Disruption of postsynaptic GABAA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons (original) (raw)
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GABA A receptors can initiate the formation of functional inhibitory GABAergic synapses
European Journal of Neuroscience, 2013
The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA A receptors (GABA A Rs) themselvesthe essential functional postsynaptic components of GABAergic synapsescan be sufficient to initiate formation of synaptic contacts, a novel co-culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. a1/b2/c2-GABA A Rs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse-like contacts in these co-cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle-luminal domain-specific antibody; and they supported spontaneous and action potential-driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N-methyl-D-aspartate receptor-expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin-2 was co-expressed with GABA A Rs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA A Rs can promote the adhesion of inhibitory axons and the development of functional synapses.
Journal of Biological Chemistry, 2012
Background: GABA A receptor ␥2 and ␦ subunits are thought to be responsible for synaptic and extrasynaptic targeting. Results: We demonstrate here that ␣2 and ␣6 subunits can target ␦/␥2 chimeras to synaptic and extrasynaptic sites. Conclusion: The ␣ subunits play a direct role in GABA A receptor targeting. Significance: Different subunits of GABA A receptors encode intrinsic signals to control subcellular targeting. GABA A receptors (GABA A-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of ␥2 and ␦ subunits in synaptic versus extrasynaptic targeting of GABA A-Rs. Here, we demonstrate differential function of ␣2 and ␣6 subunits in guiding the localization of GABA A-Rs. To study the targeting of specific subtypes of GABA A-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA A-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by ␣23␥2 receptors were very fast (rise time ϳ2 ms), whereas events mediated by ␣63␦ receptors were very slow (rise time ϳ20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by ␣63 or ␣63␥2 receptors were significantly slower than those mediated by ␣23 or ␣23␥2 receptors, suggesting a differential role of ␣ subunit in receptor targeting. This was confirmed by differential targeting of the same ␦-␥2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the ␣2 or ␣6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of ␣6 and ␦ subunits brought ␣63␦ receptors closer to synaptic sites. Therefore, the ␣ subunits, together with the ␥2 and ␦ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA A-Rs, possibly through differential interactions with gephyrin. Neural inhibition in the brain is mostly mediated by GABA A receptors (GABA A-Rs). 2 To date, 19 isoforms of GABA A-R subunits have been identified as follows: ␣1-6, 1-3, ␥1-3, ␦, ⑀, , , and 1-3 (1, 2). Most GABA A-Rs expressed in the brain are composed of two ␣, two , and one ␥ subunits, of which the ␥ subunit can be substituted by ␦, ⑀, , or (3, 4). There are two forms of GABAergic inhibition, phasic and tonic (5, 6). Phasic inhibition is mediated by postsynaptically clustered GABA A-Rs composed of ␣1-3, 2-3, and ␥2 subunits. Tonic inhibition is mediated by extrasynaptic GABA A-Rs typically composed of ␣4/6 (and possibly ␣1),  and ␦ subunits (7-9), as well as ␣5␥2 subunits (10-12). Blocking tonic inhibition significantly enhanced neuronal excitability (5, 13-15). Malfunction of tonic inhibition is implicated in epilepsy, abnormal cognition and memory, sleep disorders, anxiety, depression, schizophrenia, and alcohol addiction (16-23). Although the mechanisms for synaptic receptor targeting have been extensively studied, little is known about the molecular mechanisms specifying extrasynaptic targeting of ␦ subunit-containing GABA A-Rs. Neurons deficient in the ␣1 or ␣2 or ␣3 subunits showed diminished postsynaptic GABA A-R clusters in different subcellular localizations (24-27). The ␥2 subunit, and particularly its intracellular loop (IL) and the fourth transmembrane domain (TM4), plays a critical role in synaptic clustering of GABA A-Rs (28-31). In contrast, the ␦ subunit-containing GABA A-Rs are mainly localized at extrasynaptic membranes (7, 8). Thus, the ␥2 and ␦ subunits have been thought to be involved in the synaptic versus extrasynaptic targeting of GABA A-Rs. However, the mostly extrasynaptic ␣5␥2 and punctated ␣1␦ GABA A-Rs suggest that ␥2 and ␦ subunits cannot be solely responsible for guiding GABA A-R targeting (9, 10, 12). Here, we employed a molecularly engineered synapse model to investigate the mechanism of ␦-GABA A-R targeting. We
