Synaptic Functions of Astroglial Hemichannels

*Juan A. Orellana*

### **Abstract**

In recent decades, astrocytes have gained ground in their protagonist role at the synapses, challenging the old-historic idea that neurons are the unique functional units in the nervous system. Although for a long time considered merely supportive elements, astrocytes are now recognized as a source of gliotransmitter release that regulates synaptic transmission and plasticity. Despite the initial evidence that supported gliotransmission depends on intracellular Ca2+-mediated vesicular release, recent data indicate that hemichannels may constitute an alternative non-vesicular route for gliotransmitter efflux. These channels are plasma membrane channels formed by the oligomerization of six connexins around a central pore. Hemichannels are permeable to ions and signaling molecules—such as ATP, glutamate, and Ca2+—constituting a pathway of diffusional interchange between the cytoplasm and the extracellular milieu. Connexin 43 is the main hemichannelforming protein in astrocytes and is highly regulated under physiological and pathological conditions. In this chapter, the available data supporting the idea that hemichannels are chief components in tuning the synaptic gain in either resting or stimulated conditions is discussed.

**Keywords:** connexin 43, astrocyte, gliotransmission, brain, neuron

### **1. Introduction**

In order to ensure a proper response to external stimuli, organisms have created complex and coordinated neural structures that allow the sophisticated analysis of information. As the central nervous system (CNS) evolved from a basic network structure to compacted ganglia and centralized brains, two types of connections emerged as specialized structures favoring the integration of neural networks [1]. In 1897, Sherrington proposed the point of functional contact between neurons as the specific area at which transfer of information takes place and named it "synapsis," soon shortened to the "synapse," from the Greek word *sunáptō* (to clasp) [2]. This specialized structure is known today as the chemical synapse and transfers electrical information unidirectionally from presynaptic to postsynaptic neurons through the release of neurotransmitters, which, acting upon postsynaptic receptors, initiate a second electrical signal [1]. In the late 1950s, Furshpan and Potter reported a series of experiments revealing that synaptic transmission in the crayfish is bidirectional and voltage-dependent, two properties substantially out of range of the criteria established for chemical transmission [3]. This study revealed the pioneer evidence in favor of the existence of electrical synaptic transmission. Unlike chemical synapse, the electrical synapse permits the bidirectional

flow of ions between coupled neurons that come markedly close at intercellular specializations called gap junctions [4] (**Figure 1**). Nowadays, a growing body of evidence indicates that both mechanisms of synaptic transmission—chemical and electrical—are complementary and highly intermodulated to ensure proper brain development and function [1].

The traditional notion of neurons being the only functional elements in the synapse has been questioned with the finding that intracellular Ca2+ ([Ca2+]i) waves within and among astrocytes underlie the regenerative (nondissipative) transfer of biological signals [5–7]. Although astrocytes are not electrically silent cells [8], [Ca2+]i signals are their principal fast time-scale mechanism for allowing intra- and intercellular signaling [9]. These signals base their origin on the extracellular influx of Ca2+ via ion channels and through Ca2+ release from intracellular stores, resulting in [Ca2+]i transients that differ in frequency, kinetics, and spatial spread depending on the astroglial anatomical region [10]. Endowed with this machinery and along with pre- and postsynaptic neuronal elements, astrocytes embrace the "tripartite synapse"—the Rosetta stone of the chemical synaptic transmission—in where they sense neurotransmission and respond to it by releasing biomolecules that regulate neuronal activity called "gliotransmitters" (i.e., glutamate, D-serine, and ATP) [11]. Intracellular [Ca2+]i waves can spread among astrocytes to finally reach the terminal processes or "endfeet" of specialized astrocytes that contact the endothelium [12]. There, vasoactive molecules are released, permitting astrocytes to modulate the cerebral blood flow (CBF) and delivery of energy substances (i.e., glucose and lactate) with potentially significant consequences for neuronal firing and higher brain functions [13]. Indeed, a single astrocyte may contact over 100,000 synapses in rodents and up to 2,000,000 synapses in humans, revealing that they actually form a syncytium with multiple connections [14].

Nowadays, diverse mechanisms have been proposed to lead to gliotransmitter release (**Figure 1**), including Ca2+-dependent exocytosis [15–17], carrier membrane transport [18], and opening of a wide range of channels. Among the latter group, volume-regulated anion channels [19–21], P2X7 receptors [22–24], Ca2+-dependent Cl<sup>−</sup> channel bestrophin 1 [25, 26], and hemichannels [27–30] are included. This

### **Figure 1.**

*Basic structure of connexin-based channels. Connexins have four α-helical transmembrane domains connected by two extracellular loops and one cytoplasmic loop; both the amino- and carboxy-termini are intracellular. The relative positions of the extracellular loop cysteines (red balls) are also shown. Hemichannels (also known as connexons) are formed by the oligomerization of six subunit connexins around a central pore. Under resting conditions, hemichannels remain preferentially closed, but they may be activated by diverse physiological and pathological conditions and offer a diffuse transmembrane route between the intra- and extracellular milieu. Hemichannels dock each other to form functional cell-to-cell channels termed gap junction channels (right panel). Gap junction channels aggregate in well-known anatomical structures called gap junctions to facilitate the intercellular cytoplasmic exchange of metabolites, second messengers, and ions.*

**5**

[27, 28, 40–44].

