Presynaptic GABA(B) receptors inhibit synaptic inputs to rat subthalamic neurons.

Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude.These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.     

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.     

Synaptically released GABA activates both pre- and postsynaptic GABA(B) receptors in the rat globus pallidus

The globus pallidus (GP) contains abundant GABAergic synapses and GABA(B) receptors. To investigate whether synaptically released GABA can activate pre- and postsynaptic GABA(B) receptors in the GP, physiological recordings were performed using rat brain slice preparations. Cell-attached recordings from GABA(A) antagonist-treated preparations revealed that repetitive local stimulation induced a GABA(B) antagonist-sensitive pause in spontaneous firings of GP neurons. Whole cell recordings revealed that the repetitive stimulation evoked fast excitatory postsynaptic potentials followed by a slow inhibitory postsynaptic potential (IPSP) in GP neurons. The slow IPSP was insensitive to a GABA(A) receptor antagonist, increased in amplitude with the application of ionotropic glutamate receptor antagonists, and was suppressed by the GABA(B) antagonist CGP55845. The reversal potential of the slow IPSP was close to the potassium equilibrium potential. These results suggest that synaptically released GABA activated postsynaptic GABA(B) receptors and induced the pause and the slow IPSP. On the other hand, in the neurons that were treated to block postsynaptic GABA(B) responses, CGP55845 increased the amplitudes of repetitive local stimulation-induced GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) but not the ionotropic glutamate-mediated excitatory postsynaptic currents. Moreover, the GABA(B) receptor specific agonist baclofen reduced the frequency of miniature IPSCs without altering their amplitude distributions. These results suggest that synaptically released GABA also activated presynaptic GABA(B) autoreceptors, resulting in decreased GABA release in the GP. Together, we infer that both pre- and postsynaptic GABA(B) receptors may play crucial roles in the control of GP neuronal activity.     

Presynaptic facilitation of excitatory postsynaptic potential by glucagon in neurons of rat ventromedial hypothalamic slices.

Intracellular recordings were made from neurons in rat ventromedial hypothalamus (VMH), in vitro. Application of glucagon (100 nM to 5 microM) for 2-5 min increased the amplitude of excitatory postsynaptic potential (EPSP) lasting for 10-20 min. Forskolin and 8-bromo-cyclic AMP mimicked glucagon in producing a long-lasting facilitation of the EPSP. These drugs did not affect depolarizing response produced by glutamate. 3-Isobutyl-1-methylxanthine (IBMX) potentiated the time course of glucagon-induced facilitation of the EPSP. These results suggest that glucagon facilitates the EPSP probably by increasing transmitter release through activation of adenylate cyclase.     

Horseradish peroxidase study in rat of the neural connections of the organum vasculosum of the lamina terminalis

Using highly localized injections by a transbuccal approach a study of the afferent and efferent connections of the organum vasculosum laminae terminalis (OVLT) with horseradish peroxidase (HRP) methods was carried out. The results showed that the OVLT has direct connections to several hypothalamic nuclei (anterior, preoptic, lateral preoptic, ventromedial nucleus) and to extrahypothalamic (central gray, locus coeruleus, subfornical organ) regions. There was not a direct projection from the supraoptic nucleus. Some of these connections may be involved in the functional actions of the OVLT observed after central injections of angiotensin II.     

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN), the brain's circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.     

Ultrastructural study of the cellular components of the organum vasculosum lamina terminalis of the rabbit (Oryctolagus cuniculus)

We studied the organum vasculosum of the lamina terminalis (OVLT), under the electron microscope (transmission and scanning) to determine its ultrastructural features. Two cell types are differentiated. Type I cells are typical ependymal cells bearing few cilia. Type II cells present a tanycytic morphology. The latter present a long basal process which travels from the perikarya and intermingles with other neuronal components, finally reaching the perivascular spaces where it comes into close contact with fenestrated capillaries.     

