents activated by hyperpolarizing steps in normal saline and in high K+, respectively. C – Currents activated by hyperpolarization in high K+ and after blockage of the KIR component with Ba2+ 0.5 mM. D – Same as C after addition of a blocker of the h-current (Cs+ 1 mM) E – Representative current traces for the analysis of activation. The membrane was held at -40 mV, depolarised to test voltages from - 60 to -130 mV in 10 mV increments. Ih tails were elicited in response to a second pulse to −130 mV, following test voltages (see methods for explanation) F – I/V relationship of the hcurrent for a group of 15 cells. G – Fractional activation of the h-current as a function of

After block of the h-current with ivabradine 10MA, and using high concentration of K+ in the external saline in order to enhance this KIR-current, a family of almost pure tracings could be evoked by hyperpolarising steps ranging from -50 to -130 mV in 10 mV increments, staring from a holding potential of -40 mV (Fig. 10A); the current could be rapidly and reversibly blocked by Ba2+ 0.2 mM (Fig. 10B), and from the I/V curves (Fig. 10C) we calculated a reversal potential of -44 mV (EK -33 mV), giving conductance of 2 nS at the

The presence of autorhythmic activity was the most salient feature of DA cells in the olfactory bulb, so the first efforts were aimed at elucidating the underlying mechanisms. We first tried to understand if the spontaneous activity was due to the presence of pacemaker currents or to synaptic mechanisms reverberating excitation from one cell to the other. Dissociated TH-GFP cells conserved their capacity of generating rhythmic activity, clearly indicating that this is an intrinsic property of these cells. Dissociated TH-GFP cells showed a spontaneous frequency of firing of 13.57 1.79 (n=24) and 15.75 3.12 (n=14) in whole-cell

voltage.

steady state.

**2.8 Spontaneous activity** 

Fig. 8. Properties of the LVA calcium current. A – Voltage-clamp ramps were performed from –80 to +40 mV at a speed of 7 V/s in the presence of TTX (1M), and after substitution of Sr2+ for Ca2+; the traces were corrected for leakage. Under these conditions, a distinct bump can be seen preceding the HVA calcium current (here peaking at –10 mV), which is selectively suppressed by Ni2+ 100M. B - Transient Ca-current generated using the protocol described in A calculated by subtracting the current-voltage curves in control and in the presence of 100M Ni2+. The low-Ni2+ sensitive current (average from three cells) is activated at membrane potentials more positive than –60 mV. C – Conductance-voltage relationship of the low-Ni2+ sensitive current. The continuous line is a Boltzmann fit, with a midpoint at –45.3 mV and a maximum conductance of 0.35 nS. All recordings in this figure were performed from spontaneously bursting dissociated TH-GFP cells.

Ih was evoked by a family of seven hyperpolarizing voltage steps (step amplitude -10 mV; step duration 3 s) from the holding potential of -40 to - 130 mV. The steps were applied in 7 s intervals. *I*h was calculated as the difference between the membrane current at the end of the voltage step (*I*ss) and the instantaneous current (*I*inst) measured after the settling of the capacitative transient. To determine *I*h voltage dependence, activation curves were constructed from *I*h tail currents, which were calculated by subtracting the pre-step holding current from the peak of the tail current. The activation curves were fitted by the Boltzmann function to estimate the potential of half-activation (*V*50 , -91 mV) and the slope factor (*k,* 5 mV).

*I*h time dependence was studied by fitting current traces (from -100 to -130 mV) with a single exponential function. The activation time constant gave values ranging from 361 ± 112 ms at -100 mV to 155 ± 23 ms at -130 mV (n=10).

The reversal potential, calculated from the reversal of the tail currents (not shown) was -47 mV, from which the maximal conductance could be calculated, giving a value of 1.37 nS.

#### **2.7 KIR current**

KIR 2.1 channel subunits are highly expressed in the olfactory bulb, with the higher density in the glomerular layer (Prüss et al.2005), and KIR conductances are found in many dopaminergic systems, as in nucleus accumbens (Perez et al.2006) and substantia nigra (Bausch et al.1995). Here we describe a KIR conductance in the dopaminergic neurones of the olfactory bulb.

Fig. 8. Properties of the LVA calcium current. A – Voltage-clamp ramps were performed from –80 to +40 mV at a speed of 7 V/s in the presence of TTX (1M), and after substitution of Sr2+ for Ca2+; the traces were corrected for leakage. Under these conditions, a distinct bump can be seen preceding the HVA calcium current (here peaking at –10 mV), which is selectively suppressed by Ni2+ 100M. B - Transient Ca-current generated using the protocol described in A calculated by subtracting the current-voltage curves in control and in the presence of 100M Ni2+. The low-Ni2+ sensitive current (average from three cells) is activated at membrane potentials more positive than –60 mV. C – Conductance-voltage relationship of the low-Ni2+ sensitive current. The continuous line is a Boltzmann fit, with a midpoint at –45.3 mV and a maximum conductance of 0.35 nS. All recordings in this figure

Ih was evoked by a family of seven hyperpolarizing voltage steps (step amplitude -10 mV; step duration 3 s) from the holding potential of -40 to - 130 mV. The steps were applied in 7 s intervals. *I*h was calculated as the difference between the membrane current at the end of the voltage step (*I*ss) and the instantaneous current (*I*inst) measured after the settling of the capacitative transient. To determine *I*h voltage dependence, activation curves were constructed from *I*h tail currents, which were calculated by subtracting the pre-step holding current from the peak of the tail current. The activation curves were fitted by the Boltzmann function to estimate the potential of half-activation (*V*50 , -91 mV) and the slope factor

*I*h time dependence was studied by fitting current traces (from -100 to -130 mV) with a single exponential function. The activation time constant gave values ranging from 361 ± 112 ms at

The reversal potential, calculated from the reversal of the tail currents (not shown) was -47 mV, from which the maximal conductance could be calculated, giving a value of 1.37 nS.

