**Neurochemical Communication: The Case of Endocannabinoids**

Thomas Heinbockel

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178 Neurochemistry

259-265.

7(1) 1-38.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58410

## **1. Introduction**

The brain is equipped with a magnificent diversity of molecules that allow neurons to communicate with each other. Some of these molecules have been known to function as neurotransmitters for several decades such as GABA and glutamate while for others their involvement in brain signaling has been demonstrated more recently. Cannabinoids fall into the latter group. Even though the effects of cannabinoids as active ingredients in marijuana on human psyche and behavior have been experienced by humans for centuries or possibly millennia, their existence and production in the brain was described only some thirty years ago. Even more recently, their functional role in neural circuits of the brain has been discerned. This review focuses on these endogenously produced signaling molecules, endogenous cannabinoids or endocannabinoids (eCBs). Their functional role in the nervous system and interaction with other neurotransmitter systems will be described. One hallmark feature of endocannabinoid signaling is their ability to act as retrograde messengers in neural circuits. Two examples, one from the hippocampus and one from the main olfactory bulb, illustrate in detail this intercellular communication pathway.

Several features underscore the importance to understand the endocannabinoid system. Increasing evidence demonstrates the relevance of endocannabinoids in normal behaviors, including pain reception [1] and feeding [2, 3]. The therapeutic potential of cannabinoids has received increasing attention over the past few years [4]. endocannabinoids play a role in neuroprotection against acute excitotoxicity [5] and functional recovery after brain injury [6]. Endocannabinoids regulate human airway function and provide a means to treat respiratory pathologies [1]. Cannabinoids are in widespread use recreationally as psychoactive drugs and interact with other drugs of abuse. This fact emphasizes even more the need to understand the endocannabinoid system and the neurobiological substrate of their mood-altering capacity [7,

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

8]. Furthermore, the endocannabinoid system is crucially involved in processes of learning and memory, e.g., in the extinction of aversive memories [9].

exogenously applied cannabinoids such as cannabis. Anandamide and 2-AG were discovered in the early 1990s [27-29, reviewed in 30] while their functional role in neuronal communication remained obscure for years. Since their discovery, the role of endocannabinoids as retrograde messengers that suppress both excitatory and inhibitory transmission has been well-estab‐ lished. Endocannabinoids mediate retrograde signals in the hippocampus [31-35], cerebellum [36-38], neocortex [39, 40], amygdala [41, 42], and olfactory bulb [43]. Termination of endo‐ cannabinoidsignalling is accomplished by reuptake into both neurons and glia. Subsequently, anandamide and 2-AG are hydrolyzed intracellularly by fatty acid amide hydrolase (FAAH)

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Endocannabinoids are different from conventional neurotransmitters because they are lipids that are not stored but rather are rapidly synthesized on demand at the site of need from components of the cell membrane. Upon cellular activation, they are released from places all over the cell. They are arachidonic acid-containing messengers generated by phospholipase action [45]. Stimuli that trigger release of endocannabinoids include rise of intracellular calcium levels inside the neuron or activation of certain G-protein-coupled receptors such as metabotropic glutamate receptors (mGluR5). Subsequent to their non-synaptic, non-vesicular release, endocannabinoids bind to cannabinoid receptors on nearby neurons such as presy‐ naptic interneurons where they regulate presynaptic neurotransmitter release, e.g., through

Endocannabinoids are members of a loose family of unusual and novel neurotransmitters. Similar to endocannabinoids, other novel neurotransmitters such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) do not adhere to the classic definition of neuro‐ transmitters and challenge the notion of what constitutes a neurotransmitter [46, 47]. These synaptic molecules have changed markedly the definition of a neurotransmitter. They satisfy key neurotransmitter criteria but differ radically from classical transmitters. For example, endocannabinoids, nitric oxide and carbon monoxide are neither stored in synaptic vesicles nor released by exocytosis. Nitric oxide does not act via traditional receptors on postsynaptic

Like endocannabinoids, nitric oxide can serve as an intercellular messenger in the brain [48]. It acts as a retrograde factor at synapses and presynaptically regulates both glutama‐ tergic and GABAergic synapses to alter release-probability in synaptic plasticity. Nitric oxide influences the synaptic machinery involved in transmitter release and, in a coordinated fashion, also the vesicular recycling mechanisms. Nitric oxide has a role in the coordina‐ tion of local pre-and post-synaptic function during plasticity at individual synapses. It is involved in experience-dependent plasticity in the cerebral cortex. Likewise, cannabinoids mediate a variety of forms of short-and long-term synaptic plasticity that have been

and monoacylglycerol lipase (MAGL), respectively [44].

**3. Unusual and novel neurotransmitters**

closure of specific ion channels.

reviewed in detail elsewhere [49-51].

membranes.

#### **2. The endocannabinoid system**

Endocannabinoids are small lipids that regulate various aspects of brain function such as learning and memory including synaptic transmission and different forms of short-and longterm plasticity [10]. They also influence growth and development such as synapse formation and neurogenesis. Other biological functions modulated by endocannabinoids include eating and anxiety. Principally, two endocannabinoids, N-arachidonoylethanol-amide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the natural agonists/ligands of the most widely expressed cannabinoid receptor in the brain, CB1R [11] (Figure 1).

**Figure 1.** Two endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanol-amide (anandamide, AEA), are the natural agonists/ligands of cannabinoid receptors, CB1Rs, in the brain.

Endocannabinoids, as fatty-acid derived endogenous ligands, together with their G-protein coupled cannabinoid receptors form the endocannabinoid system. This system also includes associated biochemical machinery with endocannabinoid precursors, synthetic and degrada‐ tive enzymes for these lipidic neurotransmitters, and transporters [12-15]. Two different cannabinoid receptors have been cloned, CB1 and CB2 receptors. They share 44% amino acid sequence homology [16, 17]. The expression pattern of the two cannabinoid receptors in various body parts is distinctly different. In the brain, CB1R is the most abundant G-protein coupled receptor [18]. CB2R is primarily expressed in immune cells and peripheral tissues [17]. Some level of CB2R expression has also been detected in the brainstem, cortex and cerebellar neurons and microglia [19, 20].

Cannabinoid receptors are found at high levels in the brain [21, 22], specifically at presynaptic nerve terminals [23, 24]. They can be activated by cannabis-derived drugs. Δ9-Tetrahydrocan‐ nabinol, THC, is the bioactive ingredient of the drugs marijuana and hashish [25] and can artificially activate cannabinoid receptors as exogenous cannabinoids. Cannabinoid receptors exist in all normal brains [18, 21, 22] where they subserve many essential brain functions when activated by their natural ligands. Cannabinoid receptors in the nervous system are predom‐ inantly Gi/o-protein-coupled type 1 cannabinoid receptors (CB1 receptors, CB1Rs). Their ligands, endocannabinoids are synthesized from membrane lipids [26]. Endocannabinoids can diffuse through membranes and are thus able to activate receptors in the same manner as exogenously applied cannabinoids such as cannabis. Anandamide and 2-AG were discovered in the early 1990s [27-29, reviewed in 30] while their functional role in neuronal communication remained obscure for years. Since their discovery, the role of endocannabinoids as retrograde messengers that suppress both excitatory and inhibitory transmission has been well-estab‐ lished. Endocannabinoids mediate retrograde signals in the hippocampus [31-35], cerebellum [36-38], neocortex [39, 40], amygdala [41, 42], and olfactory bulb [43]. Termination of endo‐ cannabinoidsignalling is accomplished by reuptake into both neurons and glia. Subsequently, anandamide and 2-AG are hydrolyzed intracellularly by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively [44].

## **3. Unusual and novel neurotransmitters**

8]. Furthermore, the endocannabinoid system is crucially involved in processes of learning

Endocannabinoids are small lipids that regulate various aspects of brain function such as learning and memory including synaptic transmission and different forms of short-and longterm plasticity [10]. They also influence growth and development such as synapse formation and neurogenesis. Other biological functions modulated by endocannabinoids include eating and anxiety. Principally, two endocannabinoids, N-arachidonoylethanol-amide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the natural agonists/ligands of the most widely

**Figure 1.** Two endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanol-amide (anandamide,

Endocannabinoids, as fatty-acid derived endogenous ligands, together with their G-protein coupled cannabinoid receptors form the endocannabinoid system. This system also includes associated biochemical machinery with endocannabinoid precursors, synthetic and degrada‐ tive enzymes for these lipidic neurotransmitters, and transporters [12-15]. Two different cannabinoid receptors have been cloned, CB1 and CB2 receptors. They share 44% amino acid sequence homology [16, 17]. The expression pattern of the two cannabinoid receptors in various body parts is distinctly different. In the brain, CB1R is the most abundant G-protein coupled receptor [18]. CB2R is primarily expressed in immune cells and peripheral tissues [17]. Some level of CB2R expression has also been detected in the brainstem, cortex and cerebellar

Cannabinoid receptors are found at high levels in the brain [21, 22], specifically at presynaptic nerve terminals [23, 24]. They can be activated by cannabis-derived drugs. Δ9-Tetrahydrocan‐ nabinol, THC, is the bioactive ingredient of the drugs marijuana and hashish [25] and can artificially activate cannabinoid receptors as exogenous cannabinoids. Cannabinoid receptors exist in all normal brains [18, 21, 22] where they subserve many essential brain functions when activated by their natural ligands. Cannabinoid receptors in the nervous system are predom‐ inantly Gi/o-protein-coupled type 1 cannabinoid receptors (CB1 receptors, CB1Rs). Their ligands, endocannabinoids are synthesized from membrane lipids [26]. Endocannabinoids can diffuse through membranes and are thus able to activate receptors in the same manner as

Anandamide

N

H

O

and memory, e.g., in the extinction of aversive memories [9].

expressed cannabinoid receptor in the brain, CB1R [11] (Figure 1).

2-AG

AEA), are the natural agonists/ligands of cannabinoid receptors, CB1Rs, in the brain.

O

<sup>O</sup> HO HO

neurons and microglia [19, 20].

**2. The endocannabinoid system**

180 Neurochemistry

Endocannabinoids are different from conventional neurotransmitters because they are lipids that are not stored but rather are rapidly synthesized on demand at the site of need from components of the cell membrane. Upon cellular activation, they are released from places all over the cell. They are arachidonic acid-containing messengers generated by phospholipase action [45]. Stimuli that trigger release of endocannabinoids include rise of intracellular calcium levels inside the neuron or activation of certain G-protein-coupled receptors such as metabotropic glutamate receptors (mGluR5). Subsequent to their non-synaptic, non-vesicular release, endocannabinoids bind to cannabinoid receptors on nearby neurons such as presy‐ naptic interneurons where they regulate presynaptic neurotransmitter release, e.g., through closure of specific ion channels.

Endocannabinoids are members of a loose family of unusual and novel neurotransmitters. Similar to endocannabinoids, other novel neurotransmitters such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) do not adhere to the classic definition of neuro‐ transmitters and challenge the notion of what constitutes a neurotransmitter [46, 47]. These synaptic molecules have changed markedly the definition of a neurotransmitter. They satisfy key neurotransmitter criteria but differ radically from classical transmitters. For example, endocannabinoids, nitric oxide and carbon monoxide are neither stored in synaptic vesicles nor released by exocytosis. Nitric oxide does not act via traditional receptors on postsynaptic membranes.

Like endocannabinoids, nitric oxide can serve as an intercellular messenger in the brain [48]. It acts as a retrograde factor at synapses and presynaptically regulates both glutama‐ tergic and GABAergic synapses to alter release-probability in synaptic plasticity. Nitric oxide influences the synaptic machinery involved in transmitter release and, in a coordinated fashion, also the vesicular recycling mechanisms. Nitric oxide has a role in the coordina‐ tion of local pre-and post-synaptic function during plasticity at individual synapses. It is involved in experience-dependent plasticity in the cerebral cortex. Likewise, cannabinoids mediate a variety of forms of short-and long-term synaptic plasticity that have been reviewed in detail elsewhere [49-51].

## **4. Depolarization-induced suppression of inhibition**

The relevance of the endocannabinoid system for neural signaling and brain function in general has been explored only recently [13]. Endocannabinoids mediate a new type of neuronal communication, called DSI, **D**epolarization-induced **S**uppression of **I**nhibition (Fig. 2) (reviewed in [10, 12, 30]. A short rise in intracellular calcium concentration in a principal neuron, e.g., a pyramidal cell of the hippocampus, results in a transient decline of incoming inhibitory signals in the form of GABA arriving from other neurons. This observation led to the hypothesis that during DSI, some unknown messenger must travel from the postsynaptic cell to the presynaptic GABA-releasing one and somehow turns off neurotransmitter release. Conventional chemical synaptic signaling between two neurons involves activation of a presynaptic neuron resulting in transmitter release and subsequent activation of the postsy‐ naptic neuron, e.g., a GABAergic inhibitory interneuron makes synaptic contacts with a glutamatergic pyramidal cell in the hippocampus. When the interneuron is activated it releases the inhibitory neurotransmitter GABA and inhibits the pyramidal cell. In contrast, during DSI, when a pyramidal cell is activated, e.g., through direct current injection, the inhibitory input onto that pyramidal cell is reduced. As a major breakthrough in our understanding of endocannabinoid signaling, endocannabinoids were found to act as retrograde signaling molecules that mediate communication between postsynaptic pyramidal cells and presynaptic inhibitory interneurons and evoke the reduction in GABA release. Since endocannabinoids are lipids, they do not diffuse over great distances in the watery extracellular environment of the brain. Rather, DSI acts as a short-lived local effect that enables individual neurons to disconnect briefly from their neighbors and encode information [12].