Reduction of glycine receptor-mediated miniature inhibitory postsynaptic currents in rat spinal lamina I neurons after peripheral inflammation

Peripheral inflammation may induce long-lasting sensitization in the central nociceptive system. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the integration and relay of pain-related information. In rats we studied whether changes in passive and active membrane properties and/or alteration of glycine receptor-mediated inhibitory control of spinal lamina I neurons may contribute to central sensitization in a model of peripheral long-lasting inflammation (complete Freund's adjuvant, hindpaw). Spontaneously occurring glycine receptor-mediated miniature inhibitory postsynaptic currents (GlyR-mediated mIPSCs) were recorded in lumbar spinal lamina I neurons. Miniature IPSC rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected by peripheral inflammation. The mean frequency of GlyR-mediated mIPSCs of lamina I neurons ipsilateral to the inflamed hindpaw was, however, significantly reduced by peripheral inflammation when compared with neurons from noninflamed animals. Principal passive and active membrane properties and firing patterns of spinal lamina I neurons were not changed by inflammation. These results indicate that long-lasting peripheral inflammation leads to a reduced glycinergic inhibitory control of spinal lamina I neurons by a presynaptic mechanism.     

Development of GABA(A) receptor-mediated inhibitory postsynaptic currents in hippocampus

Hippocampal CA1 pyramidal cells receive two kinetic classes of GABA(A) receptor-mediated inhibition: slow dendritic inhibitory postsynaptic currents (GABA(A,slow) IPSCs) and fast perisomatic (GABA(A,fast)) IPSCs. These two classes of IPSCs are likely generated by two distinct groups of interneurons, and we have previously shown that the kinetics of the IPSCs have important functional consequences for generating synchronous firing patterns. Here, we studied developmental changes in the properties of GABA(A,fast) and GABA(A,slow) spontaneous, miniature, and evoked IPSCs (sIPSCs, mIPSCs, and eIPSCs, respectively) using whole cell voltage-clamp recordings in brain slices from animals aged P10-P35. We found that the rate of GABA(A,slow) sIPSCs increased by over 70-fold between P11 and P35 (from 0.0017 to 0.12 s(-1)). Over this same age range, we observed a >3.5-fold increase in the maximal amplitude of GABA(A,slow) eIPSCs evoked by stratum lacunosum-moleculare (SL-M) stimuli. However, the rate and amplitude of GABA(A,slow) mIPSCs remained unchanged between P10 and P30, suggesting that the properties of GABA(A,slow) synapses remained stable over this age range, and that the increase in sIPSC rate and in eIPSC amplitude was due to increased excitability or excitation of GABA(A,slow) interneurons. This hypothesis was tested using bath application of norepinephrine (NE), which we found at low concentrations (1 microM) selectively increased the rate of GABA(A,slow) sIPSCs while leaving GABA(A,fast) sIPSCs unchanged. This effect was observed in animals as young as P13 and was blocked by coapplication of tetrodotoxin, suggesting that NE was acting to increase the spontaneous firing rate of GABA(A,slow) interneurons and consistent with our hypothesis that developmental changes in GABA(A,slow) IPSCs are due to changes in presynaptic excitability. In contrast to the changes we observed in GABA(A,slow) IPSCs, the properties of GABA(A,fast) sIPSCs remained largely constant between P11 and P35, whereas the rate, amplitude, and kinetics of GABA(A,fast) mIPSCs showed significant changes between P10 and P30, suggesting counterbalancing changes in action potential-dependent GABA(A,fast) sIPSCs. These observations suggest differential developmental regulation of the firing properties of GABA(A,fast) and GABA(A,slow) interneurons in CA1 between P10 and P35.     

Immunocytochemical localization of GABA(B) receptors in mesencephalic trigeminal nucleus neurons in the rat.

We examined immunoreactivities for gamma-aminobutyric acidB-receptor (GABA(B)R) subtypes, GABA(B)R1 and GABA(B)R2, in the mesencephalic trigeminal nucleus neurons (MTN neurons) of the rat. Immunoreactivity for GABA(B)R1 was prominent in cell bodies of MTN, whereas that for GABA(B)R2 was very weak, if existed. For electron microscopy, the immunogold-silver method for GABA(B)R1 was combined with the immunoperoxidase method for glutamic acid decarboxylase (GAD: the synthetic enzyme of GABA). Immunogold-silver particles indicating GABA(B)R1 immunoreactivity were distributed widely in the cytoplasm of the cell bodies postsynaptic to GAD-immunoreactive axon terminals, but were rarely associated with synaptic membrane specialization or extrasynaptic sites of plasma membrane. It has been indicated that GABA(B)R1 may not be transported to plasma membrane when no GABA(B)R2 exists. Thus, it was presumed that GABA(B)R1 in the cell body of the rat MTN neurons might not be involved in the synaptic transmission.     

GABA(B) receptors are the first target of released GABA at lamina I inhibitory synapses in the adult rat spinal cord

We have previously provided functional evidence that glycine and GABA are contained in the same synaptic vesicles and coreleased at the same synapses in lamina I of the rat spinal dorsal horn. However, whereas both glycine receptors (GlyRs) and GABA(A) receptors (GABA(A)Rs) are expressed on the postsynaptic target, under certain conditions inhibitory events appeared to be mediated by GlyRs only. We therefore wanted to test whether GABA(B) receptors could be activated in conditions where GABA released was insufficient to activate GABA(A)Rs. Focal stimulation in the vicinity of visually identified lamina I neurons elicited monosynaptic IPSCs in the presence of ionotropic glutamate receptor antagonists. Pairs of stimuli were given at different interstimulus intervals (ISI), ranging from 25 ms to 1 s to study the depression of the second of evoked IPSCs (paired pulse depression; PPD). Maximal PPD of IPSCs was 60 +/- 14% (SE) (of the conditioning pulse amplitude), at ISI between 150 and 200 ms. PPD was observed with IPSCs evoked at stimulus intensities where they had no GABA(A)R component. PPD of small evoked IPSCs was not affected by the GABA(A)R antagonist bicuculline but significantly attenuated by 10-30 microM CGP52432, a specific GABA(B) receptor antagonist. These data indicate that, under conditions where GABA released is insufficient to affect postsynaptic GABA(A)Rs at lamina I inhibitory synapses, significant activation of presynaptic GABA(B) receptors can occur.     

Presynaptic modulation of GABAergic inhibition by GABA(B) receptors in the rat's inferior colliculus

Whole-cell patch clamp recordings were made from neurons in a brain slice preparation of the inferior colliculus in 11-15-day-old rat pups. Synaptic responses were elicited by applying a current pulse to the lateral lemniscus just below the central nucleus of the inferior colliculus. To examine GABAergic inhibition in the inferior colliculus all excitatory postsynaptic potentials and glycinergic inhibitory postsynaptic potentials were blocked by bath application of their respective antagonists and the contribution of GABA(B) receptors was determined for the remaining inhibitory postsynaptic potentials. For most cells the isolated inhibitory postsynaptic potential was completely blocked by the GABA(A) receptor antagonist, bicuculline, but was unaffected by the GABA(B) receptor antagonist, phaclofen. The GABA(B) receptor agonist, baclofen (10-20 microM), decreased the amplitude of the inhibitory postsynaptic potentials. This effect was completely blocked by phaclofen. Baclofen did not increase the cell membrane conductance or alter the rate of firing produced by depolarization of the cell membrane. In contrast, muscimol, a GABA(A) receptor agonist, greatly increased membrane conductance and lowered the firing rate produced by depolarization. Our results indicate that GABAergic inhibition in the auditory midbrain can be reduced by the activation of GABA(B) receptors and suggest that the effects are presynaptic.     

Noradrenergic innervation of vasopression-containing neurons in the rat hypothalamic supraoptic nucleus

The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron microscopically by using double-labeling immunocytochemistry combining the pre-embedding peroxidase-anti-peroxidase method with post-embedding immunocolloidal gold staining. Noradrenaline-like immunoreactive axon terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neurons in the SON. This study provides morphological evidence for noradrenergic control of neuronal activity of VP-containing neurons at the SON level.     

Histamine suppresses non-NMDA excitatory synaptic currents in rat supraoptic nucleus neurons