KIR 2.1 channel subunits are highly expressed in the olfactory bulb, with the higher density in the glomerular layer (Prüss et al.2005), and KIR conductances are found in many dopaminergic systems, as in nucleus accumbens (Perez et al.2006) and substantia nigra (Bausch et al.1995). Here we describe a KIR conductance in the dopaminergic neurones of

were performed from spontaneously bursting dissociated TH-GFP cells.

(*k,* 5 mV).

**2.7 KIR current** 

the olfactory bulb.


Fig. 9. Properties of the h-current. A,B – Currents activated by hyperpolarizing steps in normal saline and in high K+, respectively. C – Currents activated by hyperpolarization in high K+ and after blockage of the KIR component with Ba2+ 0.5 mM. D – Same as C after addition of a blocker of the h-current (Cs+ 1 mM) E – Representative current traces for the analysis of activation. The membrane was held at -40 mV, depolarised to test voltages from - 60 to -130 mV in 10 mV increments. Ih tails were elicited in response to a second pulse to −130 mV, following test voltages (see methods for explanation) F – I/V relationship of the hcurrent for a group of 15 cells. G – Fractional activation of the h-current as a function of voltage.

After block of the h-current with ivabradine 10MA, and using high concentration of K+ in the external saline in order to enhance this KIR-current, a family of almost pure tracings could be evoked by hyperpolarising steps ranging from -50 to -130 mV in 10 mV increments, staring from a holding potential of -40 mV (Fig. 10A); the current could be rapidly and reversibly blocked by Ba2+ 0.2 mM (Fig. 10B), and from the I/V curves (Fig. 10C) we calculated a reversal potential of -44 mV (EK -33 mV), giving conductance of 2 nS at the steady state.

#### **2.8 Spontaneous activity**

The presence of autorhythmic activity was the most salient feature of DA cells in the olfactory bulb, so the first efforts were aimed at elucidating the underlying mechanisms. We first tried to understand if the spontaneous activity was due to the presence of pacemaker currents or to synaptic mechanisms reverberating excitation from one cell to the other. Dissociated TH-GFP cells conserved their capacity of generating rhythmic activity, clearly indicating that this is an intrinsic property of these cells. Dissociated TH-GFP cells showed a spontaneous frequency of firing of 13.57 1.79 (n=24) and 15.75 3.12 (n=14) in whole-cell

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 39

Fig. 11. Effect of blockers of h-channels on spontaneous firing. A – Ivabradine (10M, bar) block of spontaneous activity; note the large hyperpolarization. At the times indicated by arrowheads, depolarising currents of increasing amplitudes were delivered. B - Enlargement of the response to the third injection of depolarising current (grey arrowhead) to show that the block of the h-current does not impairs the spontaneous activity, provided that the

The classical selective L-type Ca-channel antagonist nifedipine (10M), which blocked the long-lasting Ca-current by about two thirds (Fig. 7E and F), had no effect at all in the spontaneous firing frequency, either in cell-attached mode (Fig. 12C) or in whole-cell configuration, in a total of 6 cells recorded in slices. Also calcicludine (1M), a powerful although less selective HVA channel blocker (Schweitz et al.1994), which inhibited the longlasting Ca-channel component (73%, Fig. 7F), was equally ineffective, even after very long periods of application (Fig. 12E). Analogous results were obtained using other classical blockers of HVA Ca-channels, like -conotoxin GVIA (0.82M), which blocks the N-type, or spider toxin -agatoxin IVA (10 nM) which blocks P/Q-type channels. Both did suppress a fraction of the residual HVA Ca-current after nifedipine block (at –10 mV 38% and 42%, respectively), suggesting the presence of the corresponding types of HVA Ca-channels, but

Among the remaining candidates for a role in pacemaking was the LVA, T-type. Mibefradil, an anti-hypertensive drug, has been reported to inhibit T-type calcium channel current in cerebellar granule cells (Randall and Tsien1997), sensory neurones (Todorovic and

We therefore tried this drug, which proved to be considerably powerful in blocking the spontaneous activity of PG DA neurones, both in cell attached (Fig. 13A) and in whole-cell mode (Fig. 13B). Mibefradil (5 to 10M) completely and reversibly blocks the spontaneous activity, inducing an evident hyperpolarisation that on average amounted to about 15 mV (Fig. 13B). Nickel 100M, that we have shown to be a selective blocker of T-type calcium current in these cells (Fig. 8A), induced a reversible block of spontaneous firing, also accompanied by a hyperpolarisation of 10-15 mV (Fig. 13C), an effect almost superimposable to that observed with mibefradil, further confirming a role of ICa(T) in the

membrane is brought back to resting values.

none of them affected the spontaneous firing (not shown).

Lingle1998) and spinal motor neurones (Viana et al.1997).

pacemaking process.

Fig. 10. Properties of the KIR current. A – Family of responses to hyperpolarizing steps from -50 to -130 mV in 10 mV increments from the holding potential of -40 mV. The h-current was blocked by ivabradine 10M, and the external saline was modified with high K+ (32.5 mM) to enhance the KIR amplitude. B – Same as A, but with the addition of Ba2+ 0.2 mM. C - I/V relationship of the KIR-current measured at the onset (open circle) and at the steady state (filled circle), at the time points marked in A by the relative symbols; average values ± SE from 11 cells. The reversal potential, calculated from the intercept of the x-axis, was -44 mV, not too far from the EK, which was -33 mV.

and cell-attached modes respectively (Fig. 2D). This frequency was about double the corresponding value observed in thin slices, suggesting the existence in semi-intact tissue of some inhibitory control, possibly autoinhibition, which has not been further investigated in this study.

#### **2.9 The pacemaker currents**

We next tried to elucidate the ionic basis of the pacemaker current underlying the spontaneous firing.

