Synaptic and extrasynaptic GABA-A and GABA-B receptors in the globus pallidus: an electron microscopic immunogold analysis in monkeys

GABA-A and GABA-B receptors mediate differential effects in the CNS. To better understand the role of these receptors in regulating pallidal functions, we compared their subcellular and subsynaptic localization in the external and internal segments of the globus pallidus (GPe and GPi) in monkeys, using pre- and post-embedding immunocytochemistry with antibodies against GABA-A (alpha1, beta2/3 subunits) and GABA-BR1 receptor subtype. Our results demonstrate that GABA-A and GABA-B receptors display a differential pattern of subcellular and subsynaptic localization in both segments of the globus pallidus. The majority of GABA-BR1 immunolabeling is intracellular, whereas immunoreactivity for GABA-A receptor subunits is mostly bound to the plasma membrane. A significant proportion of both GABA-BR1 and GABA-A receptor immunolabeling is extrasynaptic, but GABA-A receptor subunits also aggregate in the main body of putative GABAergic symmetric synapses established by striatal- and pallidal-like terminals. GABA-BR1 immunoreactivity is expressed presynaptically in putative glutamatergic terminals, while GABA-A alpha1 and beta2/3 receptor subunits are exclusively post-synaptic and often coexist at individual symmetric synapses in both GPe and GPi. In conclusion, our findings corroborate the concept that ionotropic and metabotropic GABA receptors are located to subserve different effects in pallidal neurons. Although the aggregation of GABA-A receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission, the extrasynaptic distribution of both GABA-A and GABA-B receptors provides a substrate for complex modulatory functions that rely predominantly on the spillover of GABA.     

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors

Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1-induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.     

Balance of increases and decreases in firing rate of the spontaneous activity of basal ganglia high-frequency discharge neurons

Most neurons in the external and internal segments of the globus pallidus and the substantia nigra pars reticulata (GPe, GPi, and SNr) are characterized by a high-frequency discharge (HFD) rate (50-80 Hz) that, in most GPe neurons, is also interrupted by pauses. Almost all (approximately 90%) of the synaptic inputs to these HFD neurons are GABAergic and inhibitory. Nevertheless, their responses to behavioral events are usually dominated by increases in discharge rate. Additionally, there are no reports of prolonged bursts in the spontaneous activity of these cells that could reflect their disinhibition by GPe pauses. We recorded the spontaneous activity of 385 GPe, GPi, and SNr HFD neurons during a quiet-wakeful state from two monkeys. We developed three complementary methods to quantify the balance of increases and decreases in the spontaneous discharge of HFD neurons and validated them by simulations. Unlike the behavioral evoked responses, the spontaneous activity of pallidal and SNr neurons is not dominated by increases. Moreover, the activity of basal ganglia neurons does not include bursts that could reflect disinhibition by the spontaneous pauses of GPe neurons. These findings suggest that the discharge increase/decrease balance during a quiet-wakeful state better reflects the inhibitory input of the HFD basal ganglia neurons than during responses to behavioral events; however, the GPe pauses are not echoed by comparable bursts either in the GPe or in the output nuclei. Changes in the excitatory drive of these structures (e.g., during behavioral activity) thus may lead to a remarkable change in this balance.     

Extrastriatal D2-like receptors modulate basal ganglia pathways in normal and Parkinsonian monkeys

According to traditional models of the basal ganglia-thalamocortical network of connections, dopamine exerts D2-like receptor (D2LR)-mediated effects through actions on striatal neurons that give rise to the "indirect" pathway, secondarily affecting the activity in the internal and external pallidal segments (GPi and GPe, respectively) and the substantia nigra pars reticulata (SNr). However, accumulating evidence from the rodent literature suggests that D2LR activation also directly influences synaptic transmission in these nuclei. To further examine this issue in primates, we combined in vivo electrophysiological recordings and local intracerebral microinjections of drugs with electron microscopic immunocytochemistry to study D2LR-mediated modulation of neuronal activities in GPe, GPi, and SNr of normal and MPTP-treated (parkinsonian) monkeys. D2LR activation with quinpirole increased firing in most GPe neurons, likely due to a reduction of striatopallidal GABAergic inputs. In contrast, local application of quinpirole reduced firing in GPi and SNr, possibly through D2LR-mediated effects on glutamatergic inputs. Injections of the D2LR antagonist sulpiride resulted in effects opposite to those of quinpirole in GPe and GPi. D2 receptor immunoreactivity was most prevalent in putative striatal-like GABAergic terminals and unmyelinated axons in GPe, GPi, and SNr, but a significant proportion of immunoreactive boutons also displayed ultrastructural features of glutamatergic terminals. Postsynaptic labeling was minimal in all nuclei. The D2LR-mediated effects and pattern of distribution of D2 receptor immunoreactivity were maintained in the parkinsonian state. Thus, in addition to their preferential effects on indirect pathway striatal neurons, extrastriatal D2LR activation in GPi and SNr also influences direct pathway elements in the primate basal ganglia under normal and parkinsonian conditions.     