Expression of Distinct Subunits of GABAA Receptor Regulates Inhibitory Synaptic Strength
Journal of Neurophysiology, 2004
Expression of distinct ␣ subunits of GABA A receptor regulates inhibitory synaptic strength. . Distinct ␣ subunit subtypes in the molecular assembly of GABA A receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and ␣1 subunit Ϫ/Ϫ mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of ␣2 and ␣3 subunits, the increase of ␣1 and ␣6 subunits expression at synapses, and changes in the action of selective ␣ subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from Ϫ/Ϫ mice together with a decrease in the expression of ␣3 subunit. In contrast, the expression of ␣2 subunit at inhibitory synapses in Ϫ/Ϫ cells was significantly higher than in ϩ/ϩ cells at DIV11-12. ␣5 subunit was not detected, and increased sensitivity to a selective ␣4/␣6 subunit agonist suggests increased expression of extrasynaptic receptors in Ϫ/Ϫ mice. 2/3 subunit expression and loreclezole sensitivity increased with development in ϩ/ϩ but not in Ϫ/Ϫ cells, supporting the preferential association of the ␣1 with the 2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the ϩ/ϩ and Ϫ/Ϫ groups until DIV11-12. Our results uncover a pattern of sequential expression of ␣ subunits underlying the changes in functional efficacy of GABAergic networks with development. RA. Effects of ␥2S subunit incorporation on GABA A receptor macroscopic kinetics. Neuropharmacology 44: 1003-1012, 2003. Bollan K, King D, Robertson LA, Brown K, Taylor PM, Moss SJ, and Connolly CN. GABA A receptor composition is determined by distinct assembly signals within ␣ and  Subunits. J Biol Chem 278: 4747-4755, 2003. Brickley SG, Cull-Candy SG, and Farrant M. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABA A receptors. J Physiol 497: 753-759, 1996. Brickley SG, Revilla V, Cull-Candy SG, Wisden W, and Farrant M. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409: 88 -92, 2001. Brown N, Kerby J, Bonnert TP, Whiting PJ, and Wafford KA. Pharmacological characterization of a novel cell line expressing human ␣43␦ GABA A receptors. Br J Pharmacol 136: 965-974, 2002. Brunig I, Scotti E, Sidler C, and Fritschy JM. Intact sorting, targeting, and clustering of GABA A receptor subtypes in hippocampal neurons in vitro. Wenthold RJ, Bredt DS, and Nicoll RA. Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408: 936 -943, 2000. Christie SB, Miralles CP, and De Blas AL GAB Aergic innervation organizes synaptic and extrasynaptic GABA A receptor clustering in cultured hippocampal neurons. J Neurosci 22: 684 -697, 2002. Ebert B, Thompson SA, Saounatsou K, McKernan R, Krogsgaard-Larsen P, and Wafford KA. Differences in agonist/antagonist binding affinity and receptor transduction using recombinant human GABA A receptors. Mol Pharmacol 52: 1150 -1156, 1997. Farrant M and Brickley SG. Properties of GABA A receptor-mediated transmission at newly formed Golgi-granule cell synapses in the cerebellum. Neuropharmacology 44: 181-189, 2003. Farrant M, Wisden W, Cull-Candy S, and Brickley SG. Absence of tonic GABA A mediated conductance in cerebellar granule cells of ␣6 Ϫ/Ϫ mice. Soc Neurosci Abstr 25: 246, 1999. Fisher JL, Hinkle DJ, and Macdonald RL. Loreclezole inhibition of recombinant ␣11␥2L GABA A receptor single channel currents. Neuropharmacology 39: 235-245, 2000. Fritschy JM, Paysan J, and Mohler H. Switch in the expression of rat GABA A receptor subtypes during postnatal development: an immunohistochemical study. J Neurosci 9: 5302-5324, 1994. Gallo V, Kingsbury A, Balazs R, and Jorgensen OS. The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J Neurosci 7: 2203-2213, 1987. Gao B and Fritschy JM. Cerebellar granule cells in vitro recapitulate the in vivo pattern of GABA A -receptor subunit expression. Brain Res Dev Brain Res 88: 1-16, 1995. Gingrich KJ, Roberts WA, and Kass RS. Dependence of the GABA A receptor gating kinetics on the ␣-subunit isoform: implications for structurefunction relations and synaptic transmission. J Physiol 489: 529 -543, 1995. Hamann M, Rossi DJ, and Attwell D. Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33: 625-633, 2002. Kaneda M, Farrant M, and Cull-Candy SG. Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.