**plasticity**

*Synaptic Functions of Astroglial Hemichannels DOI: http://dx.doi.org/10.5772/intechopen.87142*

**2. Structure and major functions of hemichannels**

mission and plasticity.

ATP, glutamate, D-serine, NAD+

stances (i.e., Ca2+ and glucose) [37].

chapter reviews and discusses recent data supporting a role for hemichannels as pathways for gliotransmission and relevant actors in that tuning of synaptic trans-

During the past decade, a growing body of evidence began to support a novel mechanism of autocrine/paracrine communication underlying gliotransmission and astrocyte-to-neuron communication: hemichannel-mediated signaling [31]. Each hemichannel is composed of the oligomerization of six protein subunits called connexins around a central pore (**Figure 1**). Connexins embrace a highly conserved protein family encoded by 21 genes in humans and 20 in mice, with orthologs in other vertebrate species [32]. These proteins are abundantly expressed in brain cells [33], including astrocytes [34], and they are named after their predicted molecular mass expressed in kDa, for instance, connexin 43 (Cx43) has a molecular mass of ~43 kDa [35]. For several years, the key function attributed to hemichannels was to constitute the building blocks of the gap junction channels, which are intercellular channels that allow the direct cytoplasmic exchange between contacting cells [35]. Nonetheless, in the 1990s, pioneering findings by Paul and colleagues revealed the presence of functional and solitary hemichannels in "nonjunctional" membranes [36]. Today, it is well accepted that these channels act like aqueous pores, providing a diffusional route of exchange for ions and molecules between the intra- and extracellular space [37]. Across the different tissues, hemichannels allow the cellular release of relevant quantities of autocrine and paracrine signaling molecules (e.g.,

Since their discovery, hemichannels have been linked with cellular damage. This idea came from early studies suggesting that osmotic and ionic imbalances induced by the uncontrolled influx of Na2+ and Cl<sup>−</sup> through hemichannels could result in further cell swelling and plasma membrane breakdown [36]. In addition, it has been proposed that because hemichannels are permeable to Ca2+, their uncontrolled opening could lead to Ca2+ overload and the consequent production of free radicals, lipid peroxidation, and plasma membrane damage [38]. Alternatively, exacerbated hemichannel activity could also induce the release of molecules that at high concentration may be toxic for neighboring cells, such as glutamate, in the case of the CNS [39]. Despite the above, in the last decade, a substantial body of studies has proposed that hemichannels may underpin pivotal neurophysiological functions, such as synaptic efficacy, neural activity, signal processing, cognition, and behavior

**3. Astroglial hemichannels and their role in synaptic transmission and** 

Although rat, mouse, and human astrocytes express abundantly Cx30 and Cx43, as well as Cx26 [45–49], at the moment, Cx43 is the only connexin probed to form functional hemichannels in astrocytes [50]. The opening of astroglial Cx43 hemichannels has been linked with the release of different gliotransmitters (e.g., glutamate, ATP, D-serine, lactate), as well as with the influx of extracellular Ca2+ and glucose. Seminal studies by Torres and colleagues demonstrated for the first time that astrocyte hemichannels may act as both sensors and modulators of synaptic

, and PGE2), as well as the influx of other sub-

*Glia in Health and Disease*

development and function [1].

form a syncytium with multiple connections [14].

flow of ions between coupled neurons that come markedly close at intercellular specializations called gap junctions [4] (**Figure 1**). Nowadays, a growing body of evidence indicates that both mechanisms of synaptic transmission—chemical and electrical—are complementary and highly intermodulated to ensure proper brain