The announcement of this breakthrough has been given the Latin term 'Dies mirabilis' (wonderful day) by Alger [10]. In March of 2001 four independent labs described in three different journals their studies culminating in the conclusion that endocannabinoids function mainly as retrograde messengers. Elphick and Egertova [52] analyzed prior pharmacological and anatomical studies of the actions of cannabinoid receptor agonists and combined this with their knowledge of the localization of cannabinoid receptors and degradative enzymes for anandamide, fatty acid amide hydrolase (FAAH) to reason that endocannabinoids act as retrograde messengers. Pivotal work by Wilson and Nicoll [34] and Ohno-Shosaku et al. [32] established that DSI was mimicked by activating cannabinoid receptors whereas blockade of cannabinoid receptors prevented DSI. A corresponding phenomenon, DSE, **D**epolarizationinduced **S**upression of **E**xcitation, mediated by retrograde action of endocannabinoids, was identified by Kreitzer and Regehr [36] at cerebellar excitatory synapses. DSI and DSE are based on a presynaptic effect as shown by an increase in calcium in the postsynaptic cells and corresponding changes in paired pulse ratio of neurotransmitter release.

calcium-dependent manner [57, 58], in the mGluR-and mAChR-dependent pathways, no clear

**Figure 2. D**epolarization-induced **S**uppression of **I**nhibition (DSI) is a model for retrograde signaling in the brain and allows assaying real time release of endocannabinoids from principal neurons as a brief cessation of GABA ouput. Ac‐ tivation of metabotropic glutamate receptors (mGluRs) on principal neurons or depolarization of postsynaptic princi‐ pal cells evokes synthesis and release of cannabinoids (CBs). Cannabinoids bind to presynaptic cannabinoid receptors (CB1R) on GABAergic interneurons and transiently reduce GABA release from synaptic terminals. As a consequence, GABAA receptor-mediated synaptic currents and GABAergic inhibition are temporarily suppressed in postsynaptic

K channel <sup>+</sup> CB1R

Ca channel 2+

be initiated even in the presence of high intracellular concentrations of calcium chelators, although endocannabinoids may nevertheless be sensitive to the ambient intracellular calcium concentration [59]. Studies by the Alger lab and others indicate that G-protein coupled receptor activation of postsynaptic cells leads to enhancement of DSI, e.g., glutamate acting on group I metabotropic glutamate receptors (mGluRs) directly generates endocannabinoids and enhances DSI ([37, 33]. It is now established that activation of many G-protein coupled receptors is linked to the use of endocannabinoids to deliver or fine-tune their messages to

The discovery of DSI has been a major advance in our understanding of the endocannabinoid system for brain function [60]. DSI is a type of short-term synaptic plasticity originally observed in the cerebellum and hippocampus [12, 14]. Endocannabinoids are retrograde signaling molecules that are released from depolarized principal neurons and travel to presynaptic inhibitory interneurons to reduce GABA release. DSI is a novel, regulatory process that manifests itself as a transient suppression of synaptic GABAA responses mediated by retro‐

[37, 55] is necessary. The release of endocananbinoids can

Principal Neuron

GABAR

mGluR

CB

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Glu

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rise in intracellular calcium [Ca2+]i

Interneuron

target cells [11].

principal neurons.

G-protein coupled receptors (GPCRs) are involved in mediating the transduction of extracel‐ lular stimuli, such as neurotransmitters, into intracellular signaling cascades. Activation of specific G-protein coupled receptors triggers the release of endocananbinoids for many minutes, e.g., dopamine [53], metabotropic glutamate [33, 37, 54] or muscarinic M1/M3 acetylcholine receptors [55, 56]. Even though endocannabinoids are typically released in a

**4. Depolarization-induced suppression of inhibition**

182 Neurochemistry

briefly from their neighbors and encode information [12].

corresponding changes in paired pulse ratio of neurotransmitter release.

The relevance of the endocannabinoid system for neural signaling and brain function in general has been explored only recently [13]. Endocannabinoids mediate a new type of neuronal communication, called DSI, **D**epolarization-induced **S**uppression of **I**nhibition (Fig. 2) (reviewed in [10, 12, 30]. A short rise in intracellular calcium concentration in a principal neuron, e.g., a pyramidal cell of the hippocampus, results in a transient decline of incoming inhibitory signals in the form of GABA arriving from other neurons. This observation led to the hypothesis that during DSI, some unknown messenger must travel from the postsynaptic cell to the presynaptic GABA-releasing one and somehow turns off neurotransmitter release. Conventional chemical synaptic signaling between two neurons involves activation of a presynaptic neuron resulting in transmitter release and subsequent activation of the postsy‐ naptic neuron, e.g., a GABAergic inhibitory interneuron makes synaptic contacts with a glutamatergic pyramidal cell in the hippocampus. When the interneuron is activated it releases the inhibitory neurotransmitter GABA and inhibits the pyramidal cell. In contrast, during DSI, when a pyramidal cell is activated, e.g., through direct current injection, the inhibitory input onto that pyramidal cell is reduced. As a major breakthrough in our understanding of endocannabinoid signaling, endocannabinoids were found to act as retrograde signaling molecules that mediate communication between postsynaptic pyramidal cells and presynaptic inhibitory interneurons and evoke the reduction in GABA release. Since endocannabinoids are lipids, they do not diffuse over great distances in the watery extracellular environment of the brain. Rather, DSI acts as a short-lived local effect that enables individual neurons to disconnect

The announcement of this breakthrough has been given the Latin term 'Dies mirabilis' (wonderful day) by Alger [10]. In March of 2001 four independent labs described in three different journals their studies culminating in the conclusion that endocannabinoids function mainly as retrograde messengers. Elphick and Egertova [52] analyzed prior pharmacological and anatomical studies of the actions of cannabinoid receptor agonists and combined this with their knowledge of the localization of cannabinoid receptors and degradative enzymes for anandamide, fatty acid amide hydrolase (FAAH) to reason that endocannabinoids act as retrograde messengers. Pivotal work by Wilson and Nicoll [34] and Ohno-Shosaku et al. [32] established that DSI was mimicked by activating cannabinoid receptors whereas blockade of cannabinoid receptors prevented DSI. A corresponding phenomenon, DSE, **D**epolarizationinduced **S**upression of **E**xcitation, mediated by retrograde action of endocannabinoids, was identified by Kreitzer and Regehr [36] at cerebellar excitatory synapses. DSI and DSE are based on a presynaptic effect as shown by an increase in calcium in the postsynaptic cells and

G-protein coupled receptors (GPCRs) are involved in mediating the transduction of extracel‐ lular stimuli, such as neurotransmitters, into intracellular signaling cascades. Activation of specific G-protein coupled receptors triggers the release of endocananbinoids for many minutes, e.g., dopamine [53], metabotropic glutamate [33, 37, 54] or muscarinic M1/M3 acetylcholine receptors [55, 56]. Even though endocannabinoids are typically released in a

**Figure 2. D**epolarization-induced **S**uppression of **I**nhibition (DSI) is a model for retrograde signaling in the brain and allows assaying real time release of endocannabinoids from principal neurons as a brief cessation of GABA ouput. Ac‐ tivation of metabotropic glutamate receptors (mGluRs) on principal neurons or depolarization of postsynaptic princi‐ pal cells evokes synthesis and release of cannabinoids (CBs). Cannabinoids bind to presynaptic cannabinoid receptors (CB1R) on GABAergic interneurons and transiently reduce GABA release from synaptic terminals. As a consequence, GABAA receptor-mediated synaptic currents and GABAergic inhibition are temporarily suppressed in postsynaptic principal neurons.

calcium-dependent manner [57, 58], in the mGluR-and mAChR-dependent pathways, no clear rise in intracellular calcium [Ca2+]i [37, 55] is necessary. The release of endocananbinoids can be initiated even in the presence of high intracellular concentrations of calcium chelators, although endocannabinoids may nevertheless be sensitive to the ambient intracellular calcium concentration [59]. Studies by the Alger lab and others indicate that G-protein coupled receptor activation of postsynaptic cells leads to enhancement of DSI, e.g., glutamate acting on group I metabotropic glutamate receptors (mGluRs) directly generates endocannabinoids and enhances DSI ([37, 33]. It is now established that activation of many G-protein coupled receptors is linked to the use of endocannabinoids to deliver or fine-tune their messages to target cells [11].

The discovery of DSI has been a major advance in our understanding of the endocannabinoid system for brain function [60]. DSI is a type of short-term synaptic plasticity originally observed in the cerebellum and hippocampus [12, 14]. Endocannabinoids are retrograde signaling molecules that are released from depolarized principal neurons and travel to presynaptic inhibitory interneurons to reduce GABA release. DSI is a novel, regulatory process that manifests itself as a transient suppression of synaptic GABAA responses mediated by retro‐ grade signaling of endocannabinoids from principal neurons (Fig. 3). Through the retrograde signaling process neurons alter the strength of synapses made onto them and thereby control their own synaptic excitability in an activity-dependent manner, which is functionally important in information processing by neuronal networks [14]. In the cerebellum, a retrograde signaling process that is similar to DSI reduces synaptic excitation by suppressing presynaptic glutamate release and is called DSE [61], see above.

to directly study physiological effects of endocannabinoids. The development of a highly water-soluble caged anandamide that is inert to lipases circumvents these problems [62]. When perfused into hippocampal slice preparations, the caged anandamide serves as a latent endocannabinoid pool, and focal photolysis rapidly liberates highly hydrophobic anandamide *in situ* to activate CB1R. Photolysis is an alternative experimental approach to chemically stimulate synapses, cells, or circuits by directly applying neurotransmitter or neuromodula‐ tors. Often pharmacological approaches yield little control of the stimulation in terms of timing, space and specificity. However, photo-uncaging of caged neurotransmitters has made the pharmacological approach more sophisticated. Photo-uncaging uses localized, patterned light and yields higher spatial and temporal resolution. One application of photostimulation, the flash photolysis technique, can be used to determine signaling kinetics of the endocanna‐ binoid system [60, 62]. The endocannabinoid system can be used as a tool for bioassaying the temporal dynamics or kinetics of lipid signaling. Combining whole-cell voltage patch-clamp recording, intracellular calcium measurements, and photorelease of caged glutamate and a novel, caged cannabinoid, anandamide (AEA) allows determining endocannabinoid signaling kinetics. Flash photolysis of caged compounds (photolysis using so-called molecular optical probes or photoprobes) is an important tool in this endeavor. Caged compounds are inert, biologically inactive (e.g., a caged cannabinoid or caged glutamate) until a flash of laser light breaks open the molecular cage, releases the caged molecule and generates a biologically active effector molecule in situ [63]. Chemically, the caged compound is a modified signal molecule. The modification of the molecule prevents its bioactivity until light absorption results in a

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photochemical change of the signal molecule such that its bioactivity is restored.

The lipid signaling pathway comprises several temporal components that can be determined to quantify the time that it takes from the DSI-inducing stimulus to the onset of DSI. These components contribute to the latency to onset of DSI (start of DSI-inducing stimulus to initial suppression of IPSCs). Among them is the rise of calcium to initiate endocannabinoid synthesis (t-Ca). The rise in intracellular calcium leads to endocannabinoid synthesis and release, followed by travel of these molecules to cannabinoid receptors on presynaptic interneurons, t-EC. The next step is the activation of CB1R and downstream effects, t-CB1R (t-DSI = t-Ca + t-EC + t-CB1R). Experiments carried out using the above-mentioned technological advances allowed determining the time for synthesis and release of eCB from the postsynaptic neuron, which was estimated to be around 150 ms at room temperature, comparable with the timescale of metabotropic signaling and at least an order of magnitude faster than previously thought. A major portion of the DSI onset time, t-DSI, reflects activation of presynaptic CB1Rs and downstream consequences. The data suggest that, far from simply serving long-term neuro‐ modulatory functions, endocannabinoid signaling is sufficiently fast to exert moment-tomoment control of synaptic transmission. The DSI onset latency after a voltage step, t-DSI, is 350 to 400 ms. t-CB1R, the direct activation of CB1R by photoreleasing anandamide which results in suppression of sIPSCs, takes ~180ms (Fig. 4). A transient rise in intracellular calcium sufficient to obtain minimal DSI, t-Ca, is evoked by a 50-ms voltage step and takes ~60 ms. The time needed for endocannabinoid synthesis and release to occur, t-EC, is about 150 ms.

**Figure 3.** Right panel: Hippocampal pyramidal cells show spontaneous inhibitory postsynaptic currents (IPSCs). Left panel: In response to a 1-s voltage pulse the pyramidal cell reveals DSI, a transient reduction in IPSC activity as a result of endocannabinoids acting on CB1R on presynaptic GABAergic interneurons.

## **5. Hippocampal depolarization-induced suppression of inhibition**

Endocannabinoids are lipids and, unlike classic neurotransmitters, are not stored but rather rapidly synthesized from components of the cell membrane. They are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The effects of these lipid signals are detected electrophysiologically as CB1Rdependent alterations in conventional synaptic transmission, which, therefore, provide a sensitive means of bioassay in gendocannabinoid levels and actions. Endocannabinoid release can be triggered through Ca2+-dependent or relatively Ca2+-independent pathways, with different down-stream effects. As discussed above, endocannabinoids are released nonsynaptically, non-vesicular from places all over cells when levels of calcium rise inside the neuron or when certain G-protein-coupled receptors are activated. After cellular release, endocannabinoids travel to cannabinoid receptors on nearby neurons and evoke a reversible, short-term depression of synaptic transmission, DSI. In activated hippocampal pyramidal cells, DSI leads to a transient reductionof GABA release from presynaptic terminals of inhibitory interneurons. Direct insights into the actions of endocannabinoids have been based primarily on pharmacological experiments. The hydrophobicity of endocannabinoids severely limits their penetration into brain tissue, and endocannabinoids are rapidly degraded by abundant endogenous lipases. These intrinsic properties of endocannabinoids make it difficult to directly study physiological effects of endocannabinoids. The development of a highly water-soluble caged anandamide that is inert to lipases circumvents these problems [62]. When perfused into hippocampal slice preparations, the caged anandamide serves as a latent endocannabinoid pool, and focal photolysis rapidly liberates highly hydrophobic anandamide *in situ* to activate CB1R. Photolysis is an alternative experimental approach to chemically stimulate synapses, cells, or circuits by directly applying neurotransmitter or neuromodula‐ tors. Often pharmacological approaches yield little control of the stimulation in terms of timing, space and specificity. However, photo-uncaging of caged neurotransmitters has made the pharmacological approach more sophisticated. Photo-uncaging uses localized, patterned light and yields higher spatial and temporal resolution. One application of photostimulation, the flash photolysis technique, can be used to determine signaling kinetics of the endocanna‐ binoid system [60, 62]. The endocannabinoid system can be used as a tool for bioassaying the temporal dynamics or kinetics of lipid signaling. Combining whole-cell voltage patch-clamp recording, intracellular calcium measurements, and photorelease of caged glutamate and a novel, caged cannabinoid, anandamide (AEA) allows determining endocannabinoid signaling kinetics. Flash photolysis of caged compounds (photolysis using so-called molecular optical probes or photoprobes) is an important tool in this endeavor. Caged compounds are inert, biologically inactive (e.g., a caged cannabinoid or caged glutamate) until a flash of laser light breaks open the molecular cage, releases the caged molecule and generates a biologically active effector molecule in situ [63]. Chemically, the caged compound is a modified signal molecule. The modification of the molecule prevents its bioactivity until light absorption results in a photochemical change of the signal molecule such that its bioactivity is restored.

grade signaling of endocannabinoids from principal neurons (Fig. 3). Through the retrograde signaling process neurons alter the strength of synapses made onto them and thereby control their own synaptic excitability in an activity-dependent manner, which is functionally important in information processing by neuronal networks [14]. In the cerebellum, a retrograde signaling process that is similar to DSI reduces synaptic excitation by suppressing presynaptic

**Figure 3.** Right panel: Hippocampal pyramidal cells show spontaneous inhibitory postsynaptic currents (IPSCs). Left panel: In response to a 1-s voltage pulse the pyramidal cell reveals DSI, a transient reduction in IPSC activity as a result

Endocannabinoids are lipids and, unlike classic neurotransmitters, are not stored but rather rapidly synthesized from components of the cell membrane. They are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The effects of these lipid signals are detected electrophysiologically as CB1Rdependent alterations in conventional synaptic transmission, which, therefore, provide a sensitive means of bioassay in gendocannabinoid levels and actions. Endocannabinoid release can be triggered through Ca2+-dependent or relatively Ca2+-independent pathways, with different down-stream effects. As discussed above, endocannabinoids are released nonsynaptically, non-vesicular from places all over cells when levels of calcium rise inside the neuron or when certain G-protein-coupled receptors are activated. After cellular release, endocannabinoids travel to cannabinoid receptors on nearby neurons and evoke a reversible, short-term depression of synaptic transmission, DSI. In activated hippocampal pyramidal cells, DSI leads to a transient reductionof GABA release from presynaptic terminals of inhibitory interneurons. Direct insights into the actions of endocannabinoids have been based primarily on pharmacological experiments. The hydrophobicity of endocannabinoids severely limits their penetration into brain tissue, and endocannabinoids are rapidly degraded by abundant endogenous lipases. These intrinsic properties of endocannabinoids make it difficult

**5. Hippocampal depolarization-induced suppression of inhibition**

glutamate release and is called DSE [61], see above.