Cell, 2001
likely the result of an ontogenetic decrease in the intra-La Jolla, California 92093 cellular Cl Ϫ concentration ([Cl Ϫ ] i ; Cherubini et al., 1990; Luhmann and Prince, 1991; Chen et al., 1996; Owens et al., 1996). Indeed, changes in the mRNA level for the Summary K ϩ -coupled Cl Ϫ transporter KCC2 have been shown to correlate with the modification of GABAergic transmis-GABA is the main inhibitory neurotransmitter in the sion (Lu et al., 1999; Rivera et al., 1999; Vu et al., 2000). adult brain. Early in development, however, GABAergic KCC2 increases the rate of Cl Ϫ extrusion, thus leading synaptic transmission is excitatory and can exert to a reduction in [Cl Ϫ ] i and a consequent shift in E GABA widespread trophic effects. During the postnatal petoward more hyperpolarized potentials (Jarolimek et al., riod, GABAergic responses undergo a switch from be-1999; Kakazu et al., 1999; Rivera et al., 1999). ing excitatory to inhibitory. Here, we show that the This conversion of GABAergic transmission from deswitch is delayed by chronic blockade of GABA A receppolarizing to hyperpolarizing is also accompanied by a tors, and accelerated by increased GABA A receptor change in GABA-mediated biochemical signaling. Only activation. In contrast, blockade of glutamatergic during this early developmental period, depolarizing transmission or action potentials has no effect. Fur-GABAergic potentials activate voltage-dependent Ca 2ϩ thermore, GABAergic activity modulated the mRNA channels (VDCCs) and elevate [Ca 2ϩ ] i (Connor et al., levels of KCC2, a K ؉ -Cl Ϫ cotransporter whose expres-1987; Yuste and Katz, 1991; Wang et al., 1994). Such sion correlates with the switch. Finally, we report that GABA-induced elevation of [Ca 2ϩ ] i is likely to play a GABA can alter the properties of depolarizationcritical role in the maturation of the nervous system. induced Ca 2؉ influx. Thus, GABA acts as a self-limiting For instance, GABA-mediated increases in [Ca 2ϩ ] i can trophic factor during neural development. induce BDNF expression (Berninger et al., 1995) and promote neuronal survival and differentiation (LoTurco In the adult central nervous system, ␥-amino-butyric et al., 1995; Marty et al., 1996; Ikeda et al., 1997). GABAacid (GABA) is the primary inhibitory neurotransmitter. induced elevation of [Ca 2ϩ ] i may also be required to It regulates a neuron's ability to fire action potentials form, stabilize, and strengthen synaptic connections either through hyperpolarization of the membrane po-(Kirsch and Betz, 1998; Caillard et al., 1999; Kneussel tential or through shunting of excitatory inputs. During and Betz, 2000). early postnatal development, however, GABAergic syn-While the developmental transformation of GABAeraptic transmission is excitatory, able to elevate the intragic transmission is well documented, little is known cellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), and even capable about signals that induce this transformation. Since neuof triggering action potentials (Mueller et al., 1984; Luhronal activity is known to increase during development, mann and Prince, 1991; Yuste and Katz, 1991; Reichling we examined in the present study whether synaptic acet al., 1994; Wang et al., 1994; Leinekugel et al., 1995; tivity can regulate the switch of GABAergic transmis-Obrietan and van den Pol, 1995; Chen et al., 1996; Owens sion. We found that the change in GABA signaling was et al., 1996; Khazipov et al., 1997). Over a limited postnalargely prevented by chronic blockade of GABA A receptal period, in the hippocampus, neocortex, and hypotors, and was accelerated by increased GABA receptor thalamus, as well as other regions of the brain, there is activation. Changes in the level of KCC2 mRNA tightly a switch of the electrophysiological (depolarization to correlated with the observed changes in GABA signalhyperpolarization) and biochemical (Ca 2ϩ -mediated siging. In addition, we found that spontaneous GABAergic naling) properties of GABAergic transmission (Mueller activity regulated the activation of voltage-dependent et al., 1984; Ben-Ari et al., 1989; Cherubini et al., 1991; Ca 2ϩ currents. These findings point to GABA as a critical Luhmann and Prince, 1991; Owens et al., 1996). maturation factor for the switch of the physiological and The GABA A receptor channel predominantly conducts biochemical properties of GABA signaling. Cl Ϫ ions. Consequently, the nature of GABAergic transmission, excitatory versus inhibitory, is determined pri-Results marily by the electrochemical gradient for Cl Ϫ , which depends on the intra-and extracellular concentrations Switch of GABAergic Transmission from Depolarizing of Cl Ϫ . This electrochemical gradient sets the reversal to Hyperpolarizing potential for GABAergic currents (E GABA ; the membrane To study the change in GABA signaling, we first monitored GABA-induced elevations of [Ca 2ϩ ] i over developchanges in fluorescence were measured using confocal Luhmann, H.J., and Prince, D.A. (1991). Postnatal maturation of the GABAergic system in rat neocortex. J. Neurophysiol. 65, 247-263. Marty, S., Berninger, B., Carroll, P., and Thoenen, H. (1996). GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor. Neuron 16, 565-570. Mueller, A.L., Taube, J.S., and Schwartzkroin, P.A. (1984). Development of hyperpolarizing inhibitory postsynaptic potentials and hyperpolarizing response to gamma-aminobutyric acid in rabbit hippocampus studied in vitro. J. Neurosci. 4, 860-867. Murphy, T.H., Worley, P.F., and Baraban, J.M. (1991). L-type voltagesensitive calcium channels mediate synaptic activation of immediate early genes. Neuron 7, 625-635. Obrietan, K., and van den Pol, A.N. (1995). GABA neurotransmission in the hypothalamus: developmental reversal from Ca 2ϩ elevating to depressing. J. Neurosci. 15, 5065-5077. Obrietan, K., and van den Pol, A.N. (1997). GABA activity mediating cytosolic Ca 2ϩ rises in developing neurons is modulated by cAMPdependent signal transduction. J. Neurosci. 17, 4785-4799.