The traditional notion of neurons being the only functional elements in the synapse has been questioned with the finding that intracellular Ca2+ ([Ca2+]i) waves within and among astrocytes underlie the regenerative (nondissipative) transfer of biological signals [5–7]. Although astrocytes are not electrically silent cells [8], [Ca2+]i signals are their principal fast time-scale mechanism for allowing intra- and intercellular signaling [9]. These signals base their origin on the extracellular influx of Ca2+ via ion channels and through Ca2+ release from intracellular stores, resulting in [Ca2+]i transients that differ in frequency, kinetics, and spatial spread depending on the astroglial anatomical region [10]. Endowed with this machinery and along with pre- and postsynaptic neuronal elements, astrocytes embrace the "tripartite synapse"—the Rosetta stone of the chemical synaptic transmission—in where they sense neurotransmission and respond to it by releasing biomolecules that regulate neuronal activity called "gliotransmitters" (i.e., glutamate, D-serine, and ATP) [11]. Intracellular [Ca2+]i waves can spread among astrocytes to finally reach the terminal processes or "endfeet" of specialized astrocytes that contact the endothelium [12]. There, vasoactive molecules are released, permitting astrocytes to modulate the cerebral blood flow (CBF) and delivery of energy substances (i.e., glucose and lactate) with potentially significant consequences for neuronal firing and higher brain functions [13]. Indeed, a single astrocyte may contact over 100,000 synapses in rodents and up to 2,000,000 synapses in humans, revealing that they actually

Nowadays, diverse mechanisms have been proposed to lead to gliotransmitter release (**Figure 1**), including Ca2+-dependent exocytosis [15–17], carrier membrane transport [18], and opening of a wide range of channels. Among the latter group, volume-regulated anion channels [19–21], P2X7 receptors [22–24], Ca2+-dependent Cl<sup>−</sup> channel bestrophin 1 [25, 26], and hemichannels [27–30] are included. This

*Basic structure of connexin-based channels. Connexins have four α-helical transmembrane domains connected by two extracellular loops and one cytoplasmic loop; both the amino- and carboxy-termini are intracellular. The relative positions of the extracellular loop cysteines (red balls) are also shown. Hemichannels (also known as connexons) are formed by the oligomerization of six subunit connexins around a central pore. Under resting conditions, hemichannels remain preferentially closed, but they may be activated by diverse physiological and pathological conditions and offer a diffuse transmembrane route between the intra- and extracellular milieu. Hemichannels dock each other to form functional cell-to-cell channels termed gap junction channels (right panel). Gap junction channels aggregate in well-known anatomical structures called gap junctions to facilitate* 

*the intercellular cytoplasmic exchange of metabolites, second messengers, and ions.*

**4**

**Figure 1.**

chapter reviews and discusses recent data supporting a role for hemichannels as pathways for gliotransmission and relevant actors in that tuning of synaptic transmission and plasticity.

### **2. Structure and major functions of hemichannels**

During the past decade, a growing body of evidence began to support a novel mechanism of autocrine/paracrine communication underlying gliotransmission and astrocyte-to-neuron communication: hemichannel-mediated signaling [31]. Each hemichannel is composed of the oligomerization of six protein subunits called connexins around a central pore (**Figure 1**). Connexins embrace a highly conserved protein family encoded by 21 genes in humans and 20 in mice, with orthologs in other vertebrate species [32]. These proteins are abundantly expressed in brain cells [33], including astrocytes [34], and they are named after their predicted molecular mass expressed in kDa, for instance, connexin 43 (Cx43) has a molecular mass of ~43 kDa [35]. For several years, the key function attributed to hemichannels was to constitute the building blocks of the gap junction channels, which are intercellular channels that allow the direct cytoplasmic exchange between contacting cells [35]. Nonetheless, in the 1990s, pioneering findings by Paul and colleagues revealed the presence of functional and solitary hemichannels in "nonjunctional" membranes [36]. Today, it is well accepted that these channels act like aqueous pores, providing a diffusional route of exchange for ions and molecules between the intra- and extracellular space [37]. Across the different tissues, hemichannels allow the cellular release of relevant quantities of autocrine and paracrine signaling molecules (e.g., ATP, glutamate, D-serine, NAD+ , and PGE2), as well as the influx of other substances (i.e., Ca2+ and glucose) [37].

Since their discovery, hemichannels have been linked with cellular damage. This idea came from early studies suggesting that osmotic and ionic imbalances induced by the uncontrolled influx of Na2+ and Cl<sup>−</sup> through hemichannels could result in further cell swelling and plasma membrane breakdown [36]. In addition, it has been proposed that because hemichannels are permeable to Ca2+, their uncontrolled opening could lead to Ca2+ overload and the consequent production of free radicals, lipid peroxidation, and plasma membrane damage [38]. Alternatively, exacerbated hemichannel activity could also induce the release of molecules that at high concentration may be toxic for neighboring cells, such as glutamate, in the case of the CNS [39]. Despite the above, in the last decade, a substantial body of studies has proposed that hemichannels may underpin pivotal neurophysiological functions, such as synaptic efficacy, neural activity, signal processing, cognition, and behavior [27, 28, 40–44].