The presence of the h-current, typically associated with the pacemaking process in a large number of autorhythmic cell (see (Wahl-Schott and Biel2009) for a review) has suggested that it could play its archetypal role also in bulbar DA neurons. The Ih blockade with ivabradine did break the spontaneous activity, but this effect was associated to a prominent hyperpolarization (Fig. 11A). It was therefore necessary to clarify if this block was the demonstration of a direct role of the h-current in the pacemaker mechanism, or only a secondary effect, due to the hyperpolarization. If, in the presence of ivabradine block, the membrane was depolarised to the original resting potential, then the spontaneous activity resumed immediately (Fig. 11B), clearly showing that the h-current had no direct role in it.

We next analysed the role of the Ca-current. Ca2+ is involved in the pacemaker process, as Cd2+ 100M completely and reversibly blocked the spontaneous firing (Fig 12B). Then, using a panel of different Cav channels inhibitors, we tried to define which types of neuronal Cav channels (L-, P/Q-, N-, R-, and T-type) contributed to the pacemaker current.

Fig. 10. Properties of the KIR current. A – Family of responses to hyperpolarizing steps from -50 to -130 mV in 10 mV increments from the holding potential of -40 mV. The h-current was blocked by ivabradine 10M, and the external saline was modified with high K+ (32.5 mM) to enhance the KIR amplitude. B – Same as A, but with the addition of Ba2+ 0.2 mM. C - I/V relationship of the KIR-current measured at the onset (open circle) and at the steady state (filled circle), at the time points marked in A by the relative symbols; average values ± SE from 11 cells. The reversal potential, calculated from the intercept of the x-axis, was -44 mV,

and cell-attached modes respectively (Fig. 2D). This frequency was about double the corresponding value observed in thin slices, suggesting the existence in semi-intact tissue of some inhibitory control, possibly autoinhibition, which has not been further investigated in

We next tried to elucidate the ionic basis of the pacemaker current underlying the

The presence of the h-current, typically associated with the pacemaking process in a large number of autorhythmic cell (see (Wahl-Schott and Biel2009) for a review) has suggested that it could play its archetypal role also in bulbar DA neurons. The Ih blockade with ivabradine did break the spontaneous activity, but this effect was associated to a prominent hyperpolarization (Fig. 11A). It was therefore necessary to clarify if this block was the demonstration of a direct role of the h-current in the pacemaker mechanism, or only a secondary effect, due to the hyperpolarization. If, in the presence of ivabradine block, the membrane was depolarised to the original resting potential, then the spontaneous activity resumed immediately (Fig. 11B), clearly showing that the h-current had no direct role in it. We next analysed the role of the Ca-current. Ca2+ is involved in the pacemaker process, as Cd2+ 100M completely and reversibly blocked the spontaneous firing (Fig 12B). Then, using a panel of different Cav channels inhibitors, we tried to define which types of neuronal

Cav channels (L-, P/Q-, N-, R-, and T-type) contributed to the pacemaker current.

not too far from the EK, which was -33 mV.

**2.9 The pacemaker currents** 

spontaneous firing.

this study.

Fig. 11. Effect of blockers of h-channels on spontaneous firing. A – Ivabradine (10M, bar) block of spontaneous activity; note the large hyperpolarization. At the times indicated by arrowheads, depolarising currents of increasing amplitudes were delivered. B - Enlargement of the response to the third injection of depolarising current (grey arrowhead) to show that the block of the h-current does not impairs the spontaneous activity, provided that the membrane is brought back to resting values.

The classical selective L-type Ca-channel antagonist nifedipine (10M), which blocked the long-lasting Ca-current by about two thirds (Fig. 7E and F), had no effect at all in the spontaneous firing frequency, either in cell-attached mode (Fig. 12C) or in whole-cell configuration, in a total of 6 cells recorded in slices. Also calcicludine (1M), a powerful although less selective HVA channel blocker (Schweitz et al.1994), which inhibited the longlasting Ca-channel component (73%, Fig. 7F), was equally ineffective, even after very long periods of application (Fig. 12E). Analogous results were obtained using other classical blockers of HVA Ca-channels, like -conotoxin GVIA (0.82M), which blocks the N-type, or spider toxin -agatoxin IVA (10 nM) which blocks P/Q-type channels. Both did suppress a fraction of the residual HVA Ca-current after nifedipine block (at –10 mV 38% and 42%, respectively), suggesting the presence of the corresponding types of HVA Ca-channels, but none of them affected the spontaneous firing (not shown).

Among the remaining candidates for a role in pacemaking was the LVA, T-type. Mibefradil, an anti-hypertensive drug, has been reported to inhibit T-type calcium channel current in cerebellar granule cells (Randall and Tsien1997), sensory neurones (Todorovic and Lingle1998) and spinal motor neurones (Viana et al.1997).

We therefore tried this drug, which proved to be considerably powerful in blocking the spontaneous activity of PG DA neurones, both in cell attached (Fig. 13A) and in whole-cell mode (Fig. 13B). Mibefradil (5 to 10M) completely and reversibly blocks the spontaneous activity, inducing an evident hyperpolarisation that on average amounted to about 15 mV (Fig. 13B). Nickel 100M, that we have shown to be a selective blocker of T-type calcium current in these cells (Fig. 8A), induced a reversible block of spontaneous firing, also accompanied by a hyperpolarisation of 10-15 mV (Fig. 13C), an effect almost superimposable to that observed with mibefradil, further confirming a role of ICa(T) in the pacemaking process.