184 Neurochemistry

of endocannabinoids acting on CB1R on presynaptic GABAergic interneurons.

The lipid signaling pathway comprises several temporal components that can be determined to quantify the time that it takes from the DSI-inducing stimulus to the onset of DSI. These components contribute to the latency to onset of DSI (start of DSI-inducing stimulus to initial suppression of IPSCs). Among them is the rise of calcium to initiate endocannabinoid synthesis (t-Ca). The rise in intracellular calcium leads to endocannabinoid synthesis and release, followed by travel of these molecules to cannabinoid receptors on presynaptic interneurons, t-EC. The next step is the activation of CB1R and downstream effects, t-CB1R (t-DSI = t-Ca + t-EC + t-CB1R). Experiments carried out using the above-mentioned technological advances allowed determining the time for synthesis and release of eCB from the postsynaptic neuron, which was estimated to be around 150 ms at room temperature, comparable with the timescale of metabotropic signaling and at least an order of magnitude faster than previously thought. A major portion of the DSI onset time, t-DSI, reflects activation of presynaptic CB1Rs and downstream consequences. The data suggest that, far from simply serving long-term neuro‐ modulatory functions, endocannabinoid signaling is sufficiently fast to exert moment-tomoment control of synaptic transmission. The DSI onset latency after a voltage step, t-DSI, is 350 to 400 ms. t-CB1R, the direct activation of CB1R by photoreleasing anandamide which results in suppression of sIPSCs, takes ~180ms (Fig. 4). A transient rise in intracellular calcium sufficient to obtain minimal DSI, t-Ca, is evoked by a 50-ms voltage step and takes ~60 ms. The time needed for endocannabinoid synthesis and release to occur, t-EC, is about 150 ms.

Anandamide can be released from its caged form by a UV-laser flash and rapidly activates presynaptic CB1Rs to suppress the release of GABA [62]. A specific CB1R antagonist, AM 251, blocks the suppression of spontaneous IPSCs. This establishes that uncagedanandamide can be used as a CB1R agonist to study activation of CB1R in the brain. Similarly, uncaged glutamate acts at mGluRs on hippocampal pyramidal cells to evoke cannabinoid release and subsequent suppression of presynaptic GABA release [62]. The data provide the first detailed attempt to determine the minimal time required for activation of an intercellular neuronal lipid messenger system. This signaling system requires a major portion of DSI onset time, t-DSI, for activation of presynaptic CB1R and downstream consequences. Endocannabinoids, and by extension similar lipid messengers, can be mobilized and evoke responses as quickly as conventional metabotropic, G-protein receptor-coupled neurotransmitters. The speed with which neuromodulators such as endocannabinoids act places critical constraints on the physiological roles they can play. Endocannabinoids and other lipids function in brain signaling not simply in homeostatic processes or slowly-activating forms of regulation, but rather lipids can affect neuronal excitability in moment-to-moment information processing.

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**6. Depolarization-induced suppression of inhibition in glomerular circuits**

The olfactory bulb is the first relay station in the CNS for processing of sensory information that comes from olfactory receptor cells in the nasal epithelium. Cannabinoid receptors are expressed at high levels in the olfactory bulb, specifically in the input region, the glomerular layer [21, 64-66]. Neurons in the glomerular layer are immunoreactive for enzymes that synthesize endocannabinoids [67-69]. Our understanding of the physiological role of endo‐ cannabinoids and cannabinoid receptors for neural signaling in the olfactory system is just emerging. Recent electrophysiological evidence has established that the endocannabinoid system plays a functional role in regulating neuronal activity and signaling in olfactory bulb

Neurons in the glomerular fall into three subpopulations: periglomerular (PG), external tufted (eTC), and short-axon (SA) cells. Periglomerular cells are neurochemically and functionally heterogeneous [70-72]. Periglomerular cells are GABAergic, short-axon cells express both GABA and dopamine, and external tufted cells are glutamatergic [72, 73]. Periglomerular cells receive input from the olfactory nerve or dendrodendritic glutamatergic input from external tufted or mitral cells, e.g., as spontaneous bursts of EPSCs [70, 73-74]. Periglomerular cells presynaptically inhibit olfactory receptor neurons through GABAergic transmission [76, 77]. External tufted cells receive spontaneous bursts of inhibitory postsynaptic currents (sIPSCs) from periglomerular cells at inhibitory GABAergic synapses as well as spontaneous glutama‐ tergic EPSCs [74; 78]. In the glomerular layer, external tufted cells can be a potential source of

Cannabinoid receptors directly regulate membrane properties of periglomerular cells as shown by the effects of CB1R antagonist AM251 and agonist WIN in the presence of ionotropic

**of the olfactory bulb**

glomeruli [43].

endocannabinoids.

**Figure 4.** Left panel: Photolysis of caged anandamide yields bioactive anandamide. Right panel: Photorelease of anan‐ damide suppresses sIPSCs after a delay of ~180 ms. Modified from [62] with permission of the Society for Neuro‐ science.

Voltage-activated DSI works through a rise in intracellular calcium concentration. However, release of endocannabinoids can be triggered even in the presence of high intracellular concentrations of calcium chelators, although they may nevertheless be sensitive to ambient intracellular calcium [59]. To test if these two pathways function on the same time scale, the dynamic components of the mGluR-induced endocannabionoid response on sIPSC frequency in pyramidal cells are compared (Fig. 5) [62]. The mean onset latency, duration and magnitude of the IPSC suppression evoked by uncaged glutamate are similar to that caused by uncaged AEA (Figs. 4, 5). No reduction in sIPSCs occurred for 221 ms (determined by extrapolation of the exponential fit to the control sIPSC level). The time-to-onset of IPSC suppression evoked by the mGluR-induced endocannabinoid process (time to mGluR-dependent suppression of inhibition, tmGluRSI) is described by: t-mGluR-SI = 221 ms = t-eCB(mGluR) + t-CB1R, where teCB(mGluR) is the time for activation of the mGluR-dependent endocannabinoid synthesis and release, and t-CB1R is ~180 ms (see above). This leaves t-eCB(mGluR) to be < 50 ms, which is even faster than endocannabinoid synthesis and released evoked by a voltage step.

**Figure 5.** Dynamics of mGluR-dependent endocannabinoid suppression of sIPSCs in cultured hippocampal slices. Left panel: Photorelease of glutamate. Right panel: Recording from a pyramidal cell illustrates the transient reduction in spontaneous (s) IPSC frequency of CA1 pyramidal cells after flash photorelease of caged glutamate (photolysis in‐ duced suppression of inhibition, PSI). Arrow indicates laser flash. From [62] with permission of the Society for Neuro‐ science.

Anandamide can be released from its caged form by a UV-laser flash and rapidly activates presynaptic CB1Rs to suppress the release of GABA [62]. A specific CB1R antagonist, AM 251, blocks the suppression of spontaneous IPSCs. This establishes that uncagedanandamide can be used as a CB1R agonist to study activation of CB1R in the brain. Similarly, uncaged glutamate acts at mGluRs on hippocampal pyramidal cells to evoke cannabinoid release and subsequent suppression of presynaptic GABA release [62]. The data provide the first detailed attempt to determine the minimal time required for activation of an intercellular neuronal lipid messenger system. This signaling system requires a major portion of DSI onset time, t-DSI, for activation of presynaptic CB1R and downstream consequences. Endocannabinoids, and by extension similar lipid messengers, can be mobilized and evoke responses as quickly as conventional metabotropic, G-protein receptor-coupled neurotransmitters. The speed with which neuromodulators such as endocannabinoids act places critical constraints on the physiological roles they can play. Endocannabinoids and other lipids function in brain signaling not simply in homeostatic processes or slowly-activating forms of regulation, but rather lipids can affect neuronal excitability in moment-to-moment information processing.

**Figure 4.** Left panel: Photolysis of caged anandamide yields bioactive anandamide. Right panel: Photorelease of anan‐ damide suppresses sIPSCs after a delay of ~180 ms. Modified from [62] with permission of the Society for Neuro‐

Voltage-activated DSI works through a rise in intracellular calcium concentration. However, release of endocannabinoids can be triggered even in the presence of high intracellular concentrations of calcium chelators, although they may nevertheless be sensitive to ambient intracellular calcium [59]. To test if these two pathways function on the same time scale, the dynamic components of the mGluR-induced endocannabionoid response on sIPSC frequency in pyramidal cells are compared (Fig. 5) [62]. The mean onset latency, duration and magnitude of the IPSC suppression evoked by uncaged glutamate are similar to that caused by uncaged AEA (Figs. 4, 5). No reduction in sIPSCs occurred for 221 ms (determined by extrapolation of the exponential fit to the control sIPSC level). The time-to-onset of IPSC suppression evoked by the mGluR-induced endocannabinoid process (time to mGluR-dependent suppression of inhibition, tmGluRSI) is described by: t-mGluR-SI = 221 ms = t-eCB(mGluR) + t-CB1R, where teCB(mGluR) is the time for activation of the mGluR-dependent endocannabinoid synthesis and release, and t-CB1R is ~180 ms (see above). This leaves t-eCB(mGluR) to be < 50 ms, which

is even faster than endocannabinoid synthesis and released evoked by a voltage step.

**Figure 5.** Dynamics of mGluR-dependent endocannabinoid suppression of sIPSCs in cultured hippocampal slices. Left panel: Photorelease of glutamate. Right panel: Recording from a pyramidal cell illustrates the transient reduction in spontaneous (s) IPSC frequency of CA1 pyramidal cells after flash photorelease of caged glutamate (photolysis in‐ duced suppression of inhibition, PSI). Arrow indicates laser flash. From [62] with permission of the Society for Neuro‐

science.

186 Neurochemistry

science.

## **6. Depolarization-induced suppression of inhibition in glomerular circuits of the olfactory bulb**

The olfactory bulb is the first relay station in the CNS for processing of sensory information that comes from olfactory receptor cells in the nasal epithelium. Cannabinoid receptors are expressed at high levels in the olfactory bulb, specifically in the input region, the glomerular layer [21, 64-66]. Neurons in the glomerular layer are immunoreactive for enzymes that synthesize endocannabinoids [67-69]. Our understanding of the physiological role of endo‐ cannabinoids and cannabinoid receptors for neural signaling in the olfactory system is just emerging. Recent electrophysiological evidence has established that the endocannabinoid system plays a functional role in regulating neuronal activity and signaling in olfactory bulb glomeruli [43].

Neurons in the glomerular fall into three subpopulations: periglomerular (PG), external tufted (eTC), and short-axon (SA) cells. Periglomerular cells are neurochemically and functionally heterogeneous [70-72]. Periglomerular cells are GABAergic, short-axon cells express both GABA and dopamine, and external tufted cells are glutamatergic [72, 73]. Periglomerular cells receive input from the olfactory nerve or dendrodendritic glutamatergic input from external tufted or mitral cells, e.g., as spontaneous bursts of EPSCs [70, 73-74]. Periglomerular cells presynaptically inhibit olfactory receptor neurons through GABAergic transmission [76, 77]. External tufted cells receive spontaneous bursts of inhibitory postsynaptic currents (sIPSCs) from periglomerular cells at inhibitory GABAergic synapses as well as spontaneous glutama‐ tergic EPSCs [74; 78]. In the glomerular layer, external tufted cells can be a potential source of endocannabinoids.

Cannabinoid receptors directly regulate membrane properties of periglomerular cells as shown by the effects of CB1R antagonist AM251 and agonist WIN in the presence of ionotropic glutamate and GABAA receptor blockers (synaptic blockers: CNQX, APV, gabazine) [43]. This indicates that the actions of cannabinoids on periglomerular cells are mediated through CB1R expressed by periglomerular cells. AM251 directly activates periglomerular cells and enhances their GABA release. Periglomerular cells are synaptically connected to external tufted cells. Therefore, any CB1R-mediated regulation of activity of periglomerular cells could affect GABA release and synaptic transmission to external tufted cells. CB1R is also expressed in external tufted cells and may participate in modulating external tufted cell activity.

In external tufted cells, neither AM251 nor WIN influences firing frequency or membrane potential [43]. However, in the presence of synaptic blockers cannabinoid drugs have a modest effect on external tufted cells. In this condition, AM251 slightly increases the firing rate of external tufted cells without membrane depolarization. In synaptic blockers, WIN slightly decreases firing of external tufted cells without a clear change in membrane potential. The effects of AM251 and WIN in the presence of synaptic blockers, i.e., during pharmacological isolation of external tufted cells, indicate that CB1R mediates a direct effect on external tufted cells. The direct excitatory effect of a CB1R antagonist on external tufted cells is opposed by increased GABAergic synaptic input from periglomerular cells onto external tufted cells, i.e., the enhanced GABA release from periglomerular cells triggered by a CB1R antagonist may dominate and mask the CB1R antagonist-evoked direct excitation of external tufted cells.

The CB1R effects on periglomerular and external tufted cell prompt the questions if DSI is present in the glomerular layer of the olfactory bulb. In external tufted cells, DSI can be induced with a 5-sec depolarizing voltage step from a holding potential of -60 mV to 0 mV (Fig. 5). In external tufted cells DSI is visible as a decrease in the amplitude and frequency of sIPSCs. The response to a single depolarizing step is a suppression of sIPSC area by ~40 % of control which then gradually recoveres. External tufted cells exhibit a distinct intrinsic bursting pattern [74]. In order to mimic spontaneous rhythmic bursting of an external tufted cell a train of depola‐ rizing steps can be applied to the cell. This experimental paradigm allows determining a possible functional role of DSI in glomeruli. A train of depolarizing steps results in a transient 60% reduction in sIPSC area (20 steps, 0.75 Hz) (Fig. 4B, F). DSI can be completely eliminated in the presence of AM251, indicating that DSI is mediated by CB1R (Fig. 5C, F). The bursting frequency of external tufted cells ranges from 0.5 to 6.5 Hz with a mean frequency of 2.7 bursts/ sec [74]. Depolarizing voltage pulses at 2 Hz (20 steps, pulse duration: 250 ms) evoke DSI as a reduction of sIPSCs in external tufted cells, similar to the results obtained with voltage steps at 0.75 Hz to 0 mV. In external tufted cells, single depolarizing voltage steps as well as a train of voltage steps evoke suppression of inhibition (DSI). This suggests that spontaneous rhythmic bursting of these cells triggers the release of endocannabinoids which function as retrograde messengers to reduce GABA release from periglomerular cells which in turn, regulates the activity of periglomerular cell synaptic targets such as external tufted cells.

