Subcellular Localization of the GABAA Receptor γ2 Subunit in the Rat Spinal Cord

The fine subcellular organization of the GABA_A receptor complex in the adult rat spinal ventral horn was analysed by immunocytochemistry using a specific polyclonal antiserum raised against the γ subunit. This subunit confers benzodiazepine sensitivity on the chloride channel of the GABA_A receptor. With both fluorescent and peroxidase staining, the immunoreactivity was mainly observed in the grey matter and more specifically in the dorsal and ventral horns on medium and large neurons. A high number of immunostained somata were clustered in regions corresponding to motor nuclei. On the neuronal surface, labelling appeared as fluorescent dots over the more diffuse staining that was present on the soma and proximal part of dendrites. At the ultrastructural level, peroxidase end product was in most cases associated with the internal side of postsynaptic differentiations facing terminal boutons enriched with pleiomorphic small clear vesicles. The positively stained synapses were encountered on proximal dendrites of neurons and throughout the neuropil of the ventral horn (layers VII-IX). An immunoreactivity on the postsynaptic membrane was occasionally found to decorate large pieces of membrane not directly apposed to presynaptic active zones. In addition, presynaptic labelling was observed at axoaxonic contacts and at extrasynaptic sites on membranes within boutons, sometimes themselves apposed to γ2 immunoreactivity. Finally, we also observed γ2 immunoreactivity at the cytosolic face of the plasma membrane of some glial elements. These results give morphological evidence for the involvement of GABA_A receptors in both post- and presynaptic inhibition in the rat spinal ventral horn. The presence of γ2 subunit immunoreactivity at these different synaptic contacts suggests that the two types of inhibition can be modulated by benzodiazepine drugs. The findings also provide anatomical evidence for the possible regulation of GABA release through an autoreceptor, and for GABAergic communication between neuronal and glial components.     

GABAA Receptor Function in the Early Period After Transient Forebrain Ischaemia in the Rat

The purpose of this study was to evaluate the function of the GABA_A receptor following transient forebrain ischaemia. The GABA-stimulated chloride (³⁶Cl^?) uptake into synaptoneurosomes was determined as an indicator of GABA_A receptor function. Synaptoneurosomes were isolated from control rats and rats in which the forebrain was made ischaemic by way of the two-vessel occlusion model. Animals subjected to ischaemia were killed at the end of the ischaemic insult and at 30 min or 2 or 5 h of recirculation. The results showed a reduction of 75% in GABA-mediated ³⁶Cl^? uptake in synaptoneurosomes isolated from animals shortly (<0.5 h) after the ischaemic episode (P < 0.01). After longer recirculation periods the GABA-mediated ³⁶Cl^? uptake reached preischaemic control levels. To investigate whether alterations in ³⁶Cl^? uptake were related to the synaptoneurosomal metabolic status, the synaptoneurosomal ATP content was measured. The time course of the ATP recovery correlated with the recovery of the GABA-mediated ³⁶Cl^? uptake (r= 0.7, P < 0.001). To investigate the importance of ATP in GABA-mediated ³⁶Cl^?uptake more directly, synaptoneurosomes isolated from control rats were exposed to chemically induced ATP depletion with rotenone, an inhibitor of oxidative phosphorylation. This resulted in similar reductions in both ATP level and GABA-stimulated ³⁶Cl^? uptake as observed after in vivo ischaemia. These findings indicate that GABA_A receptor function is transiently impaired in the early postischaemic period in a way which is closely related to alterations in cellular energy metabolism. The relevance of these findings to the development of ischaemic cell death is discussed.     

Effect of Anticonvulsant Felbamate on GABAA Receptor System

Felbamate (FBM, 2-phenyl-1,3-propanediol dicarbamate), a potential antiepileptic drug (AED), has an unknown mechanism of action. We examined possible interaction of FBM with GABAA ergic transmission. FBM did not alter specific binding of ligands to GABA. benzodiazepine, and picrotoxin sites of the oligomeric GABAA receptor complex to rat brain membranes, nor did it enhance the effect of GABA on 36Cl-influx in well-characterized cultured spinal cord neurons. These results suggest that the anticonvulsant effect of FBM does not involve GABAA ergic transmission.     

Activation of the GABAA Receptor Inhibits the Proliferative Effects of bFGF in Cortical Progenitor Cells

Recent studies have localized γ-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABA_A receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABA_A receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the α₁ subunit of the GABA_A receptor but not for the α₂, α₃ or α₅ subunits. The α₁ subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABA_A receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABA_A receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.     

Expression of NMDA and High-affinity Kainate Receptor Subunit mRNAs in the Adult Rat Retina

The expression patterns of nine genes encoding the N -methyl- d -aspartate (NMDA) receptor subunits NR1 and NR2A, NR2B, NR2C and NR2D, and the high-affinity kainate receptor subunits KA1, KA2, GluR6 and GluR7, were studied in the adult rat retina by in situ hybridization. Hybridization with [³⁵S]dATP-labelled oligonucleotide probes revealed the expression of four of the NMDA receptor subunits (NR1, NR2A, NR2B and NR2C) and three of the high-affinity kainate receptor subunits (KA2, GluR6 and GluR7) in the retina. The NMDA receptor subunit NR2D and the high-affinity kainate receptor subunit KA1 could not be detected. In the ganglion cell layer, virtually every ganglion cell or displaced amacrine cell expressed the receptor subunits NR1, NR2A, NR2B, NR2C, KA2 and GluR7. The GluR6 subunit was expressed in a more restricted manner in the ganglion cell layer. In the inner nuclear layer, the receptor subunits NR1 and KA2 were homogeneously distributed, and therefore are most likely expressed by all cell types in this layer. The GluR6, NR2A, NR2B and NR2C subunits were expressed by subsets of amacrine cells. Labelling for NR2C was also found above the middle of the inner nuclear layer, corresponding to the location of bipolar cell somata. The GluR7 subunit was expressed by most amacrine and bipolar cells. These findings suggest that NMDA and high-affinity kainate receptor subunits could be present at a majority of glutamatergic retinal synapses.     