### **3. Astroglial hemichannels and their role in synaptic transmission and plasticity**

Although rat, mouse, and human astrocytes express abundantly Cx30 and Cx43, as well as Cx26 [45–49], at the moment, Cx43 is the only connexin probed to form functional hemichannels in astrocytes [50]. The opening of astroglial Cx43 hemichannels has been linked with the release of different gliotransmitters (e.g., glutamate, ATP, D-serine, lactate), as well as with the influx of extracellular Ca2+ and glucose. Seminal studies by Torres and colleagues demonstrated for the first time that astrocyte hemichannels may act as both sensors and modulators of synaptic

activity [43]. Using UV-photolysis of caged MNI-glutamate in hippocampal slices, they found that specific deletion of Cx43 abrogates ATP-dependent spreading of slow Ca2+ waves among astrocytes. Furthermore, these slow Ca2+ waves were potentiated when authors used slices from transgenic mice with an astrocyte-targeted point mutation (Cx43G138R) that leads to an increased Cx43 hemichannel opening [51]. In addition, they observed that depolarization of inhibitory interneurons from the stratum radiatum reduced CA1 excitatory transmission via the astroglial Cx43 hemichannel-mediated release of ATP and subsequent stimulation of interneuronal P2Y1 receptors [43]. These data shed light for the first time about how astrocyte Cx43 hemichannels may underpin a negative feedback mechanism elicited during sustained excitation to prevent excitotoxicity (**Figure 2**).

Although in normal astrocytes few Cx43 hemichannels are in the plasma membrane and most of them with a low open probability, recent findings have described that they facilitate the release of ATP under basal conditions [27, 41]. Chever and co-workers observed that basal release of ATP via astroglial Cx43 hemichannels is enough to boost the CA1 synaptic transmission triggered by stimulation of Schaffer collaterals, an effect mediated by purinergic receptors [27] (**Figure 2**). Likely the insertion of postsynaptic AMPA receptors as a result of the activation of P2X7 receptors could explain the ATP-dependent potentiation of glutamatergic transmission, as reported before in other brain areas [52]. Astroglial hemichannels also have been found to regulate neuronal activity in the olfactory bulb (OB) [41]. There, the group of Giaume demonstrated that pharmacological inhibition of Cx43 hemichannels decreased the firing and amplitude of depolarized states in mitral cells. Similar findings were observed in mitral cells of OB slices with specific astroglial deletion of Cx43 [41] or in slices treated with A1 adenosine receptor antagonists. These findings denote that likely astrocyte Cx43 hemichannels enhance the amplitude of depolarized states of mitral cells through the release of ATP and its further breakdown to

### **Figure 2.**

*Possible role of astroglial hemichannels in hippocampal synaptic transmission. During the basal firing of glutamatergic neurons in the hippocampus, Ca2+ influx into neurons results in a localized reduction in [Ca2+]e, which in turn opens Cx43 hemichannels (HCs) on astrocytes [43]. The latter lead to the release of ATP, being this crucial for sustaining basal excitatory synaptic transmission [27]. Likely this phenomenon takes place via the activation of P2X7 receptors and further insertion of AMPA receptors in postsynaptic terminals [52]. Alternatively, the conversion of ATP to ADP could depolarize and augment firing in interneurons via P2Y1 receptors, therefore, enhancing inhibitory transmission [43].*

**7**

**Figure 4.**

**Figure 3.**

*olfactory bulb.*

*Synaptic Functions of Astroglial Hemichannels DOI: http://dx.doi.org/10.5772/intechopen.87142*

rons, as occurred in other brain areas [54] (**Figure 3**).

adenosine (**Figure 3**). Usually, A1 receptors induce the presynaptic inhibition of glutamate release, reduced postsynaptic NMDAR activation, and decreased Ca2+ influx [53]. Therefore, it is possible that the adenosine-mediated enhancement of depolarized states is due to the suppression of inhibitory juxtaglomerular interneu-

*Implications of astroglial hemichannel activity in neuronal oscillations of the olfactory bulb. Spontaneous neuronal activity in the glomerular layer of the olfactory bulb requires glutamatergic transmission. In this scenario, astrocytes display a basal release of ATP via Cx43 hemichannels (HCs) [41]. The adenosine derived from ATP may reduce the activity of GABAergic inhibitory juxtaglomerular neurons through the stimulation of A1 adenosine receptors. This permits the basal slow oscillations of up and down states of mitral cells in the* 

*Astroglial hemichannels and their impact on synaptic plasticity in the prefrontal cortex. In the prefrontal cortex, continuous stimulation of layer 2/3 neurons induces long-term potentiation (LTP) of NMDA and AMPA receptor currents in layer 5 pyramidal neurons. In this context, [Ca2+]i is needed for the opening of Cx43 hemichannels (HCs) in astrocytes [28], which cause release of D-serine. This gliotransmitter facilitates* 