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 41

Finally, we investigated the role of the KIR-current on spontaneous activity. The block of the KIR channels with Ba2+ 1 mM induced a large depolarization (on average 12 mV),

The effect is illustrated in Fig. 14: a progressive depolarization leads to a complete break of spontaneous activity with a stop at a very depolarised potential (-30 mV in the example shown in figure). In these conditions, the injection of hyperpolarizing currents allows a complete recovery of spontaneous firing, suggesting that the KIR currents plays a relevant role in determining the resting membrane potential, but not in the

Fig. 14. Effect of blockers of KIR-channels on spontaneous firing. Ba2+ (0.5 mM) was applied in the bath 60 s before the beginning of the record; note the progressive depolarization and the stop of any activity in a depolarised state. In this condition, a hyperpolarizing current of

Finally, we have modelled the bulbar DA neurones in Hodgkin-Huxley terms (Hodgkin and Huxley1952), considering the cell as a single electrical and spatial compartment. As for the conductances considered, we incorporated the two sodium currents (fast transient and persistent), the L-type Ca-current, the delayed rectifier K-current, all according to the experimental data presented above, and the T-type calcium current. Since our characterisation of this current was incomplete (and because of the difficulty in obtaining a complete kinetic description of this current), in our model we have integrated our data with others derived from the literature (Wang et al.1996; Perez-Reyes2003), and consistent with

All the equations and parameters used, as well as the assumptions made, are listed in the appendix. The solution of the set of differential equations describing the kinetics of the currents considered lead to the tracings reported in Fig. 15. The model we set up shows intrinsic spiking capabilities with full-size action potentials at the same frequencies observed in TH-GFP cells. The model behaves much like the cells studied: each action potential is followed by a slow depolarisation which brings the model cell at the fast sodium channel

8 pA restores the resting potential, restarting the spontaneous activity.

**2.10 Modelling the natural burst firing in bulbar DA neurones** 

accompanied by a complete stopover of the spontaneous firing.

pacemaking process.

our experimental data.

threshold, and initiating a new Hodgkin cycle.

Fig. 12. Effect of blockers of HVA Ca-channels on spontaneous firing. A-B – Effect of the Cd2+ (100M) on action currents recorded in cell attached mode. C – Effect of L-type Cachannel antagonist, nifedipine (10M). D-E – Effect of L-type Ca-channel antagonist calcicludine (1M), recorded 15 min after application.

Fig. 13. Effect of blockers of LVA Ca-channels on spontaneous firing. A – Effect of the T-type Ca-channel blocker mibefradil (10M) on action currents recorded in cell attached mode. B – Effect of mibefradil in a different TH-GFP cell, recorded in whole-cell configuration. C – Effect of nickel 100M on spontaneous firing.

Fig. 12. Effect of blockers of HVA Ca-channels on spontaneous firing. A-B – Effect of the Cd2+ (100M) on action currents recorded in cell attached mode. C – Effect of L-type Cachannel antagonist, nifedipine (10M). D-E – Effect of L-type Ca-channel antagonist

Fig. 13. Effect of blockers of LVA Ca-channels on spontaneous firing. A – Effect of the T-type Ca-channel blocker mibefradil (10M) on action currents recorded in cell attached mode. B – Effect of mibefradil in a different TH-GFP cell, recorded in whole-cell configuration. C –

calcicludine (1M), recorded 15 min after application.

Effect of nickel 100M on spontaneous firing.

Finally, we investigated the role of the KIR-current on spontaneous activity. The block of the KIR channels with Ba2+ 1 mM induced a large depolarization (on average 12 mV), accompanied by a complete stopover of the spontaneous firing.

The effect is illustrated in Fig. 14: a progressive depolarization leads to a complete break of spontaneous activity with a stop at a very depolarised potential (-30 mV in the example shown in figure). In these conditions, the injection of hyperpolarizing currents allows a complete recovery of spontaneous firing, suggesting that the KIR currents plays a relevant role in determining the resting membrane potential, but not in the pacemaking process.

Fig. 14. Effect of blockers of KIR-channels on spontaneous firing. Ba2+ (0.5 mM) was applied in the bath 60 s before the beginning of the record; note the progressive depolarization and the stop of any activity in a depolarised state. In this condition, a hyperpolarizing current of 8 pA restores the resting potential, restarting the spontaneous activity.

#### **2.10 Modelling the natural burst firing in bulbar DA neurones**

Finally, we have modelled the bulbar DA neurones in Hodgkin-Huxley terms (Hodgkin and Huxley1952), considering the cell as a single electrical and spatial compartment. As for the conductances considered, we incorporated the two sodium currents (fast transient and persistent), the L-type Ca-current, the delayed rectifier K-current, all according to the experimental data presented above, and the T-type calcium current. Since our characterisation of this current was incomplete (and because of the difficulty in obtaining a complete kinetic description of this current), in our model we have integrated our data with others derived from the literature (Wang et al.1996; Perez-Reyes2003), and consistent with our experimental data.

All the equations and parameters used, as well as the assumptions made, are listed in the appendix. The solution of the set of differential equations describing the kinetics of the currents considered lead to the tracings reported in Fig. 15. The model we set up shows intrinsic spiking capabilities with full-size action potentials at the same frequencies observed in TH-GFP cells. The model behaves much like the cells studied: each action potential is followed by a slow depolarisation which brings the model cell at the fast sodium channel threshold, and initiating a new Hodgkin cycle.

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 43

Fifth, small changes in the parameters of INa(F) and of IK(V), such as the half activation point shift of a few millivolts, were sufficient to arrest spontaneous firing but did not affect the capacity to respond with single action potentials to depolarising stimuli. This proves that the pacemaking process is due to an interplay of conductances which are in a delicate and precise equilibrium. Furthermore it confirms the model is capable of capturing the essential

The model thus confirms that, in addition to INa(P), another component is necessary to sustain repetitive firing, a T-type calcium current. Together with the experimental finding that treatments which block ICa(T), such as mibefradil and nickel at micromolar concentrations, are both capable of preventing repetitive firing. Therefore, it appears that

This study aims at providing a description as comprehensive as possible of the functional properties of DA neurones in the mammalian olfactory bulb. The animal model used for these experiments, a strain of transgenic mice expressing a reporter protein under the TH promoter (Sawamoto et al.2001; Matsushita et al.2002), allows an easy identification of DA neurones both in thin slices and dissociated cells, proving to be a superb tool for targeting live DA neurones in electrophysiological studies. The main results obtained are the demonstration that DA neurones in the OB are autorhythmic, and the description of the

