Role of Astroglial Hemichannels and Pannexons in Memory and Neurodegenerative Diseases

Under physiological conditions, astroglial hemichannels and pannexons allow the release of gliotransmitters from astrocytes. These gliotransmitters are critical in modulating synaptic transmission, plasticity and memory. However, recent evidence suggests that under pathological conditions, they may be central in the development of various neurodegenerative diseases. Here we review current literature on the role of astroglial hemichannels and pannexons in memory, stress and the development of neurodegenerative diseases, and propose that they are not only crucial for normal brain function, including memory, but also a potential target for the treatment of neurodegenerative diseases.

Embedded in the ''tripartite synapse'', astrocytes express a plethora of receptors (reviewed in Moraga-Amaro et al., 2014) and respond locally to neurotransmitters through the above mentioned mechanisms of gliotransmitter release, including the activation of hemichannels and pannexons (Malarkey and Parpura, 2008). Indeed, several gliotransmitters such as Dserine, glutamate, ATP and lactate have been reported to be released via astrocytic hemichannels (Stout et al., 2002;Ye et al., 2003;Karagiannis et al., 2015) or pannexons (Iglesias et al., 2009;Pan et al., 2015) in vitro. This gliotransmitter release has been proposed to be necessary for different CNS functions in vivo (Orellana and Stehberg, 2014;Montero and Orellana, 2015). Other in vitro studies have reported Cx43 hemichannels to be permeable to NAD + (Bruzzone et al., 2001), glucose (Retamal et al., 2007), taurine (Stridh et al., 2008), and Ca 2+ (Schalper et al., 2010). Moreover, given that GJCs have been shown to allow for the passage of small peptides with a molecular weight of up to 1.8 kDa (Neijssen et al., 2005) and short interfering RNAs (Valiunas et al., 2005), it is possible that hemichannels may also allow the passage of such molecules, hypothesis that has not been tested so far.
Most early studies on hemichannels were performed in vitro, and suggested that Cx43 hemichannels have a low open probability in physiological conditions, requiring depolarized membrane potentials as high as +60 mV. Given that astrocytes are considered non-excitable cells in terms of membrane potential, their opening under physiological conditions was considered virtually impossible. However, later studies showed hemichannel opening at negative membrane potentials as measured by hemichannel-mediated dye uptake and ionic currents (Contreras et al., 2003;Retamal et al., 2007).
Recent in vitro studies have shown that astroglial Cx43 hemichannel activity changes in response to general anesthetics  antidepressants (Jeanson et al., 2015) and modafinil (Duchêne et al., 2016), suggesting that they may also be drug targets.

EVIDENCE FOR ASTROGLIAL HEMICHANNEL FUNCTION IN VIVO
The evidence of a role for astroglial hemichannels and pannexons in vivo in astroglial physiology and CNS function is much more limited, and is only now beginning to emerge. A recent study reported that astroglial Cx43 hemichannels are active in hippocampal slices during basal conditions and that astroglial Cx43 hemichannel-dependent release of ATP increases basal excitatory (glutamatergic) synaptic transmission through P2 receptors (Chever et al., 2014). Similar results were reported in neurons that project to the vagal nerve .
Astroglial Cx43 hemichannel opening may also contribute to neuronal oscillations. Roux et al. (2015) reported that astroglial Cx43 hemichannel opening in olfactory bulb slices increases the amplitude of slow oscillations in mitral cells, affecting their firing rate. Hemichannel activity is also necessary for maintaining spontaneous activity in the cortex during development (Moore et al., 2014). It remains unknown whether hemichannels are still relevant for spontaneous activity in the adult cortex.
Yet another example of the role of astroglial hemichannels in CNS function measured in vivo can be found in the retrotrapezoid nucleus, in which the firing rate of CO 2 /H +sensitive neurons acting as chemoreceptors (Wenker et al., 2012;Reyes et al., 2014) is modulated by ATP release from astrocytes via Cx26 hemichannels (Huckstepp et al., 2010).