Asynaptic feature and heterogeneous distribution of the cholinergic innervation of the globus pallidus in primates

The internal (GPi) and external (GPe) segments of the primate globus pallidus receive a significant cholinergic (ACh) innervation from the brainstem pedunculopontine tegmental nucleus. The present immunohistochemical study describes this innervation in the squirrel monkey (Saimiri sciureus), as visualized with an antibody raised against choline acetyltransferase (ChAT). At the light microscopic level, unbiased stereological quantification of ChAT positive (+) axon varicosities reveals a significantly lower density of innervation in GPi (0.26 ± 0.03 × 10⁶) than in GPe (0.47 ± 0.07 × 10⁶ varicosities/mm³ of tissue), with the anterior half of both segments more densely innervated than the posterior half. Neuronal density of GPi (3.00 ± 0.13 × 10³ neurons/mm³) and GPe (3.62 ± 0.22 × 10³ neurons/mm³) yields a mean ratio of ChAT+ axon varicosities per pallidal neuron of 74 ± 10 in the GPi and 128 ± 28 in the GPe. At the electron microscopic level, the pallidal ChAT+ axon varicosities are significantly smaller than their unlabeled counterparts, but are comparable in size and shape in the two pallidal segments. Only a minority of ChAT+ varicosities displays a synaptic specialization (12 % in the GPi and 17 % in the GPe); these scarce synaptic contacts are mostly of the symmetrical type and occur exclusively on pallidal dendrites. No ChAT+ axo-axonic synaptic contacts are observed, suggesting that ACh exerts its modulatory action on pallidal afferents through diffuse transmission, whereas pallidal neurons may be influenced by both volumic and synaptic delivery of ACh.     

Subtype-specific GABA transporter antagonists synergistically modulate phasic and tonic GABAA conductances in rat neocortex

GABAergic inhibition in the brain can be classified as either phasic or tonic. gamma-Aminobutyric acid (GABA) uptake by GABA transporters (GATs) can limit the time course of phasic currents arising from endogenous and exogenous GABA, as well as decrease a tonically active GABA current. GABA transporter subtypes 1 and 3 (GAT-1 and GAT-3) are the most heavily expressed of the four known GAT subtypes. The role of GATs in shaping GABA currents in the neocortex has not been explored. We obtained patch-clamp recordings from layer II/III pyramidal cells and layer I interneurons in rat sensorimotor cortex. We found that selective GAT-1 inhibition with NO711 decreased the amplitude and increased the decay time of evoked inhibitory postsynaptic currents (IPSCs) but had no effect on the tonic current or spontaneous IPSCs (sIPSCs). GAT-2/3 inhibition with SNAP-5114 had no effect on IPSCs or the tonic current. Coapplication of NO711 and SNAP-5114 substantially increased tonic currents and synergistically decreased IPSC amplitudes and increased IPSC decay times. sIPSCs were not resolvable with coapplication of NO711 and SNAP-5114. The effects of the nonselective GAT antagonist nipecotic acid were similar to those of NO711 and SNAP-5114 together. We conclude that synaptic GABA levels in neocortical neurons are controlled primarily by GAT-1, but that GAT-1 and GAT-2/3 work together extrasynaptically to limit tonic currents. Inhibition of any one GAT subtype does not increase the tonic current, presumably as a result of increased activity of the remaining transporters. Thus neocortical GAT-1 and GAT-2/3 have distinct but overlapping roles in modulating GABA conductances.     