Endocannabinoids regulate neuronal activity and signaling in olfactory bulb glomeruli. They function in the form of DSI through CB1R-mediated retrograde signaling among glomeru‐ lar neurons. Endocannabinoids are released from external tufted cells and act as retro‐ grade messengers to control the excitability of presynaptic neurons, i.e., periglomerular cells, and to regulate their transmitter release. Endocannabinoids are synthesized and

released from neuronal cell bodies as a result of cellular excitation [11]. One potential source of endocannabinoids in the olfactory bulb is neurons that synapse onto presynaptic cells, i.e., periglomerular cells, and receive feedback synaptic inputs. This profile fits external

**Figure 6. D**epolarization-induced **S**uppression of **I**nhibition (DSI) in olfactory glomeruli. **A** A depolarizing voltage step

polarization was achieved by stepping from-60 mV holding potential to 0 mV for 5 sec. **B** In the presence of CNQX and 5-AP, a train of 20 voltage steps to 0 mV (0.75 Hz; step duration: 667 ms) transiently reduced sIPSCs in an external tufted cell. Holding potential was-60 mV. **C.** In the presence of AM251, no sIPSC suppression was observed. **D** A train of 20 voltage steps to-30 mV (2 Hz; step duration: 250 ms) transiently reduced sIPSCs in an external tufted cell (in CNQX and 5-AP). **E** Normalized sIPSCs area illustrating the magnitude and time course of DSI elicited by a 5-sec depo‐ larizing pulse (*n*=7). The averaged values between 0 – 5 sec after the end of the voltage step were significantly differ‐ ent from the baseline (ANOVA and Bonferroni post-hoc analysis, *p*< 0.05). **F** Normalized sIPSC area illustrating the magnitude and time course of DSI elicited by a train of depolarizations to 0 mV (*n*=12) in control and in the presence of AM251 (*n*=10). In control conditions, the averaged values between zero to 2 5 seconds after the end of the train of voltage steps were significantly different from the baseline (ANOVA and Bonferroni post-hoc analysis, *p*< 0.05). From


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evoked DSI in a representative external tufted cell. High Cl-

[43] with permission of the Society for Neuroscience.

glutamate and GABAA receptor blockers (synaptic blockers: CNQX, APV, gabazine) [43]. This indicates that the actions of cannabinoids on periglomerular cells are mediated through CB1R expressed by periglomerular cells. AM251 directly activates periglomerular cells and enhances their GABA release. Periglomerular cells are synaptically connected to external tufted cells. Therefore, any CB1R-mediated regulation of activity of periglomerular cells could affect GABA release and synaptic transmission to external tufted cells. CB1R is also expressed in external

In external tufted cells, neither AM251 nor WIN influences firing frequency or membrane potential [43]. However, in the presence of synaptic blockers cannabinoid drugs have a modest effect on external tufted cells. In this condition, AM251 slightly increases the firing rate of external tufted cells without membrane depolarization. In synaptic blockers, WIN slightly decreases firing of external tufted cells without a clear change in membrane potential. The effects of AM251 and WIN in the presence of synaptic blockers, i.e., during pharmacological isolation of external tufted cells, indicate that CB1R mediates a direct effect on external tufted cells. The direct excitatory effect of a CB1R antagonist on external tufted cells is opposed by increased GABAergic synaptic input from periglomerular cells onto external tufted cells, i.e., the enhanced GABA release from periglomerular cells triggered by a CB1R antagonist may dominate and mask the CB1R antagonist-evoked direct excitation of external tufted cells.

The CB1R effects on periglomerular and external tufted cell prompt the questions if DSI is present in the glomerular layer of the olfactory bulb. In external tufted cells, DSI can be induced with a 5-sec depolarizing voltage step from a holding potential of -60 mV to 0 mV (Fig. 5). In external tufted cells DSI is visible as a decrease in the amplitude and frequency of sIPSCs. The response to a single depolarizing step is a suppression of sIPSC area by ~40 % of control which then gradually recoveres. External tufted cells exhibit a distinct intrinsic bursting pattern [74]. In order to mimic spontaneous rhythmic bursting of an external tufted cell a train of depola‐ rizing steps can be applied to the cell. This experimental paradigm allows determining a possible functional role of DSI in glomeruli. A train of depolarizing steps results in a transient 60% reduction in sIPSC area (20 steps, 0.75 Hz) (Fig. 4B, F). DSI can be completely eliminated in the presence of AM251, indicating that DSI is mediated by CB1R (Fig. 5C, F). The bursting frequency of external tufted cells ranges from 0.5 to 6.5 Hz with a mean frequency of 2.7 bursts/ sec [74]. Depolarizing voltage pulses at 2 Hz (20 steps, pulse duration: 250 ms) evoke DSI as a reduction of sIPSCs in external tufted cells, similar to the results obtained with voltage steps at 0.75 Hz to 0 mV. In external tufted cells, single depolarizing voltage steps as well as a train of voltage steps evoke suppression of inhibition (DSI). This suggests that spontaneous rhythmic bursting of these cells triggers the release of endocannabinoids which function as retrograde messengers to reduce GABA release from periglomerular cells which in turn, regulates the activity of periglomerular cell synaptic targets such as external tufted cells.

Endocannabinoids regulate neuronal activity and signaling in olfactory bulb glomeruli. They function in the form of DSI through CB1R-mediated retrograde signaling among glomeru‐ lar neurons. Endocannabinoids are released from external tufted cells and act as retro‐ grade messengers to control the excitability of presynaptic neurons, i.e., periglomerular cells, and to regulate their transmitter release. Endocannabinoids are synthesized and

tufted cells and may participate in modulating external tufted cell activity.

188 Neurochemistry

**Figure 6. D**epolarization-induced **S**uppression of **I**nhibition (DSI) in olfactory glomeruli. **A** A depolarizing voltage step evoked DSI in a representative external tufted cell. High Cl- -based pipette solution was used for recording sIPSCs. De‐ polarization was achieved by stepping from-60 mV holding potential to 0 mV for 5 sec. **B** In the presence of CNQX and 5-AP, a train of 20 voltage steps to 0 mV (0.75 Hz; step duration: 667 ms) transiently reduced sIPSCs in an external tufted cell. Holding potential was-60 mV. **C.** In the presence of AM251, no sIPSC suppression was observed. **D** A train of 20 voltage steps to-30 mV (2 Hz; step duration: 250 ms) transiently reduced sIPSCs in an external tufted cell (in CNQX and 5-AP). **E** Normalized sIPSCs area illustrating the magnitude and time course of DSI elicited by a 5-sec depo‐ larizing pulse (*n*=7). The averaged values between 0 – 5 sec after the end of the voltage step were significantly differ‐ ent from the baseline (ANOVA and Bonferroni post-hoc analysis, *p*< 0.05). **F** Normalized sIPSC area illustrating the magnitude and time course of DSI elicited by a train of depolarizations to 0 mV (*n*=12) in control and in the presence of AM251 (*n*=10). In control conditions, the averaged values between zero to 2 5 seconds after the end of the train of voltage steps were significantly different from the baseline (ANOVA and Bonferroni post-hoc analysis, *p*< 0.05). From [43] with permission of the Society for Neuroscience.

released from neuronal cell bodies as a result of cellular excitation [11]. One potential source of endocannabinoids in the olfactory bulb is neurons that synapse onto presynaptic cells, i.e., periglomerular cells, and receive feedback synaptic inputs. This profile fits external

tufted cells and they could be a potential endocannabinoid source in the olfactory bulb which is supported by the fact that DSI is found in external tufted cells. DSI in external tufted cells is subject to the level of cellular activation, i.e., voltage step duration and step number. DSI cannot be evoked with step durations of 1 sec or less while a step duration closer to 5 seconds evokes transient DSI. A train of depolarizing voltage steps (>3) generates particularly prominent DSI and strengthens the inhibition of sIPSCs. This suggests that excitation of external tufted cells in the form of rhythmic bursting triggers the release of endocannabinoids and regulates glomerular activity. Bursting is intrinsic to external tufted cells and mediated by several cell intrinsic conductances [79]. Bursting of neurons may modulate endocannabinoid release not only in the olfactory bulb but also in other brain systems and constitute a general phenomenon of endocannabinoidsignaling.

sion but rather on activity-dependent long-lasting hyperpolarization due to the activation of

The endocannabinoid system reciprocally modulates other neurotransmitter systems [88]. Examples include interactive cross-talk with the endogenous opioid system [89, 90]. Inciden‐ tally, like the endogenous opiate system, the endocannabinoid system was first discovered because it can be activated by a plant-derived compound – in the case of the endocannabinoids, this is Δ9-tetrahydrocannabinol, the bioactive ingredient of the drugs marijuana and hashish [25]. Other studies detected an interaction of the endocannabinoid system at the molecular and functional levels with other neurotransmitters such as the dopaminergic and adenosinergic systems [91-93]. Recent evidence has suggested cross-modulation between the endocannabi‐ noid and hypocretinergic system [88]. This idea is based on the overlap observed in the neuroanatomical distribution of both systems as well as their putative functions. Functionally, both endocannabinoids and hypocretins can contribute to the regulation of appetite, reward and analgesia. Furthermore, biochemical and functional studies have demonstrated hetero‐ dimers between CB1 cannabinoid receptor and hypocretin receptor-1. Activation of hypocretin receptor-1 stimulates the synthesis of 2-arachidonoyl glycerol which through retrograde endocannabinoid signaling results in inhibition of neighboring cells. This interaction would allow endocannabinoids to contribute to hypocretin effects and provide potential therapeutic applications to currently existing drugs targeting these systems [88]. However, these two neuromodulatory systems exert antagonistic effects in the regulation of the sleep/wake cycle and anxiety-like responses which contributes even more to the excitement of performing

This work was supported in part by U.S.-PHS grants GM08016 and MD007597. I would like to gratefully acknowledge current and former collaborators Drs. Bradley E. Alger, Joseph P.

Department of Anatomy, Howard University College of Medicine, Washington, DC, USA


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a K+

circuit in which they reside.

research targeting the endocannabinoid system.

Address all correspondence to: theinbockel@howard.edu

**Acknowledgements**

Y. Kao, and Ze-Jun Wang.

**Author details**

Thomas Heinbockel\*

Olfactory sensory neurons form direct synaptic contacts with external tufted cells. Sensory or synaptic input to external tufted cells can trigger the release of endocannabinoids which have an inhibitory effect on CB1Rs in presynaptic periglomerular cells. Endocannabinoids thus reduce inhibitory input to external tufted cells and enhance external tufted cell sensitivity to weak sensory inputs by depolarizing the membrane potential closer to spike threshold. This CB1R-mediated inhibition of periglomerular cells reduces their GABA release and, in turn, modifies the firing pattern of external tufted cells and, potentially, also reduces inhibition of mitral cells and presynaptic olfactory nerve terminals. The functional relevance of this signaling pathway lies in a potential increase of the overall sensitivity of the glomerulus to sensory inputs resulting from activation of CB1R on periglomerular cells.

## **7. Endocannabinoid-evoked physiological responses and crosstalk with other neurotransmitters**

Endocannabinoids can evoke physiological responses that are not mediated by presynaptic CB1Rs but rather by postsynaptic CB1Rs [14], e.g., via regulation of K+ conductances present on the extrasynaptic dendritic surface of neurons or modulation of postsynaptic NMDA receptors or even non-CB1R, e.g., [80]. Several conventional CB1R ligands have been reported to have CB1R unspecific effects or activate non-CB1 receptors [14]. Electrophysiological evidence suggests that the CB1R agonist WIN55,212-2 produces non-CB1R mediated effects on the excitability of principal neurons in the basolateral amygdala [81], thus providing evidence for a non-CB1R site of action of WIN55,212-2 [82, 83]. Cannabinoid drugs can activate other 'non-CB' receptors, such as GPR55, peroxisome proliferator-activated receptors (PPARs), and vanilloid type TRP channels [84, 85].

The accepted view of endocannabinoid action is based on hippocampal studies demonstrating that endocannabinoids reduce synaptic inhibition of the principal cell (DSI), see above. Endocannabinoids were found to possess other properties, namely, to mediate self-modula‐ tion of neocortical pyramidal neurons [86] or long-lasting self-inhibition in neocortical GABAcontaining interneurons [87]. This self-inhibition is mediated by autocrine release of endocannabinoids and does not depend on glutamatergic and/or GABAergic neurotransmis‐ sion but rather on activity-dependent long-lasting hyperpolarization due to the activation of a K+ -conductance. Endocannabinoids released by these interneurons target the same cells and mediate a lasting hyperpolarization that is blocked by a CB1R antagonist. Self-inhibited cells can become hyperpolarized below spike threshold and are effectively removed from the neural circuit in which they reside.

The endocannabinoid system reciprocally modulates other neurotransmitter systems [88]. Examples include interactive cross-talk with the endogenous opioid system [89, 90]. Inciden‐ tally, like the endogenous opiate system, the endocannabinoid system was first discovered because it can be activated by a plant-derived compound – in the case of the endocannabinoids, this is Δ9-tetrahydrocannabinol, the bioactive ingredient of the drugs marijuana and hashish [25]. Other studies detected an interaction of the endocannabinoid system at the molecular and functional levels with other neurotransmitters such as the dopaminergic and adenosinergic systems [91-93]. Recent evidence has suggested cross-modulation between the endocannabi‐ noid and hypocretinergic system [88]. This idea is based on the overlap observed in the neuroanatomical distribution of both systems as well as their putative functions. Functionally, both endocannabinoids and hypocretins can contribute to the regulation of appetite, reward and analgesia. Furthermore, biochemical and functional studies have demonstrated hetero‐ dimers between CB1 cannabinoid receptor and hypocretin receptor-1. Activation of hypocretin receptor-1 stimulates the synthesis of 2-arachidonoyl glycerol which through retrograde endocannabinoid signaling results in inhibition of neighboring cells. This interaction would allow endocannabinoids to contribute to hypocretin effects and provide potential therapeutic applications to currently existing drugs targeting these systems [88]. However, these two neuromodulatory systems exert antagonistic effects in the regulation of the sleep/wake cycle and anxiety-like responses which contributes even more to the excitement of performing research targeting the endocannabinoid system.