In a recent study by Orellana et al. (2015), we reported from ex-vivo hippocampal slices that acute 2 h restraint stress in mice induces opening of astroglial Cx43 hemichannels, while chronic 10-day immobilization stress-a model used to induce depression in rodents-leads to increased opening of Cx43 hemichannels, and recruitment of astroglial Panx1 channels. This increase in hemichannel activity correlated with an increase in glutamate and ATP release, being dependent on FIGURE 1 | The tripartite synapse; hemichannels, pannexons and their role in memory consolidation. (A) Astrocytes release gliotransmitters (e.g., glutamate, D-serine and ATP) through Ca 2+ -dependent exocytosis (1) and opening of connexin (Cx) and pannexin (Panx) hemichannels (2). Long-lasting activation of P2X7 by ATP may lead to large currents and release of gliotransmitters (3), effect that may be mediated by Panx1 hemichannels (4). Gliotransmitter release may also occur through volume-regulated anion channels (VRAC) (5) and different carriers and/or co-transporters acting normally or in reverse (6) (e.g., excitatory amino-acid transporters, the cysteine-glutamate antiporter and the D-serine/chloride co-transporter). Astrocytes can also communicate with neurons via the release of vesicles (e.g., exosomes, microparticles and apoptotic bodies), containing different cellular messengers (e.g., mRNA, viruses and organelles) (7). Adjacent glial cells and neurons can communicate directly through F-actin-based transient tubular connections known as tunneling nanotubes (8), via cell-to-cell contacts between membrane-bound ligand molecules and their receptors (9) or intercellular channels known as gap junctions (10). (B-D) Blockade of astroglial Cx43 hemichannel opening in the basolateral amygdala by intra-BLA microinjection of TAT-L2 mimetic peptide had (B) no effect in short term fear conditioning memory, (C) but blocked fear memory consolidation as assessed 24 h after training. This amnesic effect was also found after injection of the more unspecific hemichannel blocker GAP27, but was absent when a scrambled peptide was used (scr) or a similar peptide to TAT-L2 with two aminoacids mutated (L2-mut) to interfere with its affinity for Cx43. (D) A minimal dose of TAT-L2 still capable of blocking memory consolidation (TAT-L2) was co-injected into the basolateral amygdala with a mixture of TAT-L2 and various putative gliotransmitters (TATL2 + cocktail), including glutamate, D-serine, glycine, ATP, etc. The microinjection of the mixture prevented the amnesic effects of intra-amygdalar TAT-L2 peptide (reprinted from Stehberg et al., 2012 with permission). * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001. glutamatergic N-methyl-D-aspartate (NMDA) and purinergic receptor signaling . Moreover, Garré et al. (2016) showed that FGF-1 promotes inflammatory responses in acute spinal cord slices by a mechanism that involves release of ATP through Panx1 channels. Finally, in another study we shall discuss in more detail later, we showed that Cx43 hemichannels are necessary for fear memory consolidation in the basolateral amygdala (Stehberg et al., 2012).
As can be deduced from the above paragraph, in vivo evidence supporting a role for hemichannels and pannexons in CNS function is very recent, still limited in number but growing fast.
Astroglial hemichannels open in response to local increments in intracellular Ca 2+ . Astrocytes express receptors and respond to most neurotransmitters known to be relevant for memory (for a review see Moraga-Amaro et al., 2014) via fast and local Ca 2+ oscillations or inter-astrocytic Ca 2+ waves at speeds matching neuronal activity (Winship et al., 2007). Thus, astroglial activation may trigger the opening of hemichannels or pannexons and the concomitant release of D-serine, glutamate, ATP and lactate, among various other gliotransmitters (Orellana and Stehberg, 2014). D-serine is a co-agonist of NMDA receptors critical for synaptic plasticity (Henneberger et al., 2010). Glutamate is the main excitatory neurotransmitter in the CNS and lactate is critical for brain metabolism and acts as a neurotransmitter activating NMDA receptors (Yang et al., 2014), all of which are critical for synaptic plasticity and memory. ATP mediates propagation of inter-astrocytic Ca 2+ waves by activating astroglial purinergic receptors, whereas its conversion into adenosine may activate neuronal purinergic receptors (Zhang et al., 2003). As a consequence, it is likely that these functions are mediated by astroglial hemichannels and pannexons, but direct in vivo evidence is still lacking.