Role of ionotropic glutamatergic and GABAergic inputs on the firing activity of neurons in the external pallidum in awake monkeys

The neurons in the external segment of the pallidum (GPe) in awake animals maintain a high level of firing activity. The level and pattern of the activity change with the development of basal ganglia disorders including parkinsonism and hemiballism. The GPe projects to most of the nuclei in the basal ganglia. Thus exploring the mechanisms controlling the firing activity is essential for understanding basal ganglia function in normal and pathological conditions. To explore the role of ionotropic glutamatergic and GABAergic inputs to the GPe, unit recordings combined with local injections of receptor antagonists were performed in awake monkeys. Observations on the effects of local application of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide, the N-methyl-D-aspartic acid (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, and the GABAA antagonist gabazine as well as the effects of muscimol blockade of the subthalamic nucleus on the spontaneous firing rate, firing patterns, and cortical stimulation induced responses in the GPe suggested the following: sustained glutamatergic and GABAergic inputs control the level of the spontaneous firing of GPe neurons; both AMPA/kainate and NMDA receptors are activated by glutamatergic inputs; some GPe neurons receive glutamatergic inputs originating from areas other than the subthalamic nucleus; no GPe neurons became silent after a combined application of glutamate and GABA antagonists, suggesting that GPe neurons have intrinsic properties or nonionotropic glutamatergic tonic inputs that sustain a fast oscillatory firing or a combination of a fast and a slow oscillatory firing in GPe neurons.     

The expression of neurokinin-1 receptor at striatal and pallidal levels in normal human brain

To further our knowledge of the site of action of substance P (SP) in the human basal ganglia, we applied single- and double-antigen localization methods to human postmortem tissue to compare the distribution of SP and its high affinity receptor neurokinin-1 (NK1R) at striatal and pallidal levels. The human striatum was found to harbor numerous heterogeneously distributed aspiny neurons that expressed NK1R. Most of them were of small size, but a moderate number of large-sized neurons and a small number of medium-sized neurons also expressed NK1R. The medium-sized NK1R-positive neurons coexpressed parvalbumin and appear to represent a hitherto unknown striatal interneuron. The three types of striatal NK1R-positive neurons were preferentially localized in the peripheral region of the striosomes, which were identified by their intense immunostaining for the limbic system-associated membrane protein. Numerous NK1R expressing neurons also occurred in both external (GPe) and internal (GPi) segments of the globus pallidus, as well as in the ventral pallidum (GPv). There was a marked decreasing rostrocaudal gradient in the number of these neurons in the GPe, but not in the GPi. A multitude of smooth and highly branched SP-immunoreactive fibers pervaded the entire pallidal complex and some of these fibers were in close contact with NK1R-positive neurons in the GPi, as well as in the rostral portion of the GPe. The latter result reveals that the so-called 'direct' striatofugal pathway provides SP-immunoreactive collaterals to the GPe, a finding that is at odd with the current model of basal ganglia organization.     

Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cells

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT 1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.     

Activation of nigral and pallidal dopamine D1-like receptors modulates basal ganglia outflow in monkeys

Studies of the effects of dopamine in the basal ganglia have focused on the striatum, whereas the functions of dopamine released in the internal pallidal segment (GPi) or in the substantia nigra pars reticulata (SNr) have received less attention. Anatomic and biochemical investigations have demonstrated the presence of dopamine D1-like receptors (D1LRs) in GPi and SNr, which are primarily located on axons and axon terminals of the GABAergic striatopallidal and striatonigral afferents. Our experiments assessed the effects of D1LR ligands in GPi and SNr on local gamma-aminobutyric acid (GABA) levels and neuronal activity in these nuclei in rhesus monkeys. Microinjections of the D1LR receptor agonist SKF82958 into GPi and SNr significantly reduced discharge rates in GPi and SNr, whereas injections of the D1LR antagonist SCH23390 increased firing in the majority of GPi neurons. D1LR activation also increased bursting and oscillations in neuronal discharge in the 3- to 15-Hz band in both structures, whereas D1LR blockade had the opposite effects in GPi. Microdialysis measurements of GABA concentrations in GPi and SNr showed that the D1LR agonist increased the level of the transmitter. Both findings are compatible with the hypothesis that D1LR activation leads to GABA release from striatopallidal or striatonigral afferents, which may secondarily reduce firing of basal ganglia output neurons. The antagonist experiments suggest that a dopaminergic "tone" exists in GPi. Our results support the finding that D1LR activation may have powerful effects on GPi and SNr neurons and may mediate some of the effects of dopamine replacement therapies in Parkinson's disease.     

GABA(B) receptors in the median raphe nucleus: distribution and role in the serotonergic control of hippocampal activity.