*LTP of NMDA and AMPA excitatory synaptic currents mediated by high-frequency stimulation.*

Astroglial Cx43 hemichannels have been involved not only in synaptic function and transmission but also in synaptic plasticity. High-frequency stimulation (HFS)

### *Synaptic Functions of Astroglial Hemichannels DOI: http://dx.doi.org/10.5772/intechopen.87142*

adenosine (**Figure 3**). Usually, A1 receptors induce the presynaptic inhibition of glutamate release, reduced postsynaptic NMDAR activation, and decreased Ca2+ influx [53]. Therefore, it is possible that the adenosine-mediated enhancement of depolarized states is due to the suppression of inhibitory juxtaglomerular interneurons, as occurred in other brain areas [54] (**Figure 3**).

Astroglial Cx43 hemichannels have been involved not only in synaptic function and transmission but also in synaptic plasticity. High-frequency stimulation (HFS)

### **Figure 3.**

*Glia in Health and Disease*

activity [43]. Using UV-photolysis of caged MNI-glutamate in hippocampal slices, they found that specific deletion of Cx43 abrogates ATP-dependent spreading of slow Ca2+ waves among astrocytes. Furthermore, these slow Ca2+ waves were potentiated when authors used slices from transgenic mice with an astrocyte-targeted point mutation (Cx43G138R) that leads to an increased Cx43 hemichannel opening [51]. In addition, they observed that depolarization of inhibitory interneurons from the stratum radiatum reduced CA1 excitatory transmission via the astroglial Cx43 hemichannel-mediated release of ATP and subsequent stimulation of interneuronal P2Y1 receptors [43]. These data shed light for the first time about how astrocyte Cx43 hemichannels may underpin a negative feedback mechanism elicited during

Although in normal astrocytes few Cx43 hemichannels are in the plasma membrane and most of them with a low open probability, recent findings have described that they facilitate the release of ATP under basal conditions [27, 41]. Chever and co-workers observed that basal release of ATP via astroglial Cx43 hemichannels is enough to boost the CA1 synaptic transmission triggered by stimulation of Schaffer collaterals, an effect mediated by purinergic receptors [27] (**Figure 2**). Likely the insertion of postsynaptic AMPA receptors as a result of the activation of P2X7 receptors could explain the ATP-dependent potentiation of glutamatergic transmission, as reported before in other brain areas [52]. Astroglial hemichannels also have been found to regulate neuronal activity in the olfactory bulb (OB) [41]. There, the group of Giaume demonstrated that pharmacological inhibition of Cx43 hemichannels decreased the firing and amplitude of depolarized states in mitral cells. Similar findings were observed in mitral cells of OB slices with specific astroglial deletion of Cx43 [41] or in slices treated with A1 adenosine receptor antagonists. These findings denote that likely astrocyte Cx43 hemichannels enhance the amplitude of depolarized states of mitral cells through the release of ATP and its further breakdown to

*Possible role of astroglial hemichannels in hippocampal synaptic transmission. During the basal firing of glutamatergic neurons in the hippocampus, Ca2+ influx into neurons results in a localized reduction in [Ca2+]e, which in turn opens Cx43 hemichannels (HCs) on astrocytes [43]. The latter lead to the release of ATP, being this crucial for sustaining basal excitatory synaptic transmission [27]. Likely this phenomenon takes place via the activation of P2X7 receptors and further insertion of AMPA receptors in postsynaptic terminals [52]. Alternatively, the conversion of ATP to ADP could depolarize and augment firing in interneurons via P2Y1* 

*receptors, therefore, enhancing inhibitory transmission [43].*

sustained excitation to prevent excitotoxicity (**Figure 2**).

**6**

**Figure 2.**

*Implications of astroglial hemichannel activity in neuronal oscillations of the olfactory bulb. Spontaneous neuronal activity in the glomerular layer of the olfactory bulb requires glutamatergic transmission. In this scenario, astrocytes display a basal release of ATP via Cx43 hemichannels (HCs) [41]. The adenosine derived from ATP may reduce the activity of GABAergic inhibitory juxtaglomerular neurons through the stimulation of A1 adenosine receptors. This permits the basal slow oscillations of up and down states of mitral cells in the olfactory bulb.*