ASTROGLIAL HEMICHANNELS AND PANNEXONS IN MEMORY
As mentioned earlier, astroglial hemichannels and pannexons allow for the delivery of a wide variety of gliotransmitters to the extracellular milieu. However, the role of these channels in brain function under physiological conditions, and particularly in memory, has only recently begun to be elucidated. In 2012, we demonstrated that blockade of Cx43 hemichannels at the basolateral amygdala in vivo, using a mimetic peptide known as TAT-L2, had no effects on short term memory (Figure 1B), yet blocked memory consolidation, inducing amnesia for auditory fear conditioning 24 h after training ( Figure 1C). Interestingly, the amnesic effect of the peptide was prevented by co-injecting it together with a cocktail of gliotransmitters, including glutamate, D-serine, glycine, lactate, ATP and glutamine ( Figure 1D; Stehberg et al., 2012). This indicates that the opening of Cx43 hemichannels permits the release of gliotransmitters necessary for memory consolidation, but we were not able to identify the gliotransmitter or combination of gliotransmitters that is critical for memory. In this respect, a previous study by Henneberger et al. (2010) reported that preventing calcium oscillations in astrocytes averted long-term potentiation (LTP, a model of synaptic plasticity associated to memory) in hippocampal slices. This effect was reverted by exogenous administration of D-serine to the preparation (Henneberger et al., 2010). D-serine is a co-agonist of glutamate NMDA receptors which is secreted by astrocytes and is critical for LTP induction (Henneberger et al., 2010;Kang et al., 2013). There is currently no direct evidence that D-serine can be released via Cx43 hemichannels, but it is possible, as astroglial pannexons have been reported to release D-serine in vitro (Pan et al., 2015).
Genetic studies affecting Cx expression have had limited value in deciphering the role of hemichannels, as current genetic approaches affect the expression of both hemichannels and GJCs and do not allow for the distinction of the effects of either. Double knockout mice for both Cx43 and Cx30, the two main Cxs expressed in astrocytes, show enhanced synaptic transmission, attenuated LTP and increased long-term depression (LTD) in hippocampal CA1 pyramidal cells (Pannasch et al., 2011), which are critical for memory formation.
It is still debated whether Pannexons form GJCs in vitro (Sosinsky et al., 2011;Sahu et al., 2014). Unlike Cx43, which is expressed mainly in astrocytes (also reported in microglia, radial glia and neural progenitors; Nadarajah et al., 1997;Boucher and Bennett, 2003), Panx1 is expressed in both astrocytes and neurons (Zoidl et al., 2007;Iglesias et al., 2009;Santiago et al., 2011). Thus, Panx1 deficiency by genetic knockout or pharmacological approaches cannot distinguish neuronal from astroglial pannexons. Both pharmacological blockade and genetic deficiency of Panx1 channels induce increased excitability, enhanced LTP, reduced LTD and impairments in object recognition and spatial memory (Prochnow et al., 2012;Ardiles et al., 2014). This evidence depicts a clear role for Panx1 in synaptic plasticity and memory, regardless of whether they originate from astrocytes, neurons, or both.