Previous studies have shown that serotonergic neurons of the median raphe nucleus have a suppressive effect on theta synchronization in the hippocampus. Median raphe lesion, suppression of 5-HT neuronal activity by administration of GABA(A) receptor antagonist or by glutamate blockade or depletion produced long-lasting non-interrupted hippocampal theta in freely behaving rats independent of behavior and in rats anesthetized with urethane. Serotonergic neurons show a characteristic sleep-wake pattern of activity and there is evidence that GABAergic mechanisms play an important role in their regulation. In this study we analyzed the distribution and subcellular localization of GABA(B) receptors in the midbrain raphe complex using combined 5-HT/GABA(B) receptor immunohistochemistry at the light and electron microscopic levels and studied the effects of their pharmacological manipulation on hippocampal electroencephalographic activity in urethane-anesthetized rats. We found that sustained infusion of the GABA(B) receptor agonist baclofen into the median raphe nucleus, using the microdialysis technique, elicited lasting theta activity in the hippocampus. The effect was antagonized by selective GABA(B) receptor antagonists. The predominant localization of GABA(B) receptors in the median, as well as in dorsal raphe was found on serotonergic neurons which strongly indicates that the increase in theta occurrence after baclofen injection resulted from suppression of the serotonergic output originating from the median raphe. On the electron microscopic level, we found GABA(B) receptors located extrasynaptically indicating that these receptors are preferentially activated by strong inputs, i.e. when GABA released from the synaptic terminals is sufficient to spill over from the synaptic cleft. Such conditions might be satisfied during rapid eye movement sleep when GABAergic neurons in the raphe are firing at their highest rate and in rhythmic synchronized bursts.Our data indicate that midbrain raphe GABA(B) mechanisms play an important role in behavioral state control and in hippocampal activity, in particular.     

Distinct afferents to internal and external pallidal segments in the squirrel monkey

The use of retrograde fluorescence double-labeling method has revealed that the internal (GPi) and external (GPe) segments of globus pallidus in squirrel monkey receive projections from different cell populations in striatum and subthalamic nucleus. Striatal neurons projecting either to GPi or GPe formed wide and nonoverlapping cell bands oriented obliquely and covering large portions of putamen and caudate nucleus. Subthalamic neurons projecting to GPe were more abundant and more laterally located than those projecting to GPi. A few cells branching to GPi and GPe were found in subthalamic nucleus but not in striatum. Thus, different striatal and subthalamic neuronal populations influence GPi and GPe in primates.     

Systemic administration of adenosine A(2A) receptor antagonist reverses increased GABA release in the globus pallidus of unilateral 6-hydroxydopamine-lesioned rats: a microdialysis study

The ability of adenosine A(2A) receptor antagonists to exhibit antiparkinsonian activity has recently been reported, but the mechanisms of action are still unknown. Since A(2A) receptors have been localized to GABAergic striatopallidal neurons, it is probable that these antagonists affect the activity of these neurons. In the present study, extracellular GABA basal levels were increased in the ipsilateral striatum and globus pallidus following a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway. The A(2A) receptor-selective antagonist KW-6002 (3mg/kg, p.o.) caused a marked and sustained decrease of extracellular GABA levels in the globus pallidus of the 6-hydroxydopamine-lesioned rats, whereas no changes in GABA levels were observed in the globus pallidus of the non-lesioned rats. Microinjection of the A(2A) receptor agonist CGS21680 (0.005-0.5 microg) into the striatum of non-lesioned animals increased GABA concentrations in the globus pallidus, which was abolished by the voltage-dependent Na(+) channel blocker tetrodotoxin (1 micromol/l) delivered locally to the globus pallidus via the dialysis membrane. Furthermore, intrapallidal infusion of CGS21680 (10 micromol/l) also increased GABA levels in the globus pallidus.These data indicate that GABA release from striatopallidal neurons is regulated through A(2A) receptors in both the striatum and globus pallidus. The reversal of the 6-hydroxydopamine-induced increase in pallidal GABA levels by KW-6002 suggests that the antiparkinsonian effects of A(2A) receptor antagonists occur on the striatopallidal neurons.     