Whole cell patch-clamp recordings were obtained from supraoptic neurons to investigate the effects of histamine on excitatory postsynaptic currents evoked by electrical stimulation of areas around the posterior supraoptic nucleus. When cells were voltage-clamped at -70 mV, evoked excitatory postsynaptic currents had amplitudes of 88.4 +/- 9.6 pA and durations of 41.1 +/- 3.0 ms (mean +/- SE; n = 43). With twin stimulus pulses (20 Hz) used, paired-pulse facilitation ratios were 1.93 +/- 0.12. Bath application of 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX) abolished synaptic currents. Histamine at concentrations approximately 0.1-10 microM reversibly suppressed excitatory postsynaptic currents in all supraoptic neurons tested. Within 2 min after application of (10 microM) histamine, current amplitudes and durations decreased by 61. 5 and 31.0%, respectively, with little change in the paired-pulse facilitation ratio. Dimaprit or imetit (H(2) or H(3) receptor agonists) did not reduce synaptic currents, whereas pyrilamine (H(1) receptor antagonist) blocked histamine-induced suppression of synaptic currents. When patch electrodes containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) were used to record cells, histamine still suppressed current amplitudes by 49.1% and durations by 41.9%. Similarly, intracellular diffusion of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) and H(7) did not abolish histamine-induced suppression of synaptic currents, either. Bath perifusion of 8-bromo-quanosine 3',5'-cyclic monophosphate reduced current amplitudes by 32.3% and durations by 27.9%. After bath perfusion of slices with N(omega)-nitro-L-arginine methyl ester (L-NAME), histamine injection decreased current amplitudes only by 31.9%, much less than the inhibition rate in control (P < 0.01). In addition, histamine induced little change in current durations and paired-pulse facilitation ratios, representing a partial blockade of histamine effects on synaptic currents by L-NAME. In supraoptic neurons recorded using electrodes containing BAPTA and perifused with L-NAME, the effects of histamine on synaptic currents were completely abolished. Norepinephrine injection reversibly decreased current amplitudes by 39.1% and duration by 64.5%, with a drop in the paired-pulse facilitation ratio of 47.9%. Bath perifusion of L-NAME, as well as intracellular diffusion of GDP-beta-S, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine, or BAPTA, failed to block norepinephrine-induced suppression of evoked synaptic currents. The present results suggest that histamine suppresses non-N-methyl-D-aspartate synaptic currents in supraoptic neurons through activation of H(1) receptors. It is possible that histamine first acts at supraoptic cells (perhaps both neuronal and nonneuronal) and induces the production of nitric oxide, which then diffuses to nearby neurons and modulates synaptic transmission by a postsynaptic mechanism.     

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.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in cerebellar inhibitory interneurons.

The inhibitory interneurons in the molecular layer of the cerebellar cortex form a complex network, interconnected by both chemical and electrotonic synapses. Previous work, using voltage optical imaging in an isolated cerebellum, has indicated that these interneurons also form presynaptic inhibitory interconnections. Here we examine the participation of GABA(B) receptors in the proposed presynaptic inhibition by recording from the molecular layer interneurons (MLI) in cerebellar slices. The GABA(B) agonist, baclofen, profoundly depressed synaptic transmission; a concentration of 10 microM decreased the frequency of spontaneous inhibitory synaptic potentials by 82 +/- 15% and of miniature synaptic potentials by 75 +/- 13%. In simultaneous recording from two synaptically interconnected MLIs, baclofen (10 microM) increased the failure rate of synaptic transmission by a factor of 3, confirming a presynaptic mechanism, most likely mediated by a decrease in calcium conductance. A postsynaptic effect of baclofen was also found; 10 microM decreased the spontaneous firing rate by 55 +/- 19% even in the presence of synaptic blockers. One hundred micromolar baclofen induced an averaged hyperpolarization of 6 +/- 2 mV or an averaged 7.8 +/- 3 pA net outward current that can account for the decrease in firing rate. The outward current reflects a reduction in a tonic Ca(2+) current, since it was abolished by blocking Ca(2+) currents and remained unchanged in the presence of Ba(2+). Application of the specific GABA(B) blocker, CGP 55845A (1 microM), not only reversed the effects of baclofen but also increased the spontaneous firing rate and synaptic activity when applied alone. Thus in slice preparations, GABA(B) receptors are tonically activated by endogenous GABA. The temporal role of GABA(B) receptors was tested using the paired-pulse paradigm. Recording from two synaptically interconnected MLIs showed a 3.5 times lower probability of release for the second stimulus. In the isolated cerebellar preparation, a robust depression of the second inhibitory response was observed. This depression was partially blocked by CGP 55845A (2 microM). We conclude that both the pre- and postsynaptic effects of baclofen are mediated by GABA(B) receptors that decrease Ca(2+) currents. These can serve a modulatory role as well as participating in shaping the temporal interactions between consecutive inputs.     

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.