## **Acknowledgements**

tufted cells and they could be a potential endocannabinoid source in the olfactory bulb which is supported by the fact that DSI is found in external tufted cells. DSI in external tufted cells is subject to the level of cellular activation, i.e., voltage step duration and step number. DSI cannot be evoked with step durations of 1 sec or less while a step duration closer to 5 seconds evokes transient DSI. A train of depolarizing voltage steps (>3) generates particularly prominent DSI and strengthens the inhibition of sIPSCs. This suggests that excitation of external tufted cells in the form of rhythmic bursting triggers the release of endocannabinoids and regulates glomerular activity. Bursting is intrinsic to external tufted cells and mediated by several cell intrinsic conductances [79]. Bursting of neurons may modulate endocannabinoid release not only in the olfactory bulb but also in other brain

Olfactory sensory neurons form direct synaptic contacts with external tufted cells. Sensory or synaptic input to external tufted cells can trigger the release of endocannabinoids which have an inhibitory effect on CB1Rs in presynaptic periglomerular cells. Endocannabinoids thus reduce inhibitory input to external tufted cells and enhance external tufted cell sensitivity to weak sensory inputs by depolarizing the membrane potential closer to spike threshold. This CB1R-mediated inhibition of periglomerular cells reduces their GABA release and, in turn, modifies the firing pattern of external tufted cells and, potentially, also reduces inhibition of mitral cells and presynaptic olfactory nerve terminals. The functional relevance of this signaling pathway lies in a potential increase of the overall sensitivity of the glomerulus to

**7. Endocannabinoid-evoked physiological responses and crosstalk with**

Endocannabinoids can evoke physiological responses that are not mediated by presynaptic

on the extrasynaptic dendritic surface of neurons or modulation of postsynaptic NMDA receptors or even non-CB1R, e.g., [80]. Several conventional CB1R ligands have been reported to have CB1R unspecific effects or activate non-CB1 receptors [14]. Electrophysiological evidence suggests that the CB1R agonist WIN55,212-2 produces non-CB1R mediated effects on the excitability of principal neurons in the basolateral amygdala [81], thus providing evidence for a non-CB1R site of action of WIN55,212-2 [82, 83]. Cannabinoid drugs can activate other 'non-CB' receptors, such as GPR55, peroxisome proliferator-activated receptors (PPARs),

The accepted view of endocannabinoid action is based on hippocampal studies demonstrating that endocannabinoids reduce synaptic inhibition of the principal cell (DSI), see above. Endocannabinoids were found to possess other properties, namely, to mediate self-modula‐ tion of neocortical pyramidal neurons [86] or long-lasting self-inhibition in neocortical GABAcontaining interneurons [87]. This self-inhibition is mediated by autocrine release of endocannabinoids and does not depend on glutamatergic and/or GABAergic neurotransmis‐

conductances present

systems and constitute a general phenomenon of endocannabinoidsignaling.

sensory inputs resulting from activation of CB1R on periglomerular cells.

CB1Rs but rather by postsynaptic CB1Rs [14], e.g., via regulation of K+

**other neurotransmitters**

190 Neurochemistry

and vanilloid type TRP channels [84, 85].

This work was supported in part by U.S.-PHS grants GM08016 and MD007597. I would like to gratefully acknowledge current and former collaborators Drs. Bradley E. Alger, Joseph P. Y. Kao, and Ze-Jun Wang.

## **Author details**

Thomas Heinbockel\*

Address all correspondence to: theinbockel@howard.edu

Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

## **References**

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**Chapter 7**

**High Temporal Resolution Brain**

**Microdialysis as a Tool to Investigate**

**Olfactory Cortex and Amygdala in**

**Odor Fear Conditioning**

Chloé Hegoburu, Luc Denoroy,

http://dx.doi.org/10.5772/57490

**1. Introduction**

Anne-Marie Mouly and Sandrine Parrot

Additional information is available at the end of the chapter

vocalizations emission, or the release of stress hormones.

Fear conditioning is one of the most widely used paradigms for studying the neurobiology of emotional learning. In this paradigm, an emotionally neutral stimulus (the conditioned stimulus or CS) is paired with an aversive unconditioned stimulus (US), generally a mild foot shock. After a few trials, re-exposure to the CS alone elicits a fear response, proceeding through the necessary connecting structures, and ending with the autonomic and motoric effector outputs. This fear response is part of an anticipatory response to danger [1], initiating a range of defensive reactions that counter threats to survival [2]. The most universal response is a postural immobility, called freezing [3, 4]. Moreover, the threatening stimulus also triggers the activation of the hypothalamic subnuclei to induce an increase in blood pressure, ultrasonic

The vast majority of studies devoted to investigate the neural basis of fear conditioning have used auditory cues as conditioned stimuli and the neural pathways involved in auditory fear conditioning have been well characterized [5-8]. The information carried by the auditory CS can take one of two pathways: either directly from the thalamus to quickly reach the amygdala or the CS can travel from the auditory thalamus to the auditory cortex before reaching the amygdala. These thalamic and cortical areas send projections to the lateral nucleus of the amygdala, which is a site of CS-US convergence. The lateral nucleus, in turn, projects to the

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**the Dynamics of Interactions Between**


**Chapter 7**

**High Temporal Resolution Brain Microdialysis as a Tool to Investigate the Dynamics of Interactions Between Olfactory Cortex and Amygdala in Odor Fear Conditioning**

Chloé Hegoburu, Luc Denoroy, Anne-Marie Mouly and Sandrine Parrot

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57490

**1. Introduction**

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[91] Carriba P, Ortiz O, Patkar K, Justinova Z, Stroik J, Themann A, et al. Striatal adeno‐ sine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids. Neuropsychopharmacol 2007;

[92] Ferré S, Lluís C, Justinova Z, Quiroz C, Orru M, Navarro G, et al. Adenosine-canna‐ binoid receptor interactions. Implications for striatal function. Br J Pharmacol 2009;

[93] Fernández-Ruiz J, Hernández M, Ramos JA. Cannabinoid-dopamine interaction in the pathophysiology and treatment of CNS disorders. CNS Neurosci Ther 2010;

mediated actions. Neuropharmacol 2004; 46:115-25.

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198 Neurochemistry

gets 2010; 11:393-405.

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16:e72-91.

Fear conditioning is one of the most widely used paradigms for studying the neurobiology of emotional learning. In this paradigm, an emotionally neutral stimulus (the conditioned stimulus or CS) is paired with an aversive unconditioned stimulus (US), generally a mild foot shock. After a few trials, re-exposure to the CS alone elicits a fear response, proceeding through the necessary connecting structures, and ending with the autonomic and motoric effector outputs. This fear response is part of an anticipatory response to danger [1], initiating a range of defensive reactions that counter threats to survival [2]. The most universal response is a postural immobility, called freezing [3, 4]. Moreover, the threatening stimulus also triggers the activation of the hypothalamic subnuclei to induce an increase in blood pressure, ultrasonic vocalizations emission, or the release of stress hormones.

The vast majority of studies devoted to investigate the neural basis of fear conditioning have used auditory cues as conditioned stimuli and the neural pathways involved in auditory fear conditioning have been well characterized [5-8]. The information carried by the auditory CS can take one of two pathways: either directly from the thalamus to quickly reach the amygdala or the CS can travel from the auditory thalamus to the auditory cortex before reaching the amygdala. These thalamic and cortical areas send projections to the lateral nucleus of the amygdala, which is a site of CS-US convergence. The lateral nucleus, in turn, projects to the

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

central amygdala, which controls the expression of fear responses by way of projections to brainstem areas. The major conclusion of these studies is that the amygdala plays a critical role in linking external stimuli to defense responses through synaptic plasticity. However, although the amygdala seems to be essential to the formation and storage of fear memories, it might not store all aspects of the aversive event. Indeed, plasticity also occurs in cortical areas during fear conditioning, which could support declarative memories of the learning episode mainly through interactions with the medial temporal lobe memory system [9-13]. Thus, the amygdala may store some aspects of fear memory and facilitate the storage of other, more sensoryrelated, aspects of fear memory in cortical areas [14]. Yet few studies have investigated this question [15].

**2. Monitoring neurotransmitters using microdialysis**

Microdialysis has become a conventional technique for sampling low molecular weight molecules present in the extracellular medium of many organs from animals or humans [42]. In the brain, it has been used *in vivo* or *ex vivo* to monitor amino acids neurotransmitters as excitatory or inhibitory amino acids as glutamate and GABA, monoamines as dopamine, noradrenaline, serotonin and/or neuropeptides for instance. The microdialysis relies on a semipermeable membrane that allows free diffusion of solutes between the extracellular space and an artificial fluid. A microdialysis probe consists of two concentric tubes with the distal part (1–5 mm) covered by a dialysis hollow fibre whose cut-off ranges between 6,000 and 100,000 Da. Such a probe is inserted into a living brain tissue and is perfused by an isotonic physio‐ logical fluid. Molecules diffuse down their concentration gradient across the dialysis mem‐ brane in a bidirectional way ("dialysis" for collecting endogenous molecules or "reverse dialysis" for applying exogenous compounds) (Figure 1). In the case of collection, the relative recovery across the probe membrane, defined as the ratio between the extracellular concen‐ tration and the concentration of a compound in the dialysate collected at the outlet of the probe, depends on several factors: it increases with the surface of the membrane, it decreases with higher flow rate of the perfusion fluid, and it varies with the chemical and physical charac‐ teristics of the membrane [43]. The choice of the dialysis membrane can be crucial for com‐ pounds present at trace concentration or when the limit of detection of the analytical method is relatively moderate. For instance, a short length (i.e. 1-2 mm) of the membrane chosen to sample a very small brain area in rats, such as the periaqueductal grey matter, the locus coeruleus, the amygdala and hypothalamic nuclei leads to a low recovery of sampling, making difficult the monitoring of low concentrated compounds as monoamines or neuropeptides. Depending on the methodological parameters, recovery values usually reported in literature are between 5 and 25 % for amino acids or monoamines and can reach < 1 % for neuropeptides. Another point to take into account is the choice of the geometry of the probe tubings when considering the sampling rate, especially when designing in vivo microdialysis on awake animals. These latter peculiar points will be explained and detailed in the next section part of

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As the microdialysis probe is continuously perfused at a constant flow rate, continuous sampling of neurotransmitters is possible with no loss. Indeed, neurotransmitters present in the microdialysate can be directly analyzed without clean-up procedures as high molecular weight proteins cannot cross the dialysis membrane thanks to the cut-off of the membrane used. However, manipulation of microdialysis samples requires precaution in case of amino acids: use of sterile tubes, filtered aCSF and wear of gloves avoiding contamination due to the ubiquitous presence of free amino acids on labware and skin. Eventually, samples may be usually analyzed by a separative method like high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) [44, 45]. Microdialysates can be analyzed on-line, i.e., at the outlet of the probe, through an analytical interface, or off-line i.e., after sample collection in micro-tubes, in combination with a separative method [44, 46, 47]. Thus, the determination

**2.1. Principle of the technique and analytical considerations**

the manuscript.

Intriguingly, whereas olfaction plays a dominant role in rat behavior from birth throughout adulthood, very few studies have used odor cues as CS in fear conditioning paradigms. Yet, odors have a rather unique status for eliciting emotional memories.[16]. This particularity might be linked to the uniqueness of the anatomy of the olfactory system. Indeed, in contrast to the other sensory pathways, olfactory information has a direct access to the amygdala and olfactory (piriform) cortex with no obligatory thalamic relay [17-19]. The literature suggests that, similarly to what is observed in auditory fear conditioning, the amygdala is a key brain structure involved in the acquisition, consolidation and expression of odor fear conditioning [20-26]. Recently, some studies also suggest that the posterior piriform cortex (PPC) may play a critical role in this associative learning [23, 26, 27]. Therefore, the olfactory system constitutes a particularly relevant model for studying the relative contribution of sensory cortices and amygdalar nuclei to odor fear learning.

For several years now, the glutamatergic transmission in the amygdala is known to play a critical role in the acquisition of fear conditioning [28, 29]. Indeed, pharmacological studies show that NMDA and AMPA subclasses of glutamate receptors are crucial for synaptic plasticity and long-term potentiation to occur in the amygdala, sustaining the formation of the CS-US association [25, 30-35]. In addition, the GABAergic transmission seems to be also involved in the acquisition of fear learning. Intra-amygdala infusion of the GABAA receptor agonist muscimol before training impairs learning [36, 37]. Therefore, glutamate and GABA neurotransmission are thought to play a critical role in the acquisition and expression of fear memories (for review, see [38]).

Beside these neuropharmacological studies, a few neurochemical studies have directly measured glutamate or GABA levels in the amygdala during auditory fear conditioning, using a 10-min sampling rate [39, 40]. However, this sampling rate is too long as compared to the rapid neurobiological events underlying fear conditioning. An approach allowing rapid, subminute sampling is required to better characterize the dynamics of neurotransmitter changes evoked by the stimuli involved in fear conditioning experiments [41]. Until now, the precise time course of the differential involvement of the amygdala and sensory cortices in fear conditioning has received little investigation. The aim of this chapter is to show that intra‐ cerebral in vivo microdialysis with high temporal resolution is an interesting tool to investigate the time course of activation of amygdala and sensory cortices in this learning.

## **2. Monitoring neurotransmitters using microdialysis**

#### **2.1. Principle of the technique and analytical considerations**

central amygdala, which controls the expression of fear responses by way of projections to brainstem areas. The major conclusion of these studies is that the amygdala plays a critical role in linking external stimuli to defense responses through synaptic plasticity. However, although the amygdala seems to be essential to the formation and storage of fear memories, it might not store all aspects of the aversive event. Indeed, plasticity also occurs in cortical areas during fear conditioning, which could support declarative memories of the learning episode mainly through interactions with the medial temporal lobe memory system [9-13]. Thus, the amygdala may store some aspects of fear memory and facilitate the storage of other, more sensoryrelated, aspects of fear memory in cortical areas [14]. Yet few studies have investigated this

Intriguingly, whereas olfaction plays a dominant role in rat behavior from birth throughout adulthood, very few studies have used odor cues as CS in fear conditioning paradigms. Yet, odors have a rather unique status for eliciting emotional memories.[16]. This particularity might be linked to the uniqueness of the anatomy of the olfactory system. Indeed, in contrast to the other sensory pathways, olfactory information has a direct access to the amygdala and olfactory (piriform) cortex with no obligatory thalamic relay [17-19]. The literature suggests that, similarly to what is observed in auditory fear conditioning, the amygdala is a key brain structure involved in the acquisition, consolidation and expression of odor fear conditioning [20-26]. Recently, some studies also suggest that the posterior piriform cortex (PPC) may play a critical role in this associative learning [23, 26, 27]. Therefore, the olfactory system constitutes a particularly relevant model for studying the relative contribution of sensory cortices and

For several years now, the glutamatergic transmission in the amygdala is known to play a critical role in the acquisition of fear conditioning [28, 29]. Indeed, pharmacological studies show that NMDA and AMPA subclasses of glutamate receptors are crucial for synaptic plasticity and long-term potentiation to occur in the amygdala, sustaining the formation of the CS-US association [25, 30-35]. In addition, the GABAergic transmission seems to be also involved in the acquisition of fear learning. Intra-amygdala infusion of the GABAA receptor agonist muscimol before training impairs learning [36, 37]. Therefore, glutamate and GABA neurotransmission are thought to play a critical role in the acquisition and expression of fear

Beside these neuropharmacological studies, a few neurochemical studies have directly measured glutamate or GABA levels in the amygdala during auditory fear conditioning, using a 10-min sampling rate [39, 40]. However, this sampling rate is too long as compared to the rapid neurobiological events underlying fear conditioning. An approach allowing rapid, subminute sampling is required to better characterize the dynamics of neurotransmitter changes evoked by the stimuli involved in fear conditioning experiments [41]. Until now, the precise time course of the differential involvement of the amygdala and sensory cortices in fear conditioning has received little investigation. The aim of this chapter is to show that intra‐ cerebral in vivo microdialysis with high temporal resolution is an interesting tool to investigate

the time course of activation of amygdala and sensory cortices in this learning.

question [15].