Pre- and postsynaptic localization of GABA(B) receptors in the basal ganglia in monkeys

GABAergic neurotransmission involves ionotropic GABA(A) and metabotropic GABA(B) receptor subtypes. Although fast inhibitory transmission through GABA(A) receptors activation is commonly found in the basal ganglia, the functions as well as the cellular and subcellular localization of GABA(B) receptors are still poorly known. Polyclonal antibodies that specifically recognize the GABA(B)R1 receptor subunit were produced and used for immunocytochemical localization of these receptors at the light and electron microscope levels in the monkey basal ganglia. Western blot analysis of monkey brain homogenates revealed that these antibodies reacted specifically with two native proteins corresponding to the size of the two splice variants GABA(B)R1a and GABA(B)R1b. Preadsorption of the purified antiserum with synthetic peptides demonstrated that these antibodies recognize specifically GABA(B)R1 receptors with no cross-reactivity with GABA(B)R2 receptors. Overall, the distribution of GABA(B)R1 immunoreactivity throughout the monkey brain correlates with previous GABA(B) ligand binding studies and in situ hybridization data as well as with recent immunocytochemical studies in rodents. GABA(B)R1-immunoreactive cell bodies were found in all basal ganglia nuclei but the intensity of immunostaining varied among neuronal populations in each nucleus. In the striatum, interneurons were more strongly stained than medium-sized projection neurons while in the substantia nigra, dopaminergic neurons of the pars compacta were much more intensely labeled than GABAergic neurons of the pars reticulata. In the subthalamic nucleus, clear immunonegative neuronal perikarya were intermingled with numerous GABA(B)R1-immunoreactive cells. Moderate GABA(B)R1 immunoreactivity was observed in neuronal perikarya and dendritic processes throughout the external and internal pallidal segments. At the electron microscope level, GABA(B)R1 immunoreactivity was commonly found in neuronal cell bodies and dendrites in every basal ganglia nuclei. Many dendritic spines also displayed GABA(B)R1 immunoreactivity in the striatum. In addition to strong postsynaptic labeling, GABA(B)R1-immunoreactive preterminal axonal segments and axon terminals were frequently encountered throughout the basal ganglia components. The majority of labeled terminals displayed the ultrastructural features of glutamatergic boutons and formed asymmetric synapses. In the striatum, GABA(B)R1-containing boutons resembled terminals of cortical origin, while in the globus pallidus and substantia nigra, subthalamic-like terminals were labeled. Overall, these findings demonstrate that GABA(B) receptors are widely distributed and located to subserve both pre- and postsynaptic roles in controlling synaptic transmission in the primate basal ganglia.     

Quantifying the neural elements activated and inhibited by globus pallidus deep brain stimulation

Deep brain stimulation (DBS) of the globus pallidus pars interna (GPi) is an effective therapy option for controlling the motor symptoms of medication-refractory Parkinson's disease and dystonia. Despite the clinical successes of GPi DBS, the precise therapeutic mechanisms are unclear and questions remain on the optimal electrode placement and stimulation parameter selection strategies. In this study, we developed a three-dimensional computational model of GPi-DBS in nonhuman primates to investigate how membrane channel dynamics, synaptic inputs, and axonal collateralization contribute to the neural responses generated during stimulation. We focused our analysis on three general neural elements that surround GPi-DBS electrodes: GPi somatodendritic segments, GPi efferent axons, and globus pallidus pars externa (GPe) fibers of passage. During high-frequency electrical stimulation (136 Hz), somatic activity in the GPi showed interpulse excitatory phases at 1-3 and 4-5.5 ms. When including stimulation-induced GABA(A) and AMPA receptor dynamics into the model, the somatic firing patterns continued to be entrained to the stimulation, but the overall firing rate was reduced (78.7 to 25.0 Hz, P < 0.001). In contrast, axonal output from GPi neurons remained largely time-locked to each pulse of the stimulation train. Similar entrainment was also observed in GPe efferents, a majority of which have been shown to project through GPi en route to the subthalamic nucleus. The models suggest that pallidal DBS may have broader network effects than previously realized and the modes of therapy may depend on the relative proportion of GPi and/or GPe efferents that are directly affected by the stimulation.     

Synaptically evoked GABA transporter currents in neocortical glia

The presence, magnitude, and time course of GABA transporter currents were investigated in electrophysiologically characterized neocortical astrocytes in an in vitro slice preparation. On stimulation with a bipolar-tungsten stimulating electrode placed nearby, the majority of cells tested displayed long-lasting GABA transporter currents using both single and repetitive stimulation protocols. Using subtype-specific GABA transporter antagonists, long-lasting GABA transporter currents were identified in neocortical astrocytes that originated from at least two subtypes of GABA transporters: GAT-1 and GAT-2/3. These transporter currents displayed slow rise times and long decay times, contrasting the time course observed for glutamate transporter currents, and are indicative of a long extracellular time course of GABA as well as a role for glial GABA transporters during synaptic transmission.     