Positive modulation of δ-subunit containing GABAA receptors in mouse neurons
Neuropharmacology, 2012
a b s t r a c t d-subunit containing extrasynaptic GABA A receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of d-subunit containing GABA A receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 mM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 mM), while interneurons showed a potentiation of 2.6-fold. Moreover, AA29504 (1 mM) increased the amplitude and prolonged the decay of miniature inhibitory postsynaptic currents (mIPSCs) in granule cells, and this effect was abolished by Zn 2þ (15 mM). AA29504 (1 mM) also induced a small tonic current (12.7 AE 3.2 pA) per se, and when evaluated in a nominally GABA-free environment using Ca 2þ imaging in cultured neurons, AA29504 showed GABA A receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo.
Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
The tubulin-binding protein gephyrin, which anchors the inhibitory glycine receptor (GlyR) at postsynaptic sites, decorates GABAergic postsynaptic membranes in various brain regions, and postsynaptic gephyrin clusters are absent from cortical cultures of mice deficient for the GABA(A) receptor gamma2 subunit. Here, we investigated the postsynaptic clustering of GABA(A) receptors in gephyrin knock-out (geph -/-) mice. Both in brain sections and cultured hippocampal neurons derived from geph -/- mice, synaptic GABA(A) receptor clusters containing either the gamma2 or the alpha2 subunit were absent, whereas glutamate receptor subunits were normally localized at postsynaptic sites. Western blot analysis and electrophysiological recording revealed that normal levels of functional GABA(A) receptors are expressed in geph -/- neurons, however the pool size of intracellular GABA(A) receptors appeared increased in the mutant cells. Thus, gephyrin is required for the synaptic localization of G...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
We have studied the effects of GABAergic innervation on the clustering of GABA(A) receptors (GABA(A)Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 +/- 0.01 micrometer diameter) GABA(A)R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 +/- 0.08 micrometer breadth) GABA(A)R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA(A)R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA(A)R clusters of any neuron contained all GABA(A)R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA(A)R clusters was that a significant proportio...
Postsynaptic GABAB Receptor Activity Regulates Excitatory Neuronal Architecture and Spatial Memory
Journal of Neuroscience, 2014
Slow and persistent synaptic inhibition is mediated by metabotropic GABA B receptors (GABA B Rs). GABA B Rs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABA B Rs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABA B Rs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABA B Rs. These sorting events are paralleled by a reduction in GABA B R-dependent activation of inwardly rectifying K + channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABA B R2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABA B Rs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABA B Rs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors. endocytic | recycling | excitation-inhibition | glutamate T he availability of neurotransmitter receptors, a major determinant of synaptic efficacy, is regulated by coordinated mechanisms of intracellular trafficking that deliver newly synthesized receptors to the plasma membrane and remove them for storage, recycling, or degradation (1). The molecular mechanisms controlling the availability of GABA B receptors (GABA B Rs), which are central players in the modulation of excitatory and inhibitory synaptic activity, are unclear.
Clustered and non-clustered GABAA receptors in cultured hippocampal neurons
Molecular and Cellular Neuroscience, 2006
In cultured hippocampal neurons, ; 2 subunit-containing GABA A Rs form large postsynaptic clusters at GABAergic synapses and small clusters outside GABAergic synapses. We now show that a pool of nonclustered ; 2 subunit-containing GABA A Rs are also present at the cell surface. We also demonstrate that myc-or EGFP-tagged ; 2 , A 2 , B 3 or A 1 subunits expressed in these neurons assemble with endogenous subunits, forming GABA A Rs that target large postsynaptic clusters, small clusters outside GABAergic synapses or a pool of non-clustered surface GABA A Rs. In contrast, myc-or EGFP-tagged D subunits only form non-clustered GABA A Rs, which can be induced to form clusters by antibody capping. A myc-tagged chimeric ; 2 subunit possessing the large intracellular loop (IL) of the y-subunit IL ( myc ; 2 S/D-IL) assembled into GABA A Rs, but it did not form clusters, therefore behaving like the D subunit. Thus, the large intracellular loops of ; 2 and D play an important role in determining the synaptic clustering/ non-clustering capacity of the GABA A Rs. D