Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic γ-aminobutyric acid_A (GABA_A) and GABA_B receptors. In addition, GABA also depress transmitter release acting through presynaptic GABA_B receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABA_A receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca²⁺ ([Ca²⁺]_i) during the first postnatal week of life. During the same developmental period, the postsynaptic GABA_B-mediated inhibition is poorly developed. In contrast, the presynaptic GABA_B-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons. © 1995 John Wiley & Sons, Inc.     

A Study of the Role of GABAA- and GABAB-Receptors in Presynaptic Inhibition of Primary Afferents in the Spinal Cord of the Frog Rana ridibunda

The role of GABA_A- and GABA_B-receptors in presynaptic inhibition of primary afferent fibers was studied on an isolated preparation of the spinal cord of the frog Rana ridibunda. It is shown that the inhibitory effect of GABA on synaptic transmission from afferent fiber to motoneuron is caused by activation of both GABA_A- and GABA_B-receptors. A temporal correlation (± 5 min) was shown between the blocking action of bicuculline (a specific antagonist of GABA_A-receptors) on primary afferent fiber depolarization (PAD) and its potentiating effect on the excitatory postsynaptic potential (EPSP) at parallel intracellular recording of EPSP in motoneuron and PAD in axons of the dorsal root. As a basis of this correlation, the single GABA_A-receptor mechanism is discussed, which mediates the effect of bicuculline on PAD and EPSP. When a specific agonist of GABA_B-receptor, baclofen, and an antagonist of GABA_B-receptor, 2(OH)-saclofen, were applied, the obtained data indicated an involvement of GABA_B-receptors in inhibition of synaptic transmission from afferent fibers to the motoneuron. Analysis of parameters of the unitary synaptic responses recorded in the control experiments and of their changes under the effect of (– )-baclofen indicates that the inhibitory action caused by activation of GABA_B-receptors develops at the presynaptic level.     

Characterization of Inhibitory GABA-A Receptor Activation during Spreading Depolarization in Brain Slice

Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABA_AR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABA_AR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABA_AR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl⁻ decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABA_AR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABA_AR activation and suggest that intracellular Cl⁻ loading is insufficient to generate excitatory GABA_AR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABA_AR antagonists, and the lack of inhibitory effect of GABA_AR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABA_A modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD.     

Background Synaptic Activity in Rat Entorhinal Cortex Shows a Progressively Greater Dominance of Inhibition over Excitation from Deep to Superficial Layers

The entorhinal cortex (EC) controls hippocampal input and output, playing major roles in memory and spatial navigation. Different layers of the EC subserve different functions and a number of studies have compared properties of neurones across layers. We have studied synaptic inhibition and excitation in EC neurones, and we have previously compared spontaneous synaptic release of glutamate and GABA using patch clamp recordings of synaptic currents in principal neurones of layers II (L2) and V (L5). Here, we add comparative studies in layer III (L3). Such studies essentially look at neuronal activity from a presynaptic viewpoint. To correlate this with the postsynaptic consequences of spontaneous transmitter release, we have determined global postsynaptic conductances mediated by the two transmitters, using a method to estimate conductances from membrane potential fluctuations. We have previously presented some of this data for L3 and now extend to L2 and L5. Inhibition dominates excitation in all layers but the ratio follows a clear rank order (highest to lowest) of L2>L3>L5. The variance of the background conductances was markedly higher for excitation and inhibition in L2 compared to L3 or L5. We also show that induction of synchronized network epileptiform activity by blockade of GABA inhibition reveals a relative reluctance of L2 to participate in such activity. This was associated with maintenance of a dominant background inhibition in L2, whereas in L3 and L5 the absolute level of inhibition fell below that of excitation, coincident with the appearance of synchronized discharges. Further experiments identified potential roles for competition for bicuculline by ambient GABA at the GABA_A receptor, and strychnine-sensitive glycine receptors in residual inhibition in L2. We discuss our results in terms of control of excitability in neuronal subpopulations of EC neurones and what these may suggest for their functional roles.     

GABA Increases Electrical Excitability in a Subset of Human Unmyelinated Peripheral Axons

Background

A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established.

