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 hemichannel-forming 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.
- connexin 43
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 . 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
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, 6, 7]. Although astrocytes are not electrically silent cells , [Ca2+]i signals are their principal fast time-scale mechanism for allowing intra- and intercellular signaling . 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 . 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) . Intracellular [Ca2+]i waves can spread among astrocytes to finally reach the terminal processes or “endfeet” of specialized astrocytes that contact the endothelium . 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 . 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 .
Nowadays, diverse mechanisms have been proposed to lead to gliotransmitter release (Figure 1), including Ca2+-dependent exocytosis [15, 16, 17], carrier membrane transport , and opening of a wide range of channels. Among the latter group, volume-regulated anion channels [19, 20, 21], P2X7 receptors [22, 23, 24], Ca2+-dependent Cl− channel bestrophin 1 [25, 26], and hemichannels [27, 28, 29, 30] are included. This 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 . 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 . These proteins are abundantly expressed in brain cells , including astrocytes , and they are named after their predicted molecular mass expressed in kDa, for instance, connexin 43 (Cx43) has a molecular mass of ~43 kDa . 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 . Nonetheless, in the 1990s, pioneering findings by Paul and colleagues revealed the presence of functional and solitary hemichannels in “nonjunctional” membranes . 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 . 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) .
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− through hemichannels could result in further cell swelling and plasma membrane breakdown . 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 . 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 . 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, 41, 42, 43, 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, 46, 47, 48, 49], at the moment, Cx43 is the only connexin probed to form functional hemichannels in astrocytes . 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 . 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 . 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 . 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  (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 . Astroglial hemichannels also have been found to regulate neuronal activity in the olfactory bulb (OB) . 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  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 adenosine (Figure 3). Usually, A1 receptors induce the presynaptic inhibition of glutamate release, reduced postsynaptic NMDAR activation, and decreased Ca2+ influx . 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  (Figure 3).
Astroglial Cx43 hemichannels have been involved not only in synaptic function and transmission but also in synaptic plasticity. High-frequency stimulation (HFS) of neuronal layer 2/3 (L2/3) triggers glutamatergic synaptic transmission in pyramidal cells at layer 5 (L5) of the prefrontal cortex (PFC) . 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 . 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  (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 . 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 , significantly impairs the spatial short-term memory, as assayed with the delayed spontaneous alternation Y maze task .
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.
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 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
The author declares no conflict of interest.
Pereda AE. Electrical synapses and their functional interactions with chemical synapses. Nature Reviews. Neuroscience. 2014; 15(4):250-263. Epub 2014/03/13
Foster M, Sherrington CS. Central nervous system. In: A Textbook of Physiology. London: Macmillan; 1897. p. 930
Furshpan EJ, Potter DD. Transmission at the giant motor synapses of the crayfish. The Journal of Physiology. 1959; 145(2):289-325. Epub 1959/03/03
Bennett MV, Zukin RS. Electrical coupling and neuronal synchronization in the mammalian brain. Neuron. 2004; 41(4):495-511. Epub 2004/02/26
Cornell-Bell AH, Finkbeiner SM, Cooper MS, Smith SJ. Glutamate induces calcium waves in cultured astrocytes: Long-range glial signaling. Science. 1990; 247(4941):470-473
Nedergaard M. Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science. 1994; 263(5154):1768-1771
Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG. Glutamate-mediated astrocyte-neuron signalling. Nature. 1994; 369(6483):744-747
Dallerac G, Chever O, Rouach N. How do astrocytes shape synaptic transmission? Insights from electrophysiology. Frontiers in Cellular Neuroscience. 2013; 7:159
Rusakov DA. Disentangling calcium-driven astrocyte physiology. Nature Reviews. Neuroscience. 2015; 16(4):226-233
Bazargani N, Attwell D. Astrocyte calcium signaling: The third wave. Nature Neuroscience. 2016; 19(2):182-189
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
Allaman I, Belanger M, Magistretti PJ. Astrocyte-neuron metabolic relationships: For better and for worse. Trends in Neurosciences. 2011; 34(2):76-87
Weber B, Barros LF. The astrocyte: Powerhouse and recycling center. Cold Spring Harbor Perspectives in Biology. 2015; 7(12):a020396
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
Bezzi P, Gundersen V, Galbete JL, Seifert G, Steinhauser C, Pilati E, et al. Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nature Neuroscience. 2004; 7(6):613-620. Epub 2004/05/25