Effect of diazoxide on regulation of vesicular and plasma membrane GABA transporter genes and proteins in hippocampus of rats subjected to picrotoxin-induced kindling

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). GABA is taken up and accumulated in synaptic vesicles by the action of vesicular GABA transporter (VGAT) before its release into the synaptic cleft, and removed from synaptic regions by the action of transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, the effects of diazoxide (DIZ) on the VGAT, GAT-1 and GAT-3 mRNA and protein levels in hippocampus, and on the seizure activities of picrotoxin (PTX)-induced kindling rats were observed. DIZ caused increase in the quantity of VGAT mRNAs and proteins, and down regulation of GABA transporters GAT-1 and GAT-3 mRNAs and proteins after the PTX re-kindling. Furthermore, DIZ produced not only a prompt but also a later suppression of PTX-induced seizures. Although DIZ has effects on ATP-sensitive potassium (K(ATP)) channels when measured in vitro, our study suggests that additional mechanisms of action may involve the regulation of GABA transporters, which may aid in understanding epileptogenesis and inform investigators about future design and development of K(ATP) channel openers to treat epilepsy.     

Up-regulation of GABA transporters and GABAA receptor α1 subunit in tremor rat hippocampus

The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABA_A receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR α1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR α1 subunit were all significantly increased in TRMs hippocampus by real time PCR and Western blot, respectively; GAT-1 and GABAR α1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR α1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy.     

Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABA(A) receptors in vivo

Dopaminergic neurons express both GABA(A) and GABA(B) receptors and GABAergic inputs play a significant role in the afferent modulation of these neurons. Electrical stimulation of GABAergic pathways originating in neostriatum, globus pallidus or substantia nigra pars reticulata produces inhibition of dopaminergic neurons in vivo. Despite a number of prior studies, the identity of the GABAergic receptor subtype(s) mediating the inhibition evoked by electrical stimulation of neostriatum, globus pallidus, or the axon collaterals of the projection neurons from substantia nigra pars reticulata in vivo remain uncertain. Single-unit extracellular recordings were obtained from substantia nigra dopaminergic neurons in urethane anesthetized rats. The effects of local pressure application of the selective GABA(A) antagonists, bicuculline and picrotoxin, and the GABA(B) antagonists, saclofen and CGP-55845A, on the inhibition of dopaminergic neurons elicited by single-pulse electrical stimulation of striatum, globus pallidus, and the thalamic axon terminals of the substantia nigra pars reticulata projection neurons were recorded in vivo. Striatal, pallidal, and thalamic induced inhibition of dopaminergic neurons was always attenuated or completely abolished by local application of the GABA(A) antagonists. In contrast, the GABA(B) antagonists, saclofen or CGP-55845A, did not block or attenuate the stimulus-induced inhibition and at times even increased the magnitude and/or duration of the evoked inhibition. Train stimulation of globus pallidus and striatum also produced an inhibition of firing in dopaminergic neurons of longer duration. However this inhibition was largely insensitive to either GABA(A) or GABA(B) antagonists although the GABA(A) antagonists consistently blocked the early portion of the inhibitory period indicating the presence of a GABA(A) component. These data demonstrate that dopaminergic neurons of the substantia nigra pars compacta are inhibited by electrical stimulation of striatum, globus pallidus, and the projection neurons of substantia nigra pars reticulata in vivo. This inhibition appears to be mediated via the GABA(A) receptor subtype, and all three GABAergic afferents studied appear to possess inhibitory presynaptic GABA(B) autoreceptors that are active under physiological conditions in vivo.     

GABA(B) receptors at glutamatergic synapses in the rat striatum

Although multiple effects of GABA(B) receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABA(B) receptor subunits, GABA(B1) and GABA(B2). In addition, to investigate the relationship between GABA(B) receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABA(B1) and GABA(B2) was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABA(B) receptor subunits and vesicular glutamate transporters revealed that labeling for both GABA(B1) and GABA(B2) was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABA(B) heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABA(B) autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABA(B) receptors.