200 Neurochemistry

amygdalar nuclei to odor fear learning.

memories (for review, see [38]).

Microdialysis has become a conventional technique for sampling low molecular weight molecules present in the extracellular medium of many organs from animals or humans [42]. In the brain, it has been used *in vivo* or *ex vivo* to monitor amino acids neurotransmitters as excitatory or inhibitory amino acids as glutamate and GABA, monoamines as dopamine, noradrenaline, serotonin and/or neuropeptides for instance. The microdialysis relies on a semipermeable membrane that allows free diffusion of solutes between the extracellular space and an artificial fluid. A microdialysis probe consists of two concentric tubes with the distal part (1–5 mm) covered by a dialysis hollow fibre whose cut-off ranges between 6,000 and 100,000 Da. Such a probe is inserted into a living brain tissue and is perfused by an isotonic physio‐ logical fluid. Molecules diffuse down their concentration gradient across the dialysis mem‐ brane in a bidirectional way ("dialysis" for collecting endogenous molecules or "reverse dialysis" for applying exogenous compounds) (Figure 1). In the case of collection, the relative recovery across the probe membrane, defined as the ratio between the extracellular concen‐ tration and the concentration of a compound in the dialysate collected at the outlet of the probe, depends on several factors: it increases with the surface of the membrane, it decreases with higher flow rate of the perfusion fluid, and it varies with the chemical and physical charac‐ teristics of the membrane [43]. The choice of the dialysis membrane can be crucial for com‐ pounds present at trace concentration or when the limit of detection of the analytical method is relatively moderate. For instance, a short length (i.e. 1-2 mm) of the membrane chosen to sample a very small brain area in rats, such as the periaqueductal grey matter, the locus coeruleus, the amygdala and hypothalamic nuclei leads to a low recovery of sampling, making difficult the monitoring of low concentrated compounds as monoamines or neuropeptides. Depending on the methodological parameters, recovery values usually reported in literature are between 5 and 25 % for amino acids or monoamines and can reach < 1 % for neuropeptides. Another point to take into account is the choice of the geometry of the probe tubings when considering the sampling rate, especially when designing in vivo microdialysis on awake animals. These latter peculiar points will be explained and detailed in the next section part of the manuscript.

As the microdialysis probe is continuously perfused at a constant flow rate, continuous sampling of neurotransmitters is possible with no loss. Indeed, neurotransmitters present in the microdialysate can be directly analyzed without clean-up procedures as high molecular weight proteins cannot cross the dialysis membrane thanks to the cut-off of the membrane used. However, manipulation of microdialysis samples requires precaution in case of amino acids: use of sterile tubes, filtered aCSF and wear of gloves avoiding contamination due to the ubiquitous presence of free amino acids on labware and skin. Eventually, samples may be usually analyzed by a separative method like high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) [44, 45]. Microdialysates can be analyzed on-line, i.e., at the outlet of the probe, through an analytical interface, or off-line i.e., after sample collection in micro-tubes, in combination with a separative method [44, 46, 47]. Thus, the determination

neurotransmitter contents. As a consequence, despite constant improvements in HPLC [50, 51, 53], the required sample volume may still be too large, leading to lengthy dialysis sampling times and poor temporal resolution. Consequently, most microdialysis experiments were severely limited by the temporal resolution of microdialysis (5–30 min) compared to rapid changes occurring in the extracellular concentrations of neurotransmitters. In contrast, microdialysis coupled to CE, a more recent technique, allows the monitoring of rapid changes in the extracellular concentration of neurotransmitters by analysing nanovolume dialysates with low limits of detection. At present, it appears to be suitable for microdialysis with high sampling rate. Often coupled to laser-induced fluorescence (LIF) detection or mass spectrom‐ etry (MS), capillary electrophoresis has become one of the most powerful analytical tools for the routine determination of neurotransmitters because it offers the advantage of rapidity, high resolution and sensitivity, while requiring very small sample sizes [44, 54, 55]. In brain microdialysates, glutamate and GABA [56-64] were often analysed using CE with LIF detec‐ tion. However, as amino acid neurotransmitters are not fluorescent at wavelengths of most commercially available lasers, derivatization prior to the separation is needed. Fluorescent reagents, as naphthalene-2,3-dicarboxaldehyde (NDA), orthophtaldehyde (OPA) or fluores‐ cein isothiocyanate, reacting with the primary amine function of neurotransmitters, allow their detection following laser excitation at 442, 325, or 488 nm, respectively [63-65]. Several groups, including our own, have developed methodologies for the CE–LIF analysis of brain micro‐ dialysates after a derivatization of samples with fluorogenic agents, even on sub-microliter dialysates obtained with high temporal resolution microdialysis (5s – 1 min, [44] for mini-

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203

**2.2. Set-up for high temporal microdialysis on behaving animal**

According to the neurophysiological question asked, the experimental set-up for microdialy‐ sis experiments has to be carefully designed: the choice of microdialysis probe according to the targeted brain area, the choice of the analytical method to quantify dialysate neurotransmit‐ ters and the coupling of the experimental set-ups or approaches are all crucial to succeed in neurochemical studies. The aim of the study described here [66] was to monitor amino acid neurotransmitters as glutamate and GABA in brain areas involved in emotional learning and memory using odor fear conditioning. Indeed, while a few studies have investigated the time course of events separately in the auditory cortex and the amygdala during auditory fear conditioning [11, 12], no study up to date has been conducted to investigate simultaneously the changes in amygdala and sensory cortical areas in the same animal during fear conditioning. Our group previously reported lasting changes in electrophysiological field potential signals in both posterior piriform cortex (PPC) and amygdala (basolateral nucleus, BLA) after a session of odor fear conditioning [23]. As a consequence, we conducted an experiment using a dualmicrodialysis probe implantation, in order to compare the time courses of changes in GABA and glutamate concentrations, monitored simultaneously in BLA and PPC during odor fear conditioning. Very few groups have developed dual (or triple) implantation of microdialysis probes on the same animal [67, 68]. Indeed in most studies using brain microdialysis, dialysates collection is usually performed in one brain structure at a time, sometimes on different days. Literature on microdialysis experiments in BLA is rather abundant, mainly for monoamines.

review).

**Figure 1.** Schematic representation of a microdialysis probe. The probe is continuously perfused by an isotonic physio‐ logical fluid. Endogenous compounds (filled circles) diffuse through the dialysis membrane (filled arrow) and are sam‐ pled in the microdialysate collected at the outlet of the probe. Drugs (open circles) can be added to the perfusion medium and can diffuse out of the probe into the extracellular medium (open arrow) providing a means of local ad‐ ministration. From [109], with permission from John Wiley and Sons.

of concentrations in each sample reflects the average concentration over the defined sampling duration. Data are classically expressed as % of the average of the first samples collected, corresponding to baseline. As a consequence, microdialysis (except in very peculiar methodo‐ logical contexts not explained in this chapter) is considered as a semi-quantitative method as it permits to monitor precisely relative variations of neurotransmitters.

Analysis of microdialysates has commonly used HPLC with electrochemical [48, 49] or fluorometric detection [50, 51], and also enzymatic methods [52]. However, these analytical techniques exhibit poor mass sensitivity and require large volume samples to determine

neurotransmitter contents. As a consequence, despite constant improvements in HPLC [50, 51, 53], the required sample volume may still be too large, leading to lengthy dialysis sampling times and poor temporal resolution. Consequently, most microdialysis experiments were severely limited by the temporal resolution of microdialysis (5–30 min) compared to rapid changes occurring in the extracellular concentrations of neurotransmitters. In contrast, microdialysis coupled to CE, a more recent technique, allows the monitoring of rapid changes in the extracellular concentration of neurotransmitters by analysing nanovolume dialysates with low limits of detection. At present, it appears to be suitable for microdialysis with high sampling rate. Often coupled to laser-induced fluorescence (LIF) detection or mass spectrom‐ etry (MS), capillary electrophoresis has become one of the most powerful analytical tools for the routine determination of neurotransmitters because it offers the advantage of rapidity, high resolution and sensitivity, while requiring very small sample sizes [44, 54, 55]. In brain microdialysates, glutamate and GABA [56-64] were often analysed using CE with LIF detec‐ tion. However, as amino acid neurotransmitters are not fluorescent at wavelengths of most commercially available lasers, derivatization prior to the separation is needed. Fluorescent reagents, as naphthalene-2,3-dicarboxaldehyde (NDA), orthophtaldehyde (OPA) or fluores‐ cein isothiocyanate, reacting with the primary amine function of neurotransmitters, allow their detection following laser excitation at 442, 325, or 488 nm, respectively [63-65]. Several groups, including our own, have developed methodologies for the CE–LIF analysis of brain micro‐ dialysates after a derivatization of samples with fluorogenic agents, even on sub-microliter dialysates obtained with high temporal resolution microdialysis (5s – 1 min, [44] for minireview).

#### **2.2. Set-up for high temporal microdialysis on behaving animal**

of concentrations in each sample reflects the average concentration over the defined sampling duration. Data are classically expressed as % of the average of the first samples collected, corresponding to baseline. As a consequence, microdialysis (except in very peculiar methodo‐ logical contexts not explained in this chapter) is considered as a semi-quantitative method as

**Figure 1.** Schematic representation of a microdialysis probe. The probe is continuously perfused by an isotonic physio‐ logical fluid. Endogenous compounds (filled circles) diffuse through the dialysis membrane (filled arrow) and are sam‐ pled in the microdialysate collected at the outlet of the probe. Drugs (open circles) can be added to the perfusion medium and can diffuse out of the probe into the extracellular medium (open arrow) providing a means of local ad‐

Analysis of microdialysates has commonly used HPLC with electrochemical [48, 49] or fluorometric detection [50, 51], and also enzymatic methods [52]. However, these analytical techniques exhibit poor mass sensitivity and require large volume samples to determine

it permits to monitor precisely relative variations of neurotransmitters.

ministration. From [109], with permission from John Wiley and Sons.

202 Neurochemistry

According to the neurophysiological question asked, the experimental set-up for microdialy‐ sis experiments has to be carefully designed: the choice of microdialysis probe according to the targeted brain area, the choice of the analytical method to quantify dialysate neurotransmit‐ ters and the coupling of the experimental set-ups or approaches are all crucial to succeed in neurochemical studies. The aim of the study described here [66] was to monitor amino acid neurotransmitters as glutamate and GABA in brain areas involved in emotional learning and memory using odor fear conditioning. Indeed, while a few studies have investigated the time course of events separately in the auditory cortex and the amygdala during auditory fear conditioning [11, 12], no study up to date has been conducted to investigate simultaneously the changes in amygdala and sensory cortical areas in the same animal during fear conditioning.

Our group previously reported lasting changes in electrophysiological field potential signals in both posterior piriform cortex (PPC) and amygdala (basolateral nucleus, BLA) after a session of odor fear conditioning [23]. As a consequence, we conducted an experiment using a dualmicrodialysis probe implantation, in order to compare the time courses of changes in GABA and glutamate concentrations, monitored simultaneously in BLA and PPC during odor fear conditioning. Very few groups have developed dual (or triple) implantation of microdialysis probes on the same animal [67, 68]. Indeed in most studies using brain microdialysis, dialysates collection is usually performed in one brain structure at a time, sometimes on different days. Literature on microdialysis experiments in BLA is rather abundant, mainly for monoamines. There are only a few papers in the literature reporting microdialysis studies in the piriform cortex in an epileptic model [69] or during a feeding/diet procedure [70]. Two other studies were devoted to glutamate monitoring at 20 or 30-min sampling rate in anesthetized [71] or awake rats [72]. In order to target the centre of each brain structure and avoid excluding rats with inaccurate implantation on one hand, and in order to optimize the amount of collected glutamate and GABA in the microdialysates for analytical quantification on the other hand, 1.5-mm microdialysis probes were used for both BLA and PPC. However, due to the size of the probes and the vicinity of amygdala and olfactory cortex, we implanted one probe per hemisphere, BLA probe on one side, PPC probe contralaterally (Figure 2). As the acquisition session of odor fear conditioning was short-lasting (30 min), we monitored glutamate and GABA at high sampling rate using CE-LIFD as analytical technique. When performing high sampling rate, it is crucial to optimize the geometry of the probe tubings.

characteristics: an experimental chamber with a grid for electrical stimulation which is a mild footshock (Unconditioned Stimulus, US) and with an ventilation system bringing and removing the odor (Conditioned Stimulus, CS); besides, this experimental chamber has to be placed in a soundproof box in which we can position cameras to record the behavior of the animal. Material for microdialysis (perfusion pump) and collection have to be placed outside the box for not disturbing the animal in learning. In our set-up, the height of the box reaches almost one meter (Figure 3). The dead volume can be greatly minimized by using capillary tubings with sub-50 μm inner diameters, as previously demonstrated by our group [56]. By using a 40-μm inner diameter, we have optimized the microdialysis set-up for odor condi‐ tioning by adapting methods that our group previously used for an accurate monitoring of 30 s pharmacologically-induced increases [73] or 20-s behaviourally-induced variations [56] in extracellular levels of amino acids neurotransmitters. We showed that the experimental determination of the dead time is necessary in order to adapt each set-up to the sampling rate required (Figure 4). The final length of the outlet tubing is 120 cm with a dead time of 1 min 30 at 2μL/min as sampling rate and the final sampling resolution is 1 min. The time scale in the figures corresponds to the real time of collection of the fractions. Administration of odor

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was timed to take into account the outlet dead time of the dialysate system.

**Figure 3.** Experimental set-up for both microdialysis sampling and dialysates derivatization during the acquisition of odor fear conditioning. The dual probe-implanted animal is placed in an experimental chamber for odor (conditional stimulus) and shock (unconditional stimulus) deliveries, required in the learning procedure. This chamber is soundproof in order to prevent external noise to interfere with the animal's behaviour. Microdialysis and derivatization are performed simultaneously using one pump per brain area and four syringes delivering artificial cerebrospinal fluid (aCSF) in the inlet of the probe or reagents (NDA as derivatization agent in presence of cyanide ions at pH 8.7 and internal standard for better quantification by capillary electrophoresis with laser-induced fluorescence detection) to

tag the neurotransmitters at the outlet of the probe.