HEMICHANNELS AND PANNEXONS IN PSYCHIATRIC DISORDERS ASSOCIATED WITH COGNITIVE DYSFUNCTION
Thus far no study has reported a direct role for astroglial hemichannels in psychiatric disorders that can be associated with memory. However, in Orellana et al. (2015), we showed that astroglial Cx43 hemichannel activity in the hippocampus is increased after acute restraint stress in mice, effects that were associated with a Cx43-dependent increase in extracellular levels of glutamate and ATP . Interestingly, when mice underwent a protocol of chronic restraint stress commonly used to induce depressive-like symptoms in mice, an even greater increase in Cx43 hemichannel activity was induced, together with Panx1 channel opening, with the concomitant Cx43-and Panx1-dependent release of glutamate and ATP . This study was followed by the work of Quesseveur et al. (2015), reporting that knockdown of Cx43 in mice induced anxiolytic-and antidepressant-like effects and an increase in FIGURE 2 | The role of astrocytic hemichannels and pannexons during neurodegeneration. During the early stages of various neurodegenerative diseases, increased inflammation opens astrocytic Cx43 hemichannels and Panx1 channels (1). This results in the release of the gliotransmitters ATP and glutamate, and increases activation of neuronal NMDA and P2X7 receptors (2). It is hypothesized that NMDA and P2X7 receptor activation increases the opening of neuronal Panx1 channels through phosphorylation of Panx1 by Src family kinases (SFKs) and direct protein-to-protein interactions, respectively (3). These P2X7-related protein interactions could affect intracellular Ca 2+ homeostasis resulting in cell death. Uncontrolled activation of astrocytes may result in reactive astrogliosis and further cell death by a mechanism related to the opening of connexons and pannexons (4). In particular, dysregulated opening of Cx43 and Panx1 channels could elicit cellular damage by different mechanisms. At one end of the connexon, the entry of Ca 2+ via the Cx43 hemichannels or Panx1 channels. The added Ca 2+ activates phospholipase A 2, thus generating arachidonic acid and activating the cyclooxygenase and lipoxygenase pathways, resulting in increased free radicals, lipid peroxidation and plasma membrane damage (5). At the other end of the connexon, Na + and Cl − entry via Cx43 hemichannels or Panx1 channels may trigger cellular swelling due to an increased influx of H 2 O via aquaporins (6). Finally, given that astrocytes provide support to neurons, astroglial cell damage associated with hemichannel/pannexon opening could indirectly increase neuronal susceptibility and vulnerability due to the homeostatic imbalance occurring during neurodegeneration.
freezing in the fear-conditioning paradigm. Interestingly, it was found that chronic corticosterone administration (another model used to induce depression in rodents), caused an increase in the expression of phosphorylated Cx43 in the hippocampus, effect that was reversed by successful antidepressant treatment (Quesseveur et al., 2015). This is further supported by a recent study showing that antidepressants affect astroglial Cx43 GJC and hemichannel activity (Jeanson et al., 2015). The above findings suggest that hippocampal Cx43 hemichannel activity may be important in stress responses and for the pathophysiology of depression. How they may contribute to arousal-induced memory enhancements, post-traumatic stress disorder or depression-associated cognitive impairments are exciting questions that may be answered in the near future.

HEMICHANNELS AND PANNEXONS IN NEURODEGENERATIVE DISEASES
Many neurodegenerative diseases are characterized by destruction of memory related areas, including the hippocampus, prefrontal cortex, mediotemporal lobe, nucleus basalis, basal ganglia, etc. (for reviews on areas involved in memory see Packard and Knowlton, 2002;Jeong et al., 2015). For example, in Alzheimer's disease (AD), extensive damage to the hippocampus and cortical areas has been associated with cognitive deficits (reviewed in Pini et al., 2016). Likewise, in frontotemporal dementia, damage to frontal and anterior temporal lobes was also associated with cognitive deficits (Finger, 2016), while in Parkinson's disease (PD), damage to basal ganglia and frontal connectivity has also been correlated with cognitive deficits (Zgaljardic et al., 2003).