Methodology/Principal Findings

Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1–100 µM) increased electrical excitability in a subset (ca. 40%) of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABA_A receptors, being mimicked by bath application of the GABA_A agonist muscimol (0.1–30 µM) while the GABA_B agonist baclofen (10–30 µM) was without effect. Increases in excitability produced by GABA (10–30 µM) were blocked by the GABA_A antagonists gabazine (10–20 µM), bicuculline (10–20 µM) and picrotoxin (10–20 µM).

Conclusions/Significance

Functional GABA_A receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABA_A receptor modulation may therefore regulate segmental and peripheral components of nociception.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

Ionic mechanisms of the effect of phenibut and gaba not associated with a change in the function of the chloride channels

In experiments on isolated spinal cord of young rats 7–14 days old under conditions of takeoff of the electrical activity of the spinal roots with a sugar bridge, it was established that the GABA-mimetic phenibut induces direct depolarization of the motoneurons. In the same concentration range (10^–5-10^–4 M), GABA has a dual effect. The depolarizing component of the action of GABA in part of the experiments and the depolarizing effect of phenibut in all the experiments are preserved in the presence of picrotoxin (10^–5 M) and under conditions of superfusion of the brain with a solution with a reduced chloride concentration. This depolarizing effect of phenibut, not associated with the activation of GABA_A receptors and chloride channels coupled with them, is unchanged in a medium with Na⁺ deficiency, is enhanced during depolarization of the motoneurons due to an increased concentration of K⁺ (10 mM) and in the presence of imidazole, but is entirely eliminated in a medium with Ca²⁺ deficiency, containing 2 mM Mn²⁺, or in the presence of theophylline (10^–4 M). It is suggested that phenibut, and to some degree, GABA lower the intracellular concentration of cAMP by means of activation of the GABA_B receptors, which leads to blocking of the functional activity of the potential-dependent calcium channels and a decrease in the calcium-activated outflowing potassium currents. The ability to weaken the inflowing calcium currents may also be the basis of the presynaptic inhibiting effect of GABA and GABA-mimetics (phenibut, baclofen, etc.) on the pulsed release of mediators by the axon terminals of catecholaminergic, glutamatergic, and GABA-ergic neurons.A. M. Gor'kii Donetsk Medical Institute. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 481–489, July–August, 1985.     

Presynaptic GABAB Receptors Regulate Hippocampal Synapses during Associative Learning in Behaving Mice

GABA_B receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the central nervous system. Pharmacological activation of GABA_B receptors regulates neurotransmission and neuronal excitability at pre- and postsynaptic sites. Electrophysiological activation of GABA_B receptors in brain slices generally requires strong stimulus intensities. This raises the question as to whether behavioral stimuli are strong enough to activate GABA_B receptors. Here we show that GABA_B1a^-/- mice, which constitutively lack presynaptic GABA_B receptors at glutamatergic synapses, are impaired in their ability to acquire an operant learning task. In vivo recordings during the operant conditioning reveal a deficit in learning-dependent increases in synaptic strength at CA3-CA1 synapses. Moreover, GABA_B1a^-/- mice fail to synchronize neuronal activity in the CA1 area during the acquisition process. Our results support that activation of presynaptic hippocampal GABA_B receptors is important for acquisition of a learning task and for learning-associated synaptic changes and network dynamics.     

Decreased intracellular GABA levels contribute to spinal cord stimulation-induced analgesia in rats suffering from painful peripheral neuropathy: the role of KCC2 and GABA(A) receptor-mediated inhibition