Functional Heterogeneity of Hippocampal GABAA Receptors

γ-Aminobutyric acid type A (GABA_A) receptors were studied in cultured neurons taken from rat hippocampus at early postnatal stages. GABA-induced whole-cell currents showed a broad range of peak amplitudes and time-courses of desensitization. Dose – response curves of rapidly and slowly desensitizing cells revealed EC₅₀ values of 8.5 and 37.3 μM GABA, respectively, with the Hill coefficient being greater than unity. The main-state conductance of GABA_A receptor channels was 28 – 31 pS in all cells. GABA responses of low-affinity cells were more strongly affected by benzodiazepine receptor agonists (e.g. flunitrazepam, clonazepam) and inverse agonists (e.g. methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate), as compared to cells exhibiting high-affinity GABA responses. Currents were also potentiated by zolpidem, but were little affected by Ro 15-4513 and Zn²⁺. These data suggest the presence of physiologically and pharmacologically distinct GABA_A receptor isoforms in neurons of the early postnatal hippocampus, which may subserve different inhibitory control mechanisms in this brain region.     

Ultrastructural Localization of a Voltage-gated K+ Channel a Subunit (Kv1.2) in the Rat Cerebellum

A highly specific monoclonal antibody and pre-embedding immunocytochemistry were employed to examine the distribution of the K⁺ channel a subunit K_v1.2 in the rat cerebellum. At the light microscopic level, the heaviest immunoreactivity was seen in the basket cell pinceau at the base of Purkinje cells, with lighter staining of basket and Golgi cell bodies and a punctate pattern in the granule cell and molecular layers. Electron microscopy was performed to identify the ultrastructural location of K_v1.2 α subunit in these labelled structures. This revealed that the labelling of the pinceau was confined to the preterminal axonal plexus, the area immediately around the Purkinje axon initial segment being relatively devoid of staining. Basket cell parent axons were not immunostained, but gave rise to heavily stained fine processes. Immunoreactivity was also seen in myelinated axons in the granule cell layer and in the medial cerebellar nucleus, the staining being most concentrated at the juxtaparanodal regions of the axons. An unusual pattern of staining was seen in some mossy fibre terminals, with staining restricted to fine protuberances of mossy fibre glomeruli. Structures contacted by these protuberances included adjoining glial processes. Immunostaining was absent from Purkinje cell bodies, dendrites, their axon initial segments and their terminals in the medial cerebellar nucleus. In this study, the a subunit K_v1.2 was localized to a number of different cell types in the cerebellum. Each neuronal type displays a distinct subcellular distribution of the subunit.     

Multifaceted Interaction of Corticosteroids with the Intracellular Receptors and with Membrane GABAA Receptor Complex in the Rat Brain

In vitro assays using crude synaptosomal membrane preparations from the cortex and cytosolic fraction of hippocampus have shown that in the brain, steroids can bind to the intracellular corticosteroid receptors as well as to the membrane-bound GABA-receptor complex. Corticosteroid, deoxycorticosterone and spironolactone bound with higher affinity to the mineralocorticoid (relative binding affinity (IC₅₀ in nM) 1.2, 3.9 and 4.9, respectively) than to the glucocorticoid receptors (IC₅₀ 5.2, 14.0 and 88.0 nM, respectively) in hippocampal cytosol. They enhanced significantly the binding of [³⁵S]t-butylbicyclophosphorothionate to cortical membrane. Steroids such as 3α,5α-tetrahydroxydeoxycorticosterone and 3a-hydroxy-5α-dihydroprogesterone displaced the binding of [³⁵S]t-butylbicyclophosphorothionate with IC₅₀ (in nM) of 236.7 and 315.0, respectively. In the presence of 10 to 12.5μM added GABA, they bound with higher affinity (IC₅₀ 18.0 and 20.5 nM, respectively). Pentobarbital also bound to this site with IC₅₀ of 430,000 and 240,000 nM, respectively, in the absence and presence of GABA. These compounds also enhanced the binding of [³H]flunitrazepam which was not affected by the presence of added GABA. They showed no affinity for mineralocorticoid or glucocorticoid receptors. Data from this study showed that steroids which preferentially bound to the mineralocorticoid receptor sites (corticosterone, deoxycorticosterone and spironolactone) also enhanced the binding of [³⁵S]t-butylbicyclophosphorothionate to their recognition sites. Steroids which did not interact with the intracellular receptors (3α,5α-tetrahydroxydeoxycorticosterone and 3a-hydroxy-5α-dihydroprogesterone) displaced [³⁵S]t-butylbicyclophosphorothionate binding and enhanced [³H]flunitrazepam binding.     

Developmental Patterns in the Regulation of Chloride Homeostasis and GABAA Receptor Signaling by Seizures

Summary:  GABA_A receptors have dual functions during development. They depolarize immature neurons but hyperpolarize more mature neurons. This functional switch has been attributed to age-related differences in the relative abundance of cation chloride cotransporters, such as KCC2 and NKCC1, which regulate chloride homeostasis. Certain insults, such as trauma, ischemia, and seizures, if they occur when GABA_Aergic signaling is hyperpolarizing, such as in the adult brain, can lead to reappearance of the immature, depolarizing synaptic responses to GABA_A receptor activation. In certain cases, this has been associated with either reduced expression of KCC2 or increase in NKCC1. In epilepsy, the depolarizing effects