### **Figure 4.**

*Astroglial hemichannels and their impact on synaptic plasticity in the prefrontal cortex. In the prefrontal cortex, continuous stimulation of layer 2/3 neurons induces long-term potentiation (LTP) of NMDA and AMPA receptor currents in layer 5 pyramidal neurons. In this context, [Ca2+]i is needed for the opening of Cx43 hemichannels (HCs) in astrocytes [28], which cause release of D-serine. This gliotransmitter facilitates LTP of NMDA and AMPA excitatory synaptic currents mediated by high-frequency stimulation.*

of neuronal layer 2/3 (L2/3) triggers glutamatergic synaptic transmission in pyramidal cells at layer 5 (L5) of the prefrontal cortex (PFC) [55]. In this context and using PFC slices, Meunier and colleagues observed that genetic ablation of Cx43 or inhibition of Cx43 hemichannels strongly counteracts the NMDAR-dependent excitatory postsynaptic currents (EPSCs) and increases AMPA/NMDA current ratio induced by HSF in L5 [28]. Relevantly, the latter responses did not occur when D-serine was added at the recording media, revealing that the release of D-serine and astroglial hemichannel function are linked and modulate NMDAR-dependent synaptic transmission in PFC pyramidal cells. Furthermore, when [Ca2+]i was clamped or D-serine production was inhibited in the L5 astroglial network, HFS failed to potentiate the NMDAR-dependent synaptic currents [28] (**Figure 4**). Accordingly, the authors hypothesized that potentiation of glutamatergic transmission at the PFC relies on [Ca2+]i-mediated opening of astroglial Cx43 hemichannels and the further release of D-serine (**Figure 4**).

The impact of astroglial hemichannels on synaptic transmission and plasticity has a subsequent echo on higher brain function and behavior. Indeed, in vivo inhibition of Cx43 hemichannels at the basolateral amygdala causes transitory and specific amnesia for auditory fear conditioning [42]. Remarkably, learning capacity was restored by the co-administration of a cocktail of supposed gliotransmitters (lactate, glutamate, D-serine, glutamine, glycine, and ATP), evidencing for the first time a physiological involvement for astroglial Cx43 hemichannels in higher brain function. In the same line, a recent study found that intraventricular administration of Gap19, a specific Cx43 hemichannel blocker [56], significantly impairs the spatial short-term memory, as assayed with the delayed spontaneous alternation Y maze task [44].

### **4. Conclusions**

The impact of functional astroglial hemichannels in synaptic transmission and plasticity may depend on the number of channels available in the plasma membrane, their open probability, and their conductance and/or selectivity. Of particular relevance is to disentangle how synaptic function is modulated by regulations in gating properties of astroglial hemichannels, as well as changes in their trafficking or de novo synthesis. Elucidating the latter will allow us to understand whether hemichannel opening in astrocytes tunes the temporal outcome for sculpting either short-term (milliseconds to a few minutes) or long-term (minutes to hours) plasticity in the nervous system. One point of concern is the urgent need of developing new molecular and pharmacological tools to specifically dissect the contribution of astroglial hemichannels to the function of neural networks without affecting other hemichannel-forming proteins in other brain cells (e.g., microglia, oligodendrocytes, and endothelial cells). Finally, although growing evidence in ex vivo preparations has extended our knowledge about the role of astroglial hemichannels in gliotransmission, additional data are necessary to demonstrate whether this truly occurs in vivo.

### **Acknowledgements**

This work was supported by (i) the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 1160710 (to JAO) and (ii) the CONICYT and Programa de Investigación Asociativa (PIA) Grant Anillo de Ciencia y Tecnología

**9**

**Author details**

Juan A. Orellana

Santiago, Chile

\*Address all correspondence to: jaorella@uc.cl

provided the original work is properly cited.

*Synaptic Functions of Astroglial Hemichannels DOI: http://dx.doi.org/10.5772/intechopen.87142*

The author declares no conflict of interest.

**Conflict of interest**

ACT1411 (to JAO). The author did part of the schematics with support of the free

Departamento de Neurología, Escuela de Medicina and Centro Interdisciplinario de Neurociencias, Facultad de Medicina, Pontificia Universidad Católica de Chile,

© 2019 The Author(s). Licensee IntechOpen. 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,

online Servier Medical Art repository (https://smart.servier.com/).

ACT1411 (to JAO). The author did part of the schematics with support of the free online Servier Medical Art repository (https://smart.servier.com/).

## **Conflict of interest**

*Glia in Health and Disease*

and the further release of D-serine (**Figure 4**).

of neuronal layer 2/3 (L2/3) triggers glutamatergic synaptic transmission in pyramidal cells at layer 5 (L5) of the prefrontal cortex (PFC) [55]. In this context and using PFC slices, Meunier and colleagues observed that genetic ablation of Cx43 or inhibition of Cx43 hemichannels strongly counteracts the NMDAR-dependent excitatory postsynaptic currents (EPSCs) and increases AMPA/NMDA current ratio induced by HSF in L5 [28]. Relevantly, the latter responses did not occur when D-serine was added at the recording media, revealing that the release of D-serine and astroglial hemichannel function are linked and modulate NMDAR-dependent synaptic transmission in PFC pyramidal cells. Furthermore, when [Ca2+]i was clamped or D-serine production was inhibited in the L5 astroglial network, HFS failed to potentiate the NMDAR-dependent synaptic currents [28] (**Figure 4**). Accordingly, the authors hypothesized that potentiation of glutamatergic transmission at the PFC relies on [Ca2+]i-mediated opening of astroglial Cx43 hemichannels

The impact of astroglial hemichannels on synaptic transmission and plasticity has a subsequent echo on higher brain function and behavior. Indeed, in vivo inhibition of Cx43 hemichannels at the basolateral amygdala causes transitory and specific amnesia for auditory fear conditioning [42]. Remarkably, learning capacity was restored by the co-administration of a cocktail of supposed gliotransmitters (lactate, glutamate, D-serine, glutamine, glycine, and ATP), evidencing for the first time a physiological involvement for astroglial Cx43 hemichannels in higher brain function. In the same line, a recent study found that intraventricular administration of Gap19, a specific Cx43 hemichannel blocker [56], significantly impairs the spatial short-term memory, as assayed with the delayed spontaneous alternation Y maze

The impact of functional astroglial hemichannels in synaptic transmission and plasticity may depend on the number of channels available in the plasma membrane, their open probability, and their conductance and/or selectivity. Of particular relevance is to disentangle how synaptic function is modulated by regulations in gating properties of astroglial hemichannels, as well as changes in their trafficking or de novo synthesis. Elucidating the latter will allow us to understand whether hemichannel opening in astrocytes tunes the temporal outcome for sculpting either short-term (milliseconds to a few minutes) or long-term (minutes to hours) plasticity in the nervous system. One point of concern is the urgent need of developing new molecular and pharmacological tools to specifically dissect the contribution of astroglial hemichannels to the function of neural networks without affecting other hemichannel-forming proteins in other brain cells (e.g., microglia, oligodendrocytes, and endothelial cells). Finally, although growing evidence in ex vivo preparations has extended our knowledge about the role of astroglial hemichannels in gliotransmission, additional data are necessary to demonstrate whether this truly

This work was supported by (i) the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 1160710 (to JAO) and (ii) the CONICYT and Programa de Investigación Asociativa (PIA) Grant Anillo de Ciencia y Tecnología

**8**

task [44].

**4. Conclusions**

occurs in vivo.

**Acknowledgements**

The author declares no conflict of interest.

## **Author details**

Juan A. Orellana Departamento de Neurología, Escuela de Medicina and Centro Interdisciplinario de Neurociencias, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

\*Address all correspondence to: jaorella@uc.cl

© 2019 The Author(s). Licensee IntechOpen. 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.

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[11] Perea G, Navarrete M, Araque A. Tripartite synapses: Astrocytes process and control synaptic information. Trends in Neurosciences. 2009;**32**(8):421-431. Epub 2009/07/21

[12] Allaman I, Belanger M, Magistretti PJ. Astrocyte-neuron metabolic relationships: For better and for worse. Trends in Neurosciences. 2011;**34**(2):76-87

[13] Weber B, Barros LF. The astrocyte: Powerhouse and recycling center. Cold Spring Harbor Perspectives in Biology. 2015;**7**(12):a020396

[14] Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, et al. Uniquely hominid features of adult human astrocytes. The Journal of Neuroscience. 2009;**29**(10):3276-3287. Epub 2009/03/13

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[17] Imura Y, Morizawa Y, Komatsu R, Shibata K, Shinozaki Y, Kasai H, et al. Microglia release ATP by exocytosis. Glia. 2013;**61**(8):1320-1330. Epub 2013/07/05

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Nature. 2000;**403**(6767):316-321. Epub 2000/02/05

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[32] Leybaert L, Lampe PD, Dhein S, Kwak BR, Ferdinandy P, Beyer EC, et al. Connexins in cardiovascular and neurovascular health and disease: Pharmacological implications.

**10**

*Glia in Health and Disease*

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[42] Stehberg J, Moraga-Amaro R, Salazar C, Becerra A, Echeverria C, Orellana JA, et al. Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala. The FASEB Journal. 2012;**26**(9):3649-3657. Epub

[43] Torres A, Wang F, Xu Q, Fujita T, Dobrowolski R, Willecke K, et al. Extracellular Ca2+ acts as a mediator of communication from neurons to glia. Science Signaling. 2012;**5**(208):ra8

[44] Walrave L, Vinken M, Albertini

Smolders IJ. Inhibition of connexin43 hemichannels impairs spatial shortterm memory without affecting spatial working memory. Frontiers in Cellular Neuroscience. 2016;**10**:288. Epub

G, De Bundel D, Leybaert L,

[45] Dermietzel R, Hertberg EL, Kessler JA, Spray DC. Gap junctions between cultured astrocytes: Immunocytochemical, molecular, and electrophysiological analysis. The Journal of Neuroscience.

[46] Giaume C, Koulakoff A, Roux L, Holcman D, Rouach N. Astroglial networks: A step further in neuroglial and gliovascular interactions. Nature Reviews. Neuroscience. 2010;**11**(2):87-

1991;**11**(5):1421-1432

99. Epub 2010/01/21

20):3921-3931

2015/11/21

2012/06/06

2017/01/10

[34] De Bock M, Decrock E, Wang N, Bol M, Vinken M, Bultynck G, et al. The dual face of connexin-based astroglial Ca2+ communication: A key player in brain physiology and a prime target in pathology. Biochimica et Biophysica Acta. 2014;**1843**(10):2211-2232

[35] Sáez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: Their regulation and functions. Physiological Reviews. 2003;**83**(4):1359-1400. Epub

[36] Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA. Connexin46, a novel lens gap junction protein, induces voltagegated currents in nonjunctional plasma membrane of *Xenopus* oocytes. The Journal of Cell Biology.

[37] Saez JC, Leybaert L. Hunting for connexin hemichannels. FEBS Letters. 2014;**588**(8):1205-1211. Epub

[38] Orellana JA, von Bernhardi R, Giaume C, Saez JC. Glial hemichannels and their involvement in aging and neurodegenerative diseases. Reviews in the Neurosciences. 2012;**23**(2):163-177.

[39] Orellana JA, Retamal MA, Moraga-Amaro R, Stehberg J. Role of astroglial hemichannels and pannexons in memory and neurodegenerative diseases. Frontiers in Integrative Neuroscience. 2016;**10**:26. Epub

1991;**115**(4):1077-1089

2003/09/25

2014/03/19

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Letters. 2014;**588**(8):1259-1270

**12**

2016/08/05

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[48] Nagy JI, Patel D, Ochalski PA, Stelmack GL. Connexin30 in rodent, cat and human brain: Selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance. Neuroscience. 1999;**88**(2):447-468. Epub 1999/04/10

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[52] Gordon GR, Baimoukhametova DV, Hewitt SA, Rajapaksha WR, Fisher TE, Bains JS. Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy. Nature Neuroscience. 2005;**8**(8):1078-1086. Epub 2005/07/05

[53] Benarroch EE. Adenosine and its receptors: Multiple modulatory functions and potential therapeutic targets for neurologic disease. Neurology. 2008;**70**(3):231-236. Epub 2008/01/16

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**15**

**Chapter 2**

**Abstract**

tight junctions

**1. Introduction**

Diseases

Astrocytic S100B, Blood-Brain

Barrier and Neurodegenerative

*Anuradha Krishnan, Hao Wu and Venkat Venkataraman*

Increased life span and expectations of a better quality of life have resulted in a spotlight on neurodegenerative and cardiovascular diseases generally associated with aging. The drive toward evidence-based medicine has necessitated a constant search for objective biomarkers to assay disease onset, progress, and outcomes to make the best clinical decisions. Enhancement of their use depends on the mechanistic understanding of the biomarker's role in the disease process itself. This chapter focuses on S100B. It is a calcium sensor protein that is primarily astrocytic. While it plays a complex, interlinked role in signaling, serum levels of S100B as a biomarker for clinical decisions is also an area of intense investigation. Both aspects are presented, with an emphasis on the role of S100B in in maintaining a bloodbrain barrier, especially in the context of suggesting a unified mechanism for the

**Keywords:** S100B, calcium, blood-brain barrier, biomarker, neurodegeneration,

Rudolph Virchow first proposed the concept of neuroglia as a component of the connective tissue of the brain "nervekitt" [1]. The term "astrocyte" is attributed to Michael von Lenhosseck, coined to denote the stellate (star-like) morphology, with independent contributions also by Kolliker and Anderiezen (reviewed in [2]). The diversity of this group of cells was brought into clear focus by the excellent drawings by Cajal [3]. Glial cells, including astrocytes, were once believed to be limited to passive support in the functioning of the brain. Work over the last few decades has ushered in the understanding that they actively participate in normal metabolism and physiology of the brain, even more so during injury response and repair. They alter the microenvironment through secretion of a variety of signals including cytokines as a result of intracellular process collectively termed "activation," which operates at both ends of time scale—acute and short-term (trauma) as well as chronic and long-term (neurodegenerative diseases). While meant to be adaptive and reparative, they could also lead to exacerbation of injury or disease (for some reviews, please see [4–12]). Understanding the process of activation and its effect on the microenvironment is fundamental to devising positive interventions. One of the important signaling molecules involved in this process is S100B, a calcium sensor protein, which is secreted to act at the extracellular level but also

onset and progression of neurodegenerative diseases.

### **Chapter 2**