Zhang Z, Chen G, Zhou W, Song A, Xu T, Luo Q , et al. Regulated ATP release from astrocytes through lysosome exocytosis. Nature Cell Biology. 2007; 9(8):945-953. Epub 2007/07/10
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
Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature. 2000; 403(6767):316-321. Epub 2000/02/05
Kimelberg HK, Goderie SK, Higman S, Pang S, Waniewski RA. Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. The Journal of Neuroscience. 1990; 10(5):1583-1591. Epub 1990/05/01
Takano T, Kang J, Jaiswal JK, Simon SM, Lin JH, Yu Y, et al. Receptor-mediated glutamate release from volume sensitive channels in astrocytes. Proceedings of the National Academy of Sciences of the United States of America. 2005; 102(45):16466-16471. Epub 2005/10/29
Rudkouskaya A, Chernoguz A, Haskew-Layton RE, Mongin AA. Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes. Journal of Neurochemistry. 2008; 105(6):2260-2270. Epub 2008/03/05
Duan S, Anderson CM, Keung EC, Chen Y, Swanson RA. P2X7 receptor-mediated release of excitatory amino acids from astrocytes. The Journal of Neuroscience. 2003; 23(4):1320-1328. Epub 2003/02/25
Suadicani SO, Brosnan CF, Scemes E. P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. The Journal of Neuroscience. 2006; 26(5):1378-1385. Epub 2006/02/03
Hamilton N, Vayro S, Kirchhoff F, Verkhratsky A, Robbins J, Gorecki DC, et al. Mechanisms of ATP- and glutamate-mediated calcium signaling in white matter astrocytes. Glia. 2008; 56(7):734-749. Epub 2008/02/23
Lee S, Yoon BE, Berglund K, Oh SJ, Park H, Shin HS, et al. Channel-mediated tonic GABA release from glia. Science. 2010; 330(6005):790-796. Epub 2010/10/12
Woo DH, Han KS, Shim JW, Yoon BE, Kim E, Bae JY, et al. TREK-1 and best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell. 2012; 151(1):25-40
Chever O, Lee CY, Rouach N. Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission. The Journal of Neuroscience. 2014; 34(34):11228-11232. Epub 2014/08/22
Meunier C, Wang N, Yi C, Dallerac G, Ezan P, Koulakoff A, et al. Contribution of astroglial Cx43 hemichannels to the modulation of glutamatergic currents by D-serine in the mouse prefrontal cortex. The Journal of Neuroscience. 2017; 37(37):9064-9075. Epub 2017/08/20
Stout CE, Costantin JL, Naus CC, Charles AC. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. The Journal of Biological Chemistry. 2002; 277(12):10482-10488. Epub 2002/01/14
Ye ZC, Wyeth MS, Baltan-Tekkok S, Ransom BR. Functional hemichannels in astrocytes: A novel mechanism of glutamate release. The Journal of Neuroscience. 2003; 23(9):3588-3596. Epub 2003/05/09
Abudara V, Retamal MA, Del Rio R, Orellana JA. Synaptic functions of hemichannels and pannexons: A double-edged sword. Frontiers in Molecular Neuroscience. 2018; 11:435
Leybaert L, Lampe PD, Dhein S, Kwak BR, Ferdinandy P, Beyer EC, et al. Connexins in cardiovascular and neurovascular health and disease: Pharmacological implications. Pharmacological Reviews. 2017; 69(4):396-478
De Bock M, Vandenbroucke RE, Decrock E, Culot M, Cecchelli R, Leybaert L. A new angle on blood-CNS interfaces: A role for connexins? FEBS Letters. 2014; 588(8):1259-1270
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
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 2003/09/25
Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopusoocytes. The Journal of Cell Biology. 1991; 115(4):1077-1089
Saez JC, Leybaert L. Hunting for connexin hemichannels. FEBS Letters. 2014; 588(8):1205-1211. Epub 2014/03/19
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. Epub 2012/04/14
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 2016/08/05
Huckstepp RT, Eason R, Sachdev A, Dale N. CO2-dependent opening of connexin 26 and related beta connexins. The Journal of Physiology. 2010; 588(Pt 20):3921-3931
Roux L, Madar A, Lacroix MM, Yi C, Benchenane K, Giaume C. Astroglial connexin 43 hemichannels modulate olfactory bulb slow oscillations. The Journal of Neuroscience. 2015; 35(46):15339-15352. Epub 2015/11/21
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 2012/06/06
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
Walrave L, Vinken M, Albertini G, De Bundel D, Leybaert L, Smolders IJ. Inhibition of connexin43 hemichannels impairs spatial short-term memory without affecting spatial working memory. Frontiers in Cellular Neuroscience. 2016; 10:288. Epub 2017/01/10
Dermietzel R, Hertberg EL, Kessler JA, Spray DC. Gap junctions between cultured astrocytes: Immunocytochemical, molecular, and electrophysiological analysis. The Journal of Neuroscience. 1991; 11(5):1421-1432
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):87-99. Epub 2010/01/21
Kunzelmann P, Schroder W, Traub O, Steinhauser C, Dermietzel R, Willecke K. Late onset and increasing expression of the gap junction protein connexin30 in adult murine brain and long-term cultured astrocytes. Glia. 1999; 25(2):111-119
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
Yamamoto T, Ochalski A, Hertzberg EL, Nagy JI. LM and EM immunolocalization of the gap junctional protein connexin 43 in rat brain. Brain Research. 1990; 508(2):313-319. Epub 1990/02/05
Orellana JA, Stehberg J. Hemichannels: New roles in astroglial function. Frontiers in Physiology. 2014; 5:193. Epub 2014/07/06
Dobrowolski R, Sasse P, Schrickel JW, Watkins M, Kim JS, Rackauskas M, et al. The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans. Human Molecular Genetics. 2008; 17(4):539-554
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
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
Morairty S, Rainnie D, McCarley R, Greene R. Disinhibition of ventrolateral preoptic area sleep-active neurons by adenosine: A new mechanism for sleep promotion. Neuroscience. 2004; 123(2):451-457. Epub 2003/12/31
DeNardo LA, Berns DS, DeLoach K, Luo L. Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing. Nature Neuroscience. 2015; 18(11):1687-1697
Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, et al. Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Research in Cardiology. 2013; 108(1):309. Epub 2012/11/28