**Figure 2.** Brain sections from Paxinos & Watson's atlas with a schematic probe implanted in the left basolateral amyg‐ dala and the other implanted in the right posterior piriform cortex of the same freely-moving rat for simultaneous glutamate and GABA monitoring.

Indeed, a high sampling rate requires appropriate probe tubings since solutes could undergo more longitudinal diffusion in the outlet probe tubing if the interval of time between dialysis and collection, also called 'dead time, is superior to the sampling time [56]. As a consequence, to avoid mixing of analytes between successive samples, the dead volume of these tubings has to be minimized. This is particularly relevant when microdialysis experiments are carried out on awake animals because the setting requires long inlet and outlet probe tubings in order to let the animals move freely. In our odor fear conditioning, the set-up includes specific characteristics: an experimental chamber with a grid for electrical stimulation which is a mild footshock (Unconditioned Stimulus, US) and with an ventilation system bringing and removing the odor (Conditioned Stimulus, CS); besides, this experimental chamber has to be placed in a soundproof box in which we can position cameras to record the behavior of the animal. Material for microdialysis (perfusion pump) and collection have to be placed outside the box for not disturbing the animal in learning. In our set-up, the height of the box reaches almost one meter (Figure 3). The dead volume can be greatly minimized by using capillary tubings with sub-50 μm inner diameters, as previously demonstrated by our group [56]. By using a 40-μm inner diameter, we have optimized the microdialysis set-up for odor condi‐ tioning by adapting methods that our group previously used for an accurate monitoring of 30 s pharmacologically-induced increases [73] or 20-s behaviourally-induced variations [56] in extracellular levels of amino acids neurotransmitters. We showed that the experimental determination of the dead time is necessary in order to adapt each set-up to the sampling rate required (Figure 4). The final length of the outlet tubing is 120 cm with a dead time of 1 min 30 at 2μL/min as sampling rate and the final sampling resolution is 1 min. The time scale in the figures corresponds to the real time of collection of the fractions. Administration of odor was timed to take into account the outlet dead time of the dialysate system.

There are only a few papers in the literature reporting microdialysis studies in the piriform cortex in an epileptic model [69] or during a feeding/diet procedure [70]. Two other studies were devoted to glutamate monitoring at 20 or 30-min sampling rate in anesthetized [71] or awake rats [72]. In order to target the centre of each brain structure and avoid excluding rats with inaccurate implantation on one hand, and in order to optimize the amount of collected glutamate and GABA in the microdialysates for analytical quantification on the other hand, 1.5-mm microdialysis probes were used for both BLA and PPC. However, due to the size of the probes and the vicinity of amygdala and olfactory cortex, we implanted one probe per hemisphere, BLA probe on one side, PPC probe contralaterally (Figure 2). As the acquisition session of odor fear conditioning was short-lasting (30 min), we monitored glutamate and GABA at high sampling rate using CE-LIFD as analytical technique. When performing high

**Figure 2.** Brain sections from Paxinos & Watson's atlas with a schematic probe implanted in the left basolateral amyg‐ dala and the other implanted in the right posterior piriform cortex of the same freely-moving rat for simultaneous

Indeed, a high sampling rate requires appropriate probe tubings since solutes could undergo more longitudinal diffusion in the outlet probe tubing if the interval of time between dialysis and collection, also called 'dead time, is superior to the sampling time [56]. As a consequence, to avoid mixing of analytes between successive samples, the dead volume of these tubings has to be minimized. This is particularly relevant when microdialysis experiments are carried out on awake animals because the setting requires long inlet and outlet probe tubings in order to let the animals move freely. In our odor fear conditioning, the set-up includes specific

glutamate and GABA monitoring.

204 Neurochemistry

sampling rate, it is crucial to optimize the geometry of the probe tubings.

**Figure 3.** Experimental set-up for both microdialysis sampling and dialysates derivatization during the acquisition of odor fear conditioning. The dual probe-implanted animal is placed in an experimental chamber for odor (conditional stimulus) and shock (unconditional stimulus) deliveries, required in the learning procedure. This chamber is soundproof in order to prevent external noise to interfere with the animal's behaviour. Microdialysis and derivatization are performed simultaneously using one pump per brain area and four syringes delivering artificial cerebrospinal fluid (aCSF) in the inlet of the probe or reagents (NDA as derivatization agent in presence of cyanide ions at pH 8.7 and internal standard for better quantification by capillary electrophoresis with laser-induced fluorescence detection) to tag the neurotransmitters at the outlet of the probe.

Another analytical constraint is to tag the sample in order to detect the amino acids collected during the dialysis experiment. As glutamate and GABA are not fluorescent, we employed a validated home-made on-line system able to deliver the derivatization reagents directly in the collection tube [56], allowing to derivatize the dialysate while dialyzing, without sample loss, noticeable dilution or contamination and without increasing the dead volume of the probe. Thus, the collection tube is not only the micro-reactor of derivatization, but also the injection tube for capillary electrophoresis analysis (Figure 3).

Figure 5 presents an example of typical electropherogramm obtained from 1 minute sampling rate of brain microdialysate in freely-moving rat during the acquisition of fear conditioning.

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**Figure 5.** Typical electropherograms from microdialysates obtained in posterior piriform cortex and basolateral amyg‐ dala on the same freely-moving Long-Evans rat. Samples were collected every minute at 2µL/min and derivatized on-

line as described in Figure 3 of this chapter. Adapted from [110].

Note that the concentrations for GABA and glutamate are similar in PPC or in BLA.

**Figure 4.** Example of determination of the dead time of an 85-cm outlet home-made probe perfused at 2µL/min by plunging the probe into a known concentration of glutamate at t=0s. The dead time is determined as the time when 50% of the maximal response is reached using a sigmoid curve fitting the 30-s glutamate monitoring (up). By varying the length of the outlet tubing of customized probes (n=3), the dead time of probes can be interpolated and the final length of outlet tubing (here, 120 cm for a dead time of 1 min 30 s) can be chosen according to the height of our experimental set-up and the 1-min sampling rate (bottom). As a consequence, administration of odor was timed to take into account the outlet dead volume of the dialysate system.

Figure 5 presents an example of typical electropherogramm obtained from 1 minute sampling rate of brain microdialysate in freely-moving rat during the acquisition of fear conditioning. Note that the concentrations for GABA and glutamate are similar in PPC or in BLA.

Another analytical constraint is to tag the sample in order to detect the amino acids collected during the dialysis experiment. As glutamate and GABA are not fluorescent, we employed a validated home-made on-line system able to deliver the derivatization reagents directly in the collection tube [56], allowing to derivatize the dialysate while dialyzing, without sample loss, noticeable dilution or contamination and without increasing the dead volume of the probe. Thus, the collection tube is not only the micro-reactor of derivatization, but also the injection

**Figure 4.** Example of determination of the dead time of an 85-cm outlet home-made probe perfused at 2µL/min by plunging the probe into a known concentration of glutamate at t=0s. The dead time is determined as the time when 50% of the maximal response is reached using a sigmoid curve fitting the 30-s glutamate monitoring (up). By varying the length of the outlet tubing of customized probes (n=3), the dead time of probes can be interpolated and the final length of outlet tubing (here, 120 cm for a dead time of 1 min 30 s) can be chosen according to the height of our experimental set-up and the 1-min sampling rate (bottom). As a consequence, administration of odor was timed to

tube for capillary electrophoresis analysis (Figure 3).

206 Neurochemistry

take into account the outlet dead volume of the dialysate system.

**Figure 5.** Typical electropherograms from microdialysates obtained in posterior piriform cortex and basolateral amyg‐ dala on the same freely-moving Long-Evans rat. Samples were collected every minute at 2µL/min and derivatized online as described in Figure 3 of this chapter. Adapted from [110].

## **3. Neurochemistry of odor fear conditioning**

#### **3.1. Interactions between amygdala and piriform cortex in odor fear conditioning**

The odor fear conditioning paradigm consisted of six 20-s odor/ 2-s shock associations presented with an interval of 4 minutes between each pairing. In the amygdala, the first odorshock association is accompanied by a significant but transient +40%-increase of glutamate release. During the next trials, the concentrations returned to the baseline levels or slightly below. In contrast, in piriform cortex, each odor-shock association is followed by a transient +25%-increase in glutamate release. The comparison of the pattern of release observed in the two structures has also shown that the increase in the amygdala during the first association occurred 1-2 min before the first response in the piriform cortex (Figure 6).

early stages of the acquisition session, which could be involved in the initiation of plastic

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Glutamate collected from the BLA and PPC may have originated from both extrinsic and intrinsic sources. Indeed, the BLA receives afferences from primary olfactory cortex [76-78] and from other associative areas [19, 79], providing an extrinsic source of glutamate. Similarly, PPC receives a strong glutamatergic input from the olfactory bulb as well as inputs from the BLA, prefrontal cortex, and hippocampus [80-82]. In addition, both the BLA and PPC contain glutamatergic pyramidal cells that send axon collaterals to neighboring cells, thus providing an intrinsic source of glutamate [79, 83, 84]. Concerning GABA origin, in both BLA and PPC,

While *in vivo* microdialysis is a useful method for monitoring the neurotransmitters present in the brain extracellular fluid [86], many studies have questioned to what extent dialysate glutamate concentration reflects the amount of glutamate released by the presynaptic neuron. Indeed, due to the ubiquitous localization of its metabolism enzymes and transporters in all brain cells and its paramount role in protein synthesis and general metabolism, numerous works attempted to determine the origin of extracellular glutamate. If the proportion of extracellular glutamate taken up by astrocytes is about 80–90% of the whole glutamate pool [87], the part of the neuronal glutamate, i.e. released for the only neurotransmission purpose, is still matter of debate, because glutamate may come from many sources of effluxes as neuronal "classical" release [88], exchange via cysteine/glutamate transporters [89-92], inversion of transporters [93] and glial release via exocytosis or non-exocytosis [94-96]. Former methodological strategies used for monoamine neurotransmitters to impair the vesicular neuronal release by lowering or removing calcium in aCSF or by blocking nerve impulse Na


changes supporting the formation of the memory.

**3.2. Functional significance of neurochemical data**

GABA is released by local inhibitory interneurons [79, 85].

+

**Figure 6.** Comparison of glutamate concentration fluctuations in the amygdala and the piriform cortex during odor fear acquisition session. Glutamate concentrations were measured throughout the session and expressed as a per‐ centage of baseline calculated as the mean of concentration for the four points preceding the first pairing. Black ar‐ rowheads above the x-axis symbolize trial occurrence. Light gray vertical bars indicate the timing of the 4-min intertrial intervals. (\*) Significant difference between the two structures (p < 0.05); (#) tendency toward significant difference (p < 0.09). Adapted from [66].

The profile of GABA variations during odor fear conditioning was similar in duration and amplitude to that obtained for glutamate presented in Figure 6, i.e. with an enhancement of GABA levels in the amygdala during the first pairing and increases of GABA concentrations in piriform cortex 1-2 min after each pairing. As glutamate and GABA fluctuations were not overall significantly different throughout the experiment, it can be suggested that GABA increases could be induced by glutamate increases, as shown by [74, 75] in the prefrontal cortex.

The high temporal resolution microdialysis allowed us to highlight a differential dynamics of neurotransmitters release in the piriform cortex and amygdala during odor fear acquisition. Taken together, these results suggest that there is a temporal sequence of neurochemical events in the amygdala and piriform cortex, a very precise dynamics of neurotransmitters during the early stages of the acquisition session, which could be involved in the initiation of plastic changes supporting the formation of the memory.

#### **3.2. Functional significance of neurochemical data**

**3. Neurochemistry of odor fear conditioning**

208 Neurochemistry

< 0.09). Adapted from [66].

**3.1. Interactions between amygdala and piriform cortex in odor fear conditioning**

occurred 1-2 min before the first response in the piriform cortex (Figure 6).

The odor fear conditioning paradigm consisted of six 20-s odor/ 2-s shock associations presented with an interval of 4 minutes between each pairing. In the amygdala, the first odorshock association is accompanied by a significant but transient +40%-increase of glutamate release. During the next trials, the concentrations returned to the baseline levels or slightly below. In contrast, in piriform cortex, each odor-shock association is followed by a transient +25%-increase in glutamate release. The comparison of the pattern of release observed in the two structures has also shown that the increase in the amygdala during the first association

**Figure 6.** Comparison of glutamate concentration fluctuations in the amygdala and the piriform cortex during odor fear acquisition session. Glutamate concentrations were measured throughout the session and expressed as a per‐ centage of baseline calculated as the mean of concentration for the four points preceding the first pairing. Black ar‐ rowheads above the x-axis symbolize trial occurrence. Light gray vertical bars indicate the timing of the 4-min intertrial intervals. (\*) Significant difference between the two structures (p < 0.05); (#) tendency toward significant difference (p

The profile of GABA variations during odor fear conditioning was similar in duration and amplitude to that obtained for glutamate presented in Figure 6, i.e. with an enhancement of GABA levels in the amygdala during the first pairing and increases of GABA concentrations in piriform cortex 1-2 min after each pairing. As glutamate and GABA fluctuations were not overall significantly different throughout the experiment, it can be suggested that GABA increases could be induced by glutamate increases, as shown by [74, 75] in the prefrontal cortex.

The high temporal resolution microdialysis allowed us to highlight a differential dynamics of neurotransmitters release in the piriform cortex and amygdala during odor fear acquisition. Taken together, these results suggest that there is a temporal sequence of neurochemical events in the amygdala and piriform cortex, a very precise dynamics of neurotransmitters during the

Glutamate collected from the BLA and PPC may have originated from both extrinsic and intrinsic sources. Indeed, the BLA receives afferences from primary olfactory cortex [76-78] and from other associative areas [19, 79], providing an extrinsic source of glutamate. Similarly, PPC receives a strong glutamatergic input from the olfactory bulb as well as inputs from the BLA, prefrontal cortex, and hippocampus [80-82]. In addition, both the BLA and PPC contain glutamatergic pyramidal cells that send axon collaterals to neighboring cells, thus providing an intrinsic source of glutamate [79, 83, 84]. Concerning GABA origin, in both BLA and PPC, GABA is released by local inhibitory interneurons [79, 85].