Elevated spinal extracellular γ-aminobutyric acid (GABA) levels have been described during spinal cord stimulation (SCS)-induced analgesia in experimental chronic peripheral neuropathy. Interestingly, these increased GABA levels strongly exceeded the time frame of SCS-induced analgesia. In line with the former, pharmacologically-enhanced extracellular GABA levels by GABA_B receptor agonists in combination with SCS in non-responders to SCS solely could convert these non-responders into responders. However, similar treatment with GABA_A receptor agonists and SCS is known to be less efficient. Since K⁺ Cl⁻ cotransporter 2 (KCC2) functionality strongly determines proper GABA_A receptor-mediated inhibition, both decreased numbers of GABA_A receptors as well as reduced KCC2 protein expression might play a pivotal role in this loss of GABA_A receptor-mediated inhibition in non-responders. Here, we explored the mechanisms underlying both changes in extracellular GABA levels and impaired GABA_A receptor-mediated inhibition after 30 min of SCS in rats suffering from partial sciatic nerve ligation (PSNL). Immediately after cessation of SCS, a decreased spinal intracellular dorsal horn GABA-immunoreactivity was observed in responders when compared to non-responders or sham SCS rats. One hour later however, GABA-immunoreactivity was already increased to similar levels as those observed in non-responder or sham SCS rats. These changes did not coincide with alterations in the number of GABA-immunoreactive cells. C-Fos/GABA double-fluorescence clearly confirmed a SCS-induced activation of GABA-immunoreactive cells in responders immediately after SCS. Differences in spinal dorsal horn GABA_A receptor-immunoreactivity and KCC2 protein levels were absent between all SCS groups. However, KCC2 protein levels were significantly decreased compared to sham PSNL animals. In conclusion, reduced intracellular GABA levels are only present during the time frame of SCS in responders and strongly point to a SCS-mediated on/off GABAergic release mechanism. Furthermore, a KCC2-dependent impaired GABA_A receptor-mediated inhibition seems to be present both in responders and non-responders to SCS due to similar KCC2 and GABA_A receptor levels.     

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABA_B receptors (GABA_BRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABA_A and GABA_BRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABA_BR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED₅₀: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca²⁺ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABA_BRs and mediated by Ca²⁺ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.     

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABA_A receptors (GABA_ARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABA_ARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABA_ARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.     

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABA_A receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np^−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np^−/− hippocampal cultures or in mature CA1 and DG wild-type (Np^+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np^−/− neurons or in mature Np65-Fc-treated Np^+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np^−/− cultures. Furthermore, GABA_A receptor composition was altered at inhibitory synapses in Np^−/− neurons as the α1 to α2 GABA_A receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np^−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.     

Pharmacological study of inhibited frog olfactory bulb glomerular input produced by a puff of air on the olfactory mucosa

The effects of applying 4-aminopyridine (10^–2 M), aminooxyacetic acid (AOAA — 10^–4–10^–3 M), -alanine (10^–3–10^–2 M), and bicuculline (10^–5, 10^–4 M) to the intact frog olfactory bulb were investigated. Having measured inhibition of orthodromic potential postsynaptic components produced either by a puff of air on the olfactory mucosa (OB input inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition), it was found that 4-aminopyridine greatly intensified postsynaptic inhibition but strongly reduced that of OB input; inhibition of the latter was raised by AOAA or bicuculline and decreased by -alanine. These substances failed to exert any consistent, clear-cut effects on postsynaptic inhibition. Findings would support the hypothesis that OB input inhibition produced by a puff of air on the olfactory mucosa could occur as a result of GABA release from glial cells and subsequent binding of GABA to presynaptic GABA_B-receptors in glomeruli.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 12–20, January–February, 1987.     

GABA Release Modified by Adenosine Receptors in Mouse Hippocampal Slices under Normal and Ischemic Conditions

The excitatory glutamatergic neurons in the hippocampus are modulated by inhibitory GABA-releasing interneurons. The neuromodulator adenosine is known to inhibit the presynaptic release of neurotransmitters and to hyperpolarize postsynaptic neurons in the hippocampus, which would imply that it is an endogenous protective agent against cerebral ischemia and excitotoxic neuronal damage. Interactions of the GABAergic and adenosinergic systems in regulating neuronal excitability in the hippocampus is of crucial importance, particularly under cell-damaging conditions. We now characterized the effects of adenosine receptor agonists and antagonists on the release of preloaded [³H]GABA from hippocampal slices prepared from adult (3-month-old) mice, using a superfusion system. The effects were tested both under normal conditions and in ischemia induced by omitting glucose and oxygen from the superfusion medium. Basal and K⁺-evoked GABA release in the hippocampus were depressed by adenosinergic compounds. Under normal conditions activation of both adenosine A₁ and A_2A receptors by the agonists R(-)N⁶-(2-phenylisopropyl)adenosine and CGS 21680 inhibited the K⁺-evoked release, which effects were blocked by their specific antagonists, 8-cyclopentyl-1,3-dipropyl-xanthine and 3,7-dimethyl-1-propargylxanthine, respectively. Under ischemic conditions the release of both GABA and adenosine is markedly enhanced. The above receptor agonists then depressed both the basal and K⁺-evoked GABA release, only the action of A_2A receptors being however receptor-mediated. The demonstrated depression of GABA release by adenosine in the hippocampus could be deleterious to neurons and contribute to excitotoxicity.     