Neurones expressing high levels of the reporter protein were found in the glomerular layer, as expected from abundant literature, indicating that this is the only region of the main olfactory bulb where TH is expressed (Halász1990; Kratskin and Belluzzi2003). A previous study in the same mice strain has demonstrated an overlapping expression of the fluorescent reporter and TH protein in olfactory bulb only in those DA neurons that

DA neurones in the mice OB have a complement of voltage-dependent currents, which have been kinetically characterised in our study. Among these, the persistent Na current deserves some comment. Many neurones in the mammalian CNS have a non-inactivating component of the TTX-sensitive sodium current (Crill1996). Although its magnitude in bulbar DA neurones is about 0.5 % of the transient sodium current, INa(P) appears to have an important functional significance because it is activated at potentials 8-10 mV more negative than the transient sodium current. At this potential few voltage-gated channels are activated and the neuron input resistance is high. The conductance-voltage relationship for gNa(P) in TH-GFP DA neurones has a half-activation point at –47.7 mV, very close to the values found in hippocampal CA1 neurones (French et al.1990), and pyramidal neurones of the neocortex (Brown et al.1994). Although this current might appear small, 7.3 pA at –60 mV (Fig. 6A), it suffices to depolarise the cell membrane of these cells, which have an average input

We have considered the possibility that INa(P) is in fact a window current, the steady current predicted by the HH model and arising from overlap of the steady-state activation and

features of the excitability profile of these cells.

**3. Discussion** 

such current is an essential component of the pacemaking process.

interplay of subthreshold currents underlying intrinsic spiking.

received afferent stimulation from receptor cells (Baker et al.2003).

resistance of 700 M, by about 5 mV.

**3.1 Distribution and general properties of DA neurones** 

Fig. 15. Numerical reconstitution of spontaneous activity in TH-GFP PG cells. A - Voltage tracings. B - Current tracings including the five conductances: INa(F), INa(P), ICa(T), ICa(L), IK(V). C, D - Enlargement of the last two events of panels A-B to show the pacemaking process. In D the outward current has been omitted, and the inward currents have been amplified by a factor of about 500 with respect to panel B.

The model is effective to verify the accuracy of the kinetics calculated, and it is particularly useful to understand the details of the interplay of the currents underlying the pacemaking process. The main findings provided by the numerical simulations can be summarised as follows.

First, during the interspike interval, the currents present which cause the progressive depolarisation of the cell are, in order, the T-type calcium current, and then the persistent sodium current. These currents are amazingly small in amplitude (max 4 pA) compared with fast transient sodium and delayed rectifier potassium currents associated to the action potential (about 1 nA), but nevertheless they are sufficient to depolarise these cells, due to their rather high input resistance (about 700 M.

Second, both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity: the model cell is still capable of responding with a single action potential to the injection of a depolarising current pulse, but it fails to fire repetitively.

Third, the model suggests that it is the T-type calcium current which sets in motion the depolarising process: although essential to the rhythm generation, INa(P) replaces ICa(T) only in the second half of the depolarising phase. The T-type calcium conductance amplitude is critical in determining the firing frequency: small changes in its value (from 0.35 to 0.4 nS) are sufficient to drive the intrinsic spiking from 8 Hz to 16 Hz.

Fourth, the HVA calcium currents are unnecessary for the pacemaking process, and their suppression has no consequence on the frequency of spontaneous firing, exactly as in living TH-GFP cells.

Fifth, small changes in the parameters of INa(F) and of IK(V), such as the half activation point shift of a few millivolts, were sufficient to arrest spontaneous firing but did not affect the capacity to respond with single action potentials to depolarising stimuli. This proves that the pacemaking process is due to an interplay of conductances which are in a delicate and precise equilibrium. Furthermore it confirms the model is capable of capturing the essential features of the excitability profile of these cells.

The model thus confirms that, in addition to INa(P), another component is necessary to sustain repetitive firing, a T-type calcium current. Together with the experimental finding that treatments which block ICa(T), such as mibefradil and nickel at micromolar concentrations, are both capable of preventing repetitive firing. Therefore, it appears that such current is an essential component of the pacemaking process.

#### **3. Discussion**

42 Electrophysiology – From Plants to Heart

Fig. 15. Numerical reconstitution of spontaneous activity in TH-GFP PG cells. A - Voltage tracings. B - Current tracings including the five conductances: INa(F), INa(P), ICa(T), ICa(L), IK(V). C, D - Enlargement of the last two events of panels A-B to show the pacemaking process. In D the outward current has been omitted, and the inward currents have been amplified by a

The model is effective to verify the accuracy of the kinetics calculated, and it is particularly useful to understand the details of the interplay of the currents underlying the pacemaking process. The main findings provided by the numerical simulations can be summarised as

First, during the interspike interval, the currents present which cause the progressive depolarisation of the cell are, in order, the T-type calcium current, and then the persistent sodium current. These currents are amazingly small in amplitude (max 4 pA) compared with fast transient sodium and delayed rectifier potassium currents associated to the action potential (about 1 nA), but nevertheless they are sufficient to depolarise these cells, due to

Second, both INa(P) and ICa(T) are necessary to sustain spontaneous firing as the selective block of one or both abolish spontaneous activity: the model cell is still capable of responding with a single action potential to the injection of a depolarising current pulse, but it fails to fire

Third, the model suggests that it is the T-type calcium current which sets in motion the depolarising process: although essential to the rhythm generation, INa(P) replaces ICa(T) only in the second half of the depolarising phase. The T-type calcium conductance amplitude is critical in determining the firing frequency: small changes in its value (from 0.35 to 0.4 nS)

Fourth, the HVA calcium currents are unnecessary for the pacemaking process, and their suppression has no consequence on the frequency of spontaneous firing, exactly as in living

factor of about 500 with respect to panel B.

their rather high input resistance (about 700 M.

are sufficient to drive the intrinsic spiking from 8 Hz to 16 Hz.

follows.

repetitively.

TH-GFP cells.

This study aims at providing a description as comprehensive as possible of the functional properties of DA neurones in the mammalian olfactory bulb. The animal model used for these experiments, a strain of transgenic mice expressing a reporter protein under the TH promoter (Sawamoto et al.2001; Matsushita et al.2002), allows an easy identification of DA neurones both in thin slices and dissociated cells, proving to be a superb tool for targeting live DA neurones in electrophysiological studies. The main results obtained are the demonstration that DA neurones in the OB are autorhythmic, and the description of the interplay of subthreshold currents underlying intrinsic spiking.

#### **3.1 Distribution and general properties of DA neurones**

Neurones expressing high levels of the reporter protein were found in the glomerular layer, as expected from abundant literature, indicating that this is the only region of the main olfactory bulb where TH is expressed (Halász1990; Kratskin and Belluzzi2003). A previous study in the same mice strain has demonstrated an overlapping expression of the fluorescent reporter and TH protein in olfactory bulb only in those DA neurons that received afferent stimulation from receptor cells (Baker et al.2003).

DA neurones in the mice OB have a complement of voltage-dependent currents, which have been kinetically characterised in our study. Among these, the persistent Na current deserves some comment. Many neurones in the mammalian CNS have a non-inactivating component of the TTX-sensitive sodium current (Crill1996). Although its magnitude in bulbar DA neurones is about 0.5 % of the transient sodium current, INa(P) appears to have an important functional significance because it is activated at potentials 8-10 mV more negative than the transient sodium current. At this potential few voltage-gated channels are activated and the neuron input resistance is high. The conductance-voltage relationship for gNa(P) in TH-GFP DA neurones has a half-activation point at –47.7 mV, very close to the values found in hippocampal CA1 neurones (French et al.1990), and pyramidal neurones of the neocortex (Brown et al.1994). Although this current might appear small, 7.3 pA at –60 mV (Fig. 6A), it suffices to depolarise the cell membrane of these cells, which have an average input resistance of 700 M, by about 5 mV.

We have considered the possibility that INa(P) is in fact a window current, the steady current predicted by the HH model and arising from overlap of the steady-state activation and

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 45

potential, therefore, whose influence on the interplay of pacemaker currents can be well understood with the numerical model proposed, can be adjusted in either directions by a modulation of the two hyperpolarization-activated currents. Both currents can be modulated by a variety of neurotransmitters (see (Biel et al.2009) and (Hibino et al.2010) for recent reviews on h-current and KIR, respectively). Interestingly, some intracellular pathway, like AC/cAMP, can act on both Ih and KIR, enhancing the first and hampering the second (Podda et al.2010); the contrary effect is also observed, e.g. in the rat substantia nigra neurones, where bath application of noradrenaline or dopamine both inhibit Ih and increase the KIR conductance (Cathala and Paupardin-Tritsch1999). Elucidating these interactions will be a critical step in order to understand the functioning of these cells and, in a longer perspective, the still elusive role of dopaminergic neurones in signal processing in the

Our results, and especially the indication that DA neurones in the glomerular layer are

It is known that the glomerular neuropile, far from being a homogeneous structure, shows a complex subcompartimental organisation: within a single glomerulus, olfactory nerve (ON) islets delimit areas in which dendritic branches receive sensory input from ON terminals, and are well separated from non-ON zones, from which ON terminals are excluded (Chao et al.1997; Kasowski et al.1999; Toida et al.2000). Some TH-immunoreactive PG cells arborise on the ON islets, others in the non-ON zone (Kosaka et al.1997). Although it has been reported that in some mouse strains TH positive cells are not in contact with ON terminals (Weruaga et al.2000), in the strain used for these experiments – C57BL/6J, one of the most commons - such contacts have been demonstrated (T. Kosaka and K. Kosaka, personal

The glomerular compartmentalisation supports the hypothesis that information processing is subdivided regionally within the mammalian glomerulus (Kasowski et al.1999). DA neurones establishing contacts in ON- or in non-ON zones, possibly play different roles. Within the ON zones, dendrites of DA neurones receive excitatory synapses from ON axon terminals (Chao et al.1997; Kasowski et al.1999; Toida et al.2000). It has been reported that D2 dopaminergic receptors are located in ON terminals (Levey et al.1993; Coronas et al.1997), and electrophysiological studies have shown that their activation can reduce the probability of glutamate release, and hence the excitation of projection neurones (Duchamp-Viret et al.1997; Hsia et al.1999; Berkowicz and Trombley2000; Ennis et al.2001). In fact, the most significant impact of DA neurones is expected at the level of the synaptic triad formed by the ON and the dendrites of mitral/tufted (MT) cells and PG cells (Bardoni et al.1996), where DA neurones directly control the input of projection neurones from receptor cell axons (Brünig et al.1999). Since the ON islets appear to be further segregated from the rest of the glomerular neuropile due to the presence of "glial wraps" (Kasowski et al.1999), the spontaneous activity of DA neurones (which implies continuous release of dopamine within

In addition, DA neurones send their dendrites also in non-ON zones, where they contact dendrites of projection neurones and interneurons, and centrifugal axons. Projection

a restricted space), would create a condition of tonic inhibition of the ON.

olfactory bulb.

communication).

**3.4 Significance to olfactory function** 

autorhythmic, open the field to many speculations.

inactivation curves for sodium conductance (Attwell et al.1979; Colatsky1982). Based on the kinetic analysis of the fast Na-current described in the previous paragraph (Fig. 4H), we calculated the window current at different potentials. The numerical simulation indicates this current does provide a measurable contribution to the TTX-sensitive persistent inward current. However, this contribution, which is maximum at –55 mV, becomes virtually zero at –30 mV (Fig. 4H), the potential at which the persistent sodium current displays its maximum amplitude (Fig. 6A). In other words, INa(P) and the window current of INa(F) develop at different potentials, and therefore are distinct currents.

#### **3.2 Interaction of ionic currents to produce spontaneous firing**

The pharmacological treatments, ion substitution experiments, kinetic analysis and numerical simulations allow a rather precise understanding of mechanisms underlying spontaneous firing in TH-GFP cells. The analysis indicates that the slow depolarisation between spikes results from an interplay between the persistent, tetrodotoxin-sensitive sodium current and the T-type calcium current.

The role of a calcium current in rhythm generation is revealed by the rapid and reversible block of the spontaneous firing by Cd2+, both in slices and in dissociated cells (Fig. 12B). However, among the many HVA Ca-channels present in the DA neurones of the olfactory bulb (a large L-, and smaller N- and P/Q types), none proved to be effective in the control of spontaneous firing. On the contrary, conditions which are known to block the LVA T-type Ca-channels (mibefradil and nickel in the micromolar range) did break off the spontaneous activity.

We developed a numerical HH-type model (Hodgkin and Huxley1952), based on the kinetic characterisation of the voltage-dependent currents described above, which appears to be capable of reproducing fairly well the behaviour of TH-GFP cells. The model was designed in order to verify the accuracy of our kinetic analysis, and, moreover, in order to understand the mechanisms underlying the intrinsic spiking.

Our model reproduces the properties of real neurones, and clearly indicates that the rhythm generation requires the presence of both INa(P) and ICa(T): in the absence of any of the two currents, the model can respond with a single action potential to a depolarising pulse, but is unable to produce repetitive firing.

The model shows that the current which sets in motion the spontaneous spiking is the Ttype calcium current, which in turn brings into play the persistent sodium current. In spite of the limits inherent to this type of models, we note that small changes in the kinetic parameters lead to the loss of intrinsic spiking but not of the capacity of the cell to respond with a single action potential to the "injection" of a short depolarising current step, thus suggesting that the model has captured the essential features of the real cell.

#### **3.3 The role of hyperpolarization-activated currents**

Our results exclude a direct contribution of the h-current to the spontaneous firing and, although less conclusively, also for the KIR current. However, it is of great interest that, as we show, both currents exert a powerful control on the resting membrane potential, the first depolarising, the second hyperpolarising the membrane at rest. The resting membrane potential, therefore, whose influence on the interplay of pacemaker currents can be well understood with the numerical model proposed, can be adjusted in either directions by a modulation of the two hyperpolarization-activated currents. Both currents can be modulated by a variety of neurotransmitters (see (Biel et al.2009) and (Hibino et al.2010) for recent reviews on h-current and KIR, respectively). Interestingly, some intracellular pathway, like AC/cAMP, can act on both Ih and KIR, enhancing the first and hampering the second (Podda et al.2010); the contrary effect is also observed, e.g. in the rat substantia nigra neurones, where bath application of noradrenaline or dopamine both inhibit Ih and increase the KIR conductance (Cathala and Paupardin-Tritsch1999). Elucidating these interactions will be a critical step in order to understand the functioning of these cells and, in a longer perspective, the still elusive role of dopaminergic neurones in signal processing in the olfactory bulb.

#### **3.4 Significance to olfactory function**

44 Electrophysiology – From Plants to Heart

inactivation curves for sodium conductance (Attwell et al.1979; Colatsky1982). Based on the kinetic analysis of the fast Na-current described in the previous paragraph (Fig. 4H), we calculated the window current at different potentials. The numerical simulation indicates this current does provide a measurable contribution to the TTX-sensitive persistent inward current. However, this contribution, which is maximum at –55 mV, becomes virtually zero at –30 mV (Fig. 4H), the potential at which the persistent sodium current displays its maximum amplitude (Fig. 6A). In other words, INa(P) and the window current of INa(F)

The pharmacological treatments, ion substitution experiments, kinetic analysis and numerical simulations allow a rather precise understanding of mechanisms underlying spontaneous firing in TH-GFP cells. The analysis indicates that the slow depolarisation between spikes results from an interplay between the persistent, tetrodotoxin-sensitive

The role of a calcium current in rhythm generation is revealed by the rapid and reversible block of the spontaneous firing by Cd2+, both in slices and in dissociated cells (Fig. 12B). However, among the many HVA Ca-channels present in the DA neurones of the olfactory bulb (a large L-, and smaller N- and P/Q types), none proved to be effective in the control of spontaneous firing. On the contrary, conditions which are known to block the LVA T-type Ca-channels (mibefradil and nickel in the micromolar range) did break off the spontaneous

We developed a numerical HH-type model (Hodgkin and Huxley1952), based on the kinetic characterisation of the voltage-dependent currents described above, which appears to be capable of reproducing fairly well the behaviour of TH-GFP cells. The model was designed in order to verify the accuracy of our kinetic analysis, and, moreover, in order to understand

Our model reproduces the properties of real neurones, and clearly indicates that the rhythm generation requires the presence of both INa(P) and ICa(T): in the absence of any of the two currents, the model can respond with a single action potential to a depolarising pulse, but is

The model shows that the current which sets in motion the spontaneous spiking is the Ttype calcium current, which in turn brings into play the persistent sodium current. In spite of the limits inherent to this type of models, we note that small changes in the kinetic parameters lead to the loss of intrinsic spiking but not of the capacity of the cell to respond with a single action potential to the "injection" of a short depolarising current step, thus

Our results exclude a direct contribution of the h-current to the spontaneous firing and, although less conclusively, also for the KIR current. However, it is of great interest that, as we show, both currents exert a powerful control on the resting membrane potential, the first depolarising, the second hyperpolarising the membrane at rest. The resting membrane

suggesting that the model has captured the essential features of the real cell.

develop at different potentials, and therefore are distinct currents.

**3.2 Interaction of ionic currents to produce spontaneous firing** 

sodium current and the T-type calcium current.

the mechanisms underlying the intrinsic spiking.

**3.3 The role of hyperpolarization-activated currents** 

unable to produce repetitive firing.

activity.

Our results, and especially the indication that DA neurones in the glomerular layer are autorhythmic, open the field to many speculations.

It is known that the glomerular neuropile, far from being a homogeneous structure, shows a complex subcompartimental organisation: within a single glomerulus, olfactory nerve (ON) islets delimit areas in which dendritic branches receive sensory input from ON terminals, and are well separated from non-ON zones, from which ON terminals are excluded (Chao et al.1997; Kasowski et al.1999; Toida et al.2000). Some TH-immunoreactive PG cells arborise on the ON islets, others in the non-ON zone (Kosaka et al.1997). Although it has been reported that in some mouse strains TH positive cells are not in contact with ON terminals (Weruaga et al.2000), in the strain used for these experiments – C57BL/6J, one of the most commons - such contacts have been demonstrated (T. Kosaka and K. Kosaka, personal communication).

The glomerular compartmentalisation supports the hypothesis that information processing is subdivided regionally within the mammalian glomerulus (Kasowski et al.1999). DA neurones establishing contacts in ON- or in non-ON zones, possibly play different roles. Within the ON zones, dendrites of DA neurones receive excitatory synapses from ON axon terminals (Chao et al.1997; Kasowski et al.1999; Toida et al.2000). It has been reported that D2 dopaminergic receptors are located in ON terminals (Levey et al.1993; Coronas et al.1997), and electrophysiological studies have shown that their activation can reduce the probability of glutamate release, and hence the excitation of projection neurones (Duchamp-Viret et al.1997; Hsia et al.1999; Berkowicz and Trombley2000; Ennis et al.2001). In fact, the most significant impact of DA neurones is expected at the level of the synaptic triad formed by the ON and the dendrites of mitral/tufted (MT) cells and PG cells (Bardoni et al.1996), where DA neurones directly control the input of projection neurones from receptor cell axons (Brünig et al.1999). Since the ON islets appear to be further segregated from the rest of the glomerular neuropile due to the presence of "glial wraps" (Kasowski et al.1999), the spontaneous activity of DA neurones (which implies continuous release of dopamine within a restricted space), would create a condition of tonic inhibition of the ON.

In addition, DA neurones send their dendrites also in non-ON zones, where they contact dendrites of projection neurones and interneurons, and centrifugal axons. Projection

Pacemaker Currents in Dopaminergic Neurones of the Mice Olfactory Bulb 47

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neurones express D1 and D2 receptors (Brünig et al.1999; Davila et al.2003) , and it has been shown that dopamine exerts a complex modulatory action between them and interneurons (*ibidem*), an effect that also in this case would be amplified by the restricted space of the glomerular neuropile. Within this framework, dopamine might play a central role in the processing of olfactory information by acting at two levels: it would control the input of the sensory signal, and it would modulate the mechanism of GABAergic inhibition (Brünig et al.1999; Davila et al.2003).

It remains to be explained why DA neurones in the glomerular region of the OB are among the very few in the mammalian CNS which are generated also in adulthood. On one end this brings up series of questions concerning the mechanisms controlling their migration and differentiation, and, on the other end, it opens interesting perspectives for the exploitation of the olfactory bulb as a source of undifferentiated DA cells that could be expanded *ex vivo* and used for transplants in neurodegenerative diseases.

#### **4. Acknowledgements**

This work was supported by grants from MURST (PRIN 2009) and from Programma Medicina Rigenerativa Regione E.R. – Università, 2007–2009.

#### **5. References**


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It remains to be explained why DA neurones in the glomerular region of the OB are among the very few in the mammalian CNS which are generated also in adulthood. On one end this brings up series of questions concerning the mechanisms controlling their migration and differentiation, and, on the other end, it opens interesting perspectives for the exploitation of the olfactory bulb as a source of undifferentiated DA cells that could be expanded *ex vivo*

This work was supported by grants from MURST (PRIN 2009) and from Programma

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**3** 

Avital Schurr

*USA* 

**Hippocampal Slices and Their Electrophysiogy** 

Dorland's Illustrated Medical Dictionary in its 24th Edition (1965), describes the term "Electrophysiology" as "The Science of physiology in its relations to electricity; the study of the electric reactions of the body in health." The ability of scientists to observe and record physiology's electrical phenomena long preceded the understanding of the membranous ionic processes that are responsible for them. Consequently, for a while, electrophysiology has been considered a subfield of physiology, aiming at improving our understanding of cellular, organ and bodily functions. With the advances made in molecular biology, genetics and neuroscience, the role of electrophysiology has shifted, where today it is being employed as one of the best, most accurate and least expensive real-time monitoring tools in

The discovery in the early 1950s that brain slices can sustain certain electrophysiological characteristics typical of the intact brain opened a wide range of possibilities for studying cerebral tissue and its electrophysiology *in vitro.* Obviously, the brain slice preparation affords the experimenter both the control and manipulation of the environmental conditions under which the neural tissue is maintained. Employing electrophysiological techniques allows a continuous monitoring/recording of the neural tissue's viability, its functions and its responses to environmental and other changes brought about by the experimenter's

Thousands of papers and several books have been published over the past 30 years, where brain slices were the topic itself or where studies employed them in combination with various techniques, including electrophysiological ones. The present chapter describes some important advances made over the past three decades using brain slices and their electrophysiology in our laboratory, advances that provided us with a better understanding of cerebral energy metabolism. All the experiments detailed herewith employed the rat hippocampal slice preparation, using a continuous extracellular, real-time monitoring of the

Of the different brain slice preparations available, the rat hippocampal slice preparation is without a doubt the most studied. Henry McIlwain and colleagues were the first to use thin brain sections for metabolic studies (McIlwain et al., 1951; McIlwain & Buddle, 1953;

basic science research and clinical studies and practice alike.

electrically-evoked CA1 population spike (PS).

**1. Introduction** 

chosen manipulations.

**in the Study of Brain Energy Metabolism** 

*Department of Anesthesiology & Perioperative Medicine, University of Louisville School of Medicine, Louisville, KY,* 