While *in vivo* microdialysis is a useful method for monitoring the neurotransmitters present in the brain extracellular fluid [86], many studies have questioned to what extent dialysate glutamate concentration reflects the amount of glutamate released by the presynaptic neuron. Indeed, due to the ubiquitous localization of its metabolism enzymes and transporters in all brain cells and its paramount role in protein synthesis and general metabolism, numerous works attempted to determine the origin of extracellular glutamate. If the proportion of extracellular glutamate taken up by astrocytes is about 80–90% of the whole glutamate pool [87], the part of the neuronal glutamate, i.e. released for the only neurotransmission purpose, is still matter of debate, because glutamate may come from many sources of effluxes as neuronal "classical" release [88], exchange via cysteine/glutamate transporters [89-92], inversion of transporters [93] and glial release via exocytosis or non-exocytosis [94-96]. Former methodological strategies used for monoamine neurotransmitters to impair the vesicular neuronal release by lowering or removing calcium in aCSF or by blocking nerve impulse Na + -dependent channels with tetrodotoxine (TTX) gave contradictory answers: some studies reported decreases in basal level of glutamate in dialysate, which is in favour of the neuronal origin of basal extracellular glutamate. In contrast, other studies described no change or even increases under such experimental conditions [97]. Consequently, it was suggested that most part of neurotransmitter glutamate released into synaptic cleft in basal conditions may be taken up into surrounding glia and diffuses poorly to the dialysis probe. The same question can be asked for extracellular GABA because its metabolism is tightly linked to glutamate. As for glutamate, unsuccessful conclusions were also reported regarding the origin of dialysate GABA in basal conditions [97]. However, increasing the microdialysis sampling rate had been proposed in order to observe the rapid variations in glutamate extracellular level which are expected to occur in neurophysiological events [73, 98]. Recent studies using high sampling rate microdialysis showed that NMDA application [99] or electrical stimulation of the pre‐ frontal cortex [100] increases dialysate glutamate concentrations in brain regions receiving projections from this area and that the increase is suppressed or partly altered by TTX [99, 100]. These studies strongly suggest that the transient increase in dialysate glutamate detected under these conditions really represents evoked neurotransmitter glutamate release [101].

Extracellular (or dialysate) concentrations of glutamate and GABA may not always provide a reliable index of their synaptic exocytotic release. Indeed, a strict compartment between intrasynaptic and extra-synaptic was evidenced with poor spill-over from synaptic compartment to extra-synaptic part [102, 103], mainly due to a strong glial uptake. So that, the changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli should not be only interpreted as a consequence of the activation of specific neurochemical circuits, but as an expression of the activity of the neuron-astrocyte unit in specific circuits of the brain. Several authors proposed that dialysate changes in glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters. This hypothesis is based firstly on the assumption that the activity of neurons is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space [94, 104]; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of receptors located in the synapse [105, 106]; and thirdly, on the experimental evidence reporting specific electro‐ physiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space [107]. Thus, glutamate and GABA, once released into the extracellular compartment, can diffuse and have long-lasting effects modulating glutamatergic and/or GABAergic neuron-astrocytic networks and interact with neurons containing other neurotransmitters and located in the same areas of the brain. In conclusion, monitoring of glutamate and GABA concentrations in the extracellular space using microdialysis may provide an indirect index of amino acids synaptic neurotransmission while giving direct indications of amino acids volume neurotransmission [101].

A few studies have specifically measured extracellular glutamate or GABA in brain structures involved in fear conditioning, investigating the neurotransmitters changes in amygdala during auditory fear learning. More precisely, these microdialysis studies have described a longlasting decrease in GABA during expression of conditioned fear [39] and a small increase in glutamate during auditory fear conditioning [40]. However, these studies suffer from poor (10–20 min) temporal resolution associated with traditional microdialysis. Only one study has used high temporal resolution microdialysis to measured glutamate and GABA into the amygdala during the acquisition of auditory fear conditioning [108] and suggests that the covariations of glutamate and GABA may be explained as a glutamate-induced increase in GABA. This study also showed an increase in neurotransmitters levels in the amygdala only for the first association after which the concentration returned to baseline levels. This last result is in agreement with our microdialysis data for the amygdala in odor fear conditioning. Unfortunately, microdialysis was not performed in the auditory cortex, thus precluding any comparison with our own data on the piriform cortex. Nevertheless, there are some electro‐ physiological data which show that the amygdala is activated before the auditory cortex in auditory fear learning [9, 11]. These data are in accordance with our findings and confirm the differential activation of the amygdala and sensory cortices within the acquisition session of fear conditioning.

**Figure 7.** Functional hypotheses on the interactions between the amygdala and the piriform cortex during the acquis‐ ition of odor fear learning. An early transient response in the amygdala may allow fast signaling following the first CS-US pairing. Projections from the amygdala to the piriform cortex may provide a pathway through which the amygdala can modulate cortical processing of olfactory information and initiate the storage of the various attributes of the learned odor. The piriform cortex might store certain aspects of the conditioning experience, including the learned

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http://dx.doi.org/10.5772/57490

211

Our microdialysis data led us to formulate the following working hypothesis (Figure 7). During the first pairing, the amygdala glutamatergic transmission would allow the formation of the memory of the CS-US association as it is the case for auditory fear conditioning. The new idea brought by our data is that this could be initiated by a single trial, since no further increase in glutamate was detected in the amygdala for the next pairings. In parallel, the amygdala would trigger the first glutamate increase detected in the olfactory cortex. This would be achieved through the involvement of a direct projection pathway between the amygdala and the piriform cortex [82]. After that, the piriform cortex would progressively

build the memory of the different attributes of the learned odor across the next trials.

*What are the strengths of our data?* This work was aimed at studying the neurochemical inter‐ action between the amygdala and the piriform cortex in odor fear conditioning. We described a differential activation dynamics between the amygdala and the olfactory cortex within the acquisition session. This dynamic reveals an early and transient involvement of the amygdala, restricted to the first odor-shock association followed by the activation of the olfactory cortex

hedonic value of the CS odor.

High Temporal Resolution Brain Microdialysis as a Tool to Investigate the Dynamics of Interactions Between… http://dx.doi.org/10.5772/57490 211

Extracellular (or dialysate) concentrations of glutamate and GABA may not always provide a reliable index of their synaptic exocytotic release. Indeed, a strict compartment between intrasynaptic and extra-synaptic was evidenced with poor spill-over from synaptic compartment to extra-synaptic part [102, 103], mainly due to a strong glial uptake. So that, the changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli should not be only interpreted as a consequence of the activation of specific neurochemical circuits, but as an expression of the activity of the neuron-astrocyte unit in specific circuits of the brain. Several authors proposed that dialysate changes in glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters. This hypothesis is based firstly on the assumption that the activity of neurons is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space [94, 104]; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of receptors located in the synapse [105, 106]; and thirdly, on the experimental evidence reporting specific electro‐ physiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space [107]. Thus, glutamate and GABA, once released into the extracellular compartment, can diffuse and have long-lasting effects modulating glutamatergic and/or GABAergic neuron-astrocytic networks and interact with neurons containing other neurotransmitters and located in the same areas of the brain. In conclusion, monitoring of glutamate and GABA concentrations in the extracellular space using microdialysis may provide an indirect index of amino acids synaptic neurotransmission while giving direct

A few studies have specifically measured extracellular glutamate or GABA in brain structures involved in fear conditioning, investigating the neurotransmitters changes in amygdala during auditory fear learning. More precisely, these microdialysis studies have described a longlasting decrease in GABA during expression of conditioned fear [39] and a small increase in glutamate during auditory fear conditioning [40]. However, these studies suffer from poor (10–20 min) temporal resolution associated with traditional microdialysis. Only one study has used high temporal resolution microdialysis to measured glutamate and GABA into the amygdala during the acquisition of auditory fear conditioning [108] and suggests that the covariations of glutamate and GABA may be explained as a glutamate-induced increase in GABA. This study also showed an increase in neurotransmitters levels in the amygdala only for the first association after which the concentration returned to baseline levels. This last result is in agreement with our microdialysis data for the amygdala in odor fear conditioning. Unfortunately, microdialysis was not performed in the auditory cortex, thus precluding any comparison with our own data on the piriform cortex. Nevertheless, there are some electro‐ physiological data which show that the amygdala is activated before the auditory cortex in auditory fear learning [9, 11]. These data are in accordance with our findings and confirm the differential activation of the amygdala and sensory cortices within the acquisition session of

indications of amino acids volume neurotransmission [101].

fear conditioning.

210 Neurochemistry

**Figure 7.** Functional hypotheses on the interactions between the amygdala and the piriform cortex during the acquis‐ ition of odor fear learning. An early transient response in the amygdala may allow fast signaling following the first CS-US pairing. Projections from the amygdala to the piriform cortex may provide a pathway through which the amygdala can modulate cortical processing of olfactory information and initiate the storage of the various attributes of the learned odor. The piriform cortex might store certain aspects of the conditioning experience, including the learned hedonic value of the CS odor.

Our microdialysis data led us to formulate the following working hypothesis (Figure 7). During the first pairing, the amygdala glutamatergic transmission would allow the formation of the memory of the CS-US association as it is the case for auditory fear conditioning. The new idea brought by our data is that this could be initiated by a single trial, since no further increase in glutamate was detected in the amygdala for the next pairings. In parallel, the amygdala would trigger the first glutamate increase detected in the olfactory cortex. This would be achieved through the involvement of a direct projection pathway between the amygdala and the piriform cortex [82]. After that, the piriform cortex would progressively build the memory of the different attributes of the learned odor across the next trials.

*What are the strengths of our data?* This work was aimed at studying the neurochemical inter‐ action between the amygdala and the piriform cortex in odor fear conditioning. We described a differential activation dynamics between the amygdala and the olfactory cortex within the acquisition session. This dynamic reveals an early and transient involvement of the amygdala, restricted to the first odor-shock association followed by the activation of the olfactory cortex during the next associations, which persists until the end of the acquisition session. Until now, most studies questioning the role of the amygdala and sensory areas in fear conditioning have used local lesions or inactivation before or after the acquisition session. Our technical approach is one of the very few allowing online monitoring of neurochemical events occurring in parallel in the amygdala and olfactory cortex, using a temporal resolution compatible with the observation of very transient changes.

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## **4. Conclusion and perspectives**

The recent development of high sampling rate microdialysis coupled with high-performance separative microtechniques able to handle sub-microliter sample allows monitoring rapid changes in extracellular levels of amino acid neurotransmitters. Such a platform allows in vivo investigating neurotransmission in freely-moving animals learning fear as in odor fear conditioning. Using two microdialysis probes, we were able to study the neurochemical interactions between two major brain areas involved in the acquisition and consolidation of odor fear conditioning: the amygdala which role is crucial for the formation of fear memory and the piriform cortex which could encode other aspects of the aversive event. Our data suggest that projections from the amygdala to the piriform cortex might provide a pathway via which the amygdala could modulate the cortical processing of olfactory information and initiate the progressive storage of the different attributes of the learned odor in long-term memory.

#### **Author details**

Chloé Hegoburu1,2,4, Luc Denoroy3,4, Anne-Marie Mouly2,4 and Sandrine Parrot4,5

\*Address all correspondence to: sandrine.parrot@univ-lyon1.fr

1 Present address: Center for Psychiatric Neuroscience, Prilly, University of Lausanne, Lausanne, Switzerland

2 CNRS, UMR 5292, Lyon Neuroscience Research Center, Olfaction: From Coding to Memory Team, Lyon, France

3 University Lyon, Lyon Neuroscience Research Center, BioRaN team, Lyon, France

4 University Lyon , Lyon, France

5 INSERM, U1028, Lyon Neuroscience Research Center, NeuroDialyTics, Lyon, France

#### **References**

during the next associations, which persists until the end of the acquisition session. Until now, most studies questioning the role of the amygdala and sensory areas in fear conditioning have used local lesions or inactivation before or after the acquisition session. Our technical approach is one of the very few allowing online monitoring of neurochemical events occurring in parallel in the amygdala and olfactory cortex, using a temporal resolution compatible with the

The recent development of high sampling rate microdialysis coupled with high-performance separative microtechniques able to handle sub-microliter sample allows monitoring rapid changes in extracellular levels of amino acid neurotransmitters. Such a platform allows in vivo investigating neurotransmission in freely-moving animals learning fear as in odor fear conditioning. Using two microdialysis probes, we were able to study the neurochemical interactions between two major brain areas involved in the acquisition and consolidation of odor fear conditioning: the amygdala which role is crucial for the formation of fear memory and the piriform cortex which could encode other aspects of the aversive event. Our data suggest that projections from the amygdala to the piriform cortex might provide a pathway via which the amygdala could modulate the cortical processing of olfactory information and initiate the progressive storage of the different attributes of the learned odor in long-term

Chloé Hegoburu1,2,4, Luc Denoroy3,4, Anne-Marie Mouly2,4 and Sandrine Parrot4,5

1 Present address: Center for Psychiatric Neuroscience, Prilly, University of Lausanne,

3 University Lyon, Lyon Neuroscience Research Center, BioRaN team, Lyon, France

5 INSERM, U1028, Lyon Neuroscience Research Center, NeuroDialyTics, Lyon, France

2 CNRS, UMR 5292, Lyon Neuroscience Research Center, Olfaction: From Coding to Memory

\*Address all correspondence to: sandrine.parrot@univ-lyon1.fr

observation of very transient changes.

**4. Conclusion and perspectives**

memory.

212 Neurochemistry

**Author details**

Lausanne, Switzerland

Team, Lyon, France

4 University Lyon , Lyon, France


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**Section 3**

**Growth, Development and Differentiation**

**Growth, Development and Differentiation**

**Chapter 8**

**Participation of Neurochemical Signaling in Adult**

The revealed peculiarities of structural and neurochemical organization and description of basic histogenetic processes (proliferation, migration and neuronal cell differentiation) during the brain forming in fish, which have signs of fetal organization, widen the existing knowledge about histogenesis of these structures in postembryonic development. It seems conceivable, that during postembryonic development in teleost fishes some neurotransmitters and gaseous mediators (NO and H2S) act as factors, which initiate and regulate the cellular and the tissues processes of genetic program during the brain development. We suppose the presence of epigenetic control of adult neurogenesis in salmon brain via highly coordinated nonsynaptic cell–cell signaling. This communication engages the neurotransmitters GABA and dopamine whose extracellular concentrations depend on neuroblasts number and high affinity uptake systems in neural stem cells. Neuroblasts release GABA providing a negative feedback control of stem cell proliferation and instructing them on the size of the neuroblast pool. We suggest that in salmon brain exist strong control mechanisms of neuroblast production. The data provided by our study add to our general understanding, that peculiarities of distribution of classical neuromediators (GABA, catecholamines) and gasotransmitters (NO and H2S) are directly connected with ability of the fishes brain to grow during the animal entire life. We suggest, that some classical neuromediators (GABA, catecholamines) and gasotransmitters (NO and H2S) not only regulate functional activity of neurons and modulate synaptic trans‐ mission in mature neural networks, but also are regarded as inductors of the fishes brain development (morphogenetic factors) in postembryonic ontogenesis. We propose that dopamine and GABA act as homeostatic signals to regulate neuroblast production. This confirmation is proved by finding of the phenotypically immature elements, expressing the above mentioned molecules in proliferating brain areas, in the three-year-old salmon brain,

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Neurogenesis and Differentiation**

E.V. Pushchina, A.A. Varaksin and D.K. Obukhov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58306

**1. Introduction**