Diversity matters: combinatorial information coding by GABAA receptor subunits during spatial learning and its allosteric modulation

In the hippocampus, GABA inhibition tunes network oscillations and shapes synchronous activity during spatial learning and memory coding. Once released from the presynapse, GABA primarily binds to ionotropic GABA_A receptors (GABA_ARs), which are heteropentamers combinatorially assembled from nineteen known subunits to induce Cl^- currents postsynaptically. Dissecting GABA_AR subtype specificities in neurobiology is daunting because of differences in their developmental dynamics, regional distribution and subcellular compartmentalization. Here, we review recent data to show that the combination of single-cell mRNA-seq and neuroanatomy can reveal unprecedented cell-type and network-specificity of GABA_AR subunits and limit the permutation in subunit configurations, thus rationalizing GABA_AR physiology and pharmacology. By comparing RNA-seq data on principal cells and interneurons we discuss a tight match between GABA_AR subunit allocation, diversity in the origins of GABA inputs and operational rules at synaptic and extrasynaptic sites. We propose that coincident analysis of all GABA_AR subunits, particularly in relation to specific behaviors, could overcome existing pitfalls of the genetic and pharmacological manipulation of single subunits. By using α1 and α5 GABA_AR subunits, we single out hippocampal spatial learning as a process in which, despite the many studies available to date, neither consensus nor causality exists with regards to GABA_AR subtype requirements, curtailing a unifying concept on postsynaptic coding of GABA signals. Finally, we address the modulation of GABA_AR subunits by dopamine and endocannabinoids through receptor heteromerization, cross-modulation of signal transduction and allostery. In sum, data in this review infer that multiparametric computation gains momentum to improve knowledge on GABA_ARs function in cognition and neuropsychiatric illnesses.     

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA_A receptors (GABA_ARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA_ARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA_AR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA_AR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA_ARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.     

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABA_A receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABA_A receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABA_A receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABA_A receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y₁₁ purinoceptor. Thus, our findings reveal that postsynaptic GABA_A receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

Presynaptic inhibition and the participation of GABAB receptors at neuromuscular junctions of the crab Eriphia spinifrons

Presynaptic inhibition exerted by the common inhibitor on the closer and opener muscles and by the specific inhibitor on the opener muscle was investigated in the crab Eriphia spinifrons. In the closer muscle, activation of GABA_B receptors by baclofen reduced the mean quantal content of excitatory junctional currents by about 25%. Blocking GABA_B receptors with CGP 55845 diminished presynaptic inhibition at a similar percentage. GABA_B receptor-mediated presynaptic inhibition is linked to G proteins. Application of pertussis toxin eliminated about 25% of the inhibition exerted by the common inhibitory neuron. GABA_B receptors participate in presynaptic inhibition at release boutons of the slow and the fast closer excitor at a similar percentage. In the opener muscle, presynaptic inhibition of transmitter release from the same endings of the opener excitor was about 15% stronger with the specific inhibitor than with the common inhibitor. About 10% of the presynaptic inhibition produced by either one of the two inhibitors could be abolished by blocking GABA_B receptors. The amplitudes of the excitatory junctional currents in the opener were reduced in the presence of baclofen by about 25%, suggesting that synaptic terminals of the opener excitor are endowed with a similar percentage of GABA_B receptors as terminals of the slow and the fast closer excitors. Baclofen had no effect on postsynaptic inhibition, indicating that GABA_B receptors are not involved in postsynaptic neuromuscular inhibition. Accepted: 8 January 2000