Various studies have linked dysregulation of hemichannel and pannexon permeability and expression, with the progression of different neurodegenerative diseases (Orellana and Stehberg, 2014;Penuela et al., 2014). However, the mechanisms by which astroglial hemichannels and pannexons contribute to neuronal damage remain elusive. It is possible that enhanced reactive astrogliosis evoked by neuroinflammation may alter different astroglial functions necessary for proper astrocyte-to-neuron crosstalk and neuronal survival, including synaptic gliotransmission, Ca 2+ and NO signaling, as well as antioxidant and inflammatory responses. Hemichannels and pannexons are both affected by multiple inflammatory mediators released by reactive astrocytes and microglia (e.g., cytokines, NO and ROS; Retamal et al., 2007;Abudara et al., 2015). Inflammatory conditions could increase astroglial hemichannel/pannexon opening, leading to an uncontrolled influx of potentially toxic agents, as is the case of Ca 2+ . Because hemichannels are permeable to Ca 2+ (De Bock et al., 2012;Fiori et al., 2012), and their opening is controlled by intracellular Ca 2+ (De Bock et al., 2012), it is possible that overactivation of hemichannels/pannexons results in intracellular Ca 2+ overload and the subsequent impairment of vital functions for astroglial survival; including energy metabolism, Ca 2+ handling, osmotic regulation and antioxidant defense. Consistent with this notion, hemichannel and pannexon activity has been linked to an alteration in Ca 2+ dynamics and cell death in reactive astrocytes (Orellana et al., 2010;Abudara et al., 2015;Rovegno et al., 2015). In addition, osmotic and ionic imbalances induced by uncontrolled influx of Na + and Cl − through these channels could result in further cell swelling and plasma membrane breakdown. Given that astrocytes provide metabolic, synaptic and trophic support to neurons and maintain the extracellular microenvironment, astroglial cell damage or dysfunction associated with hemichannel and pannexon opening could increase neuronal susceptibility to different pathological conditions (Contreras et al., 2004;Orellana et al., 2009).
Alternatively, uncontrolled opening of hemichannels and pannexons induced by inflammatory conditions may trigger excessive release of molecules at toxic levels, such as glutamate and ATP. Consistent with this idea, astrocytes stimulated with amlyloid-β (Aβ) peptide exhibit increased Cx43 hemichannel-dependent release of glutamate and ATP, which are toxic for hippocampal and cortical neurons (Orellana et al., 2011a). Similarly, a follow-up work showed that astrocytes pre-treated with conditioned media from Aβ peptide-stimulated microglia release neurotoxic amounts of glutamate and ATP via Cx43 hemichannels when subjected to hypoxia in high glucose conditions (Orellana et al., 2011b). Interestingly, their release reduced neuronal survival via activation of neuronal NMDA/P2X7 receptors and Panx1 channels in neurons (Orellana et al., 2011a,b). Neurons express functional hemichannels formed by Cx36 and pannexons formed by Panx1 (Thompson et al., 2006;Zappalà et al., 2006), and the opening of Panx1 channels could occur via protein-protein interactions with activated P2X7 receptors (Iglesias et al., 2008), through increases in intracellular Ca 2+ or phosphorylation triggered by activation of P2Y (Locovei et al., 2006b) or NMDA receptors (Weilinger et al., 2012). For a scheme of proposed roles for hemichannels and pannexons in neurodegeneration, see Figure 2.

CONCLUDING REMARKS
Exciting research on astrocytes and particularly on astroglial hemichannels and pannexons characterizes the last few years. Although hemichannels and pannexons initially appeared to be one of the many cellular mechanisms used by astrocytes to share their gliotransmitters, accumulating evidence indicates that astroglial hemichannels play a key role in brain function under physiological conditions, and in pathology. In normal conditions astroglial hemichannels and pannexons are important for memory consolidation, stress response, and possibly even for the pathophysiology of depression. Given their role in NMDA-dependent plasticity, they may also prove to be relevant in depression-associated memory impairments. Yet in pathological conditions, they appear to have a central role in the development of neurodegenerative diseases. Although many questions remain unanswered regarding their role in memory and in cognitive dysfunction, it is clear that astroglial hemichannels and pannexons play essential roles, in sickness and in health, until death do us part.

AUTHOR CONTRIBUTIONS
Review was divided into equal parts, which were combined and edited by JS. JAO made Figure 2; JS and JAO made Figure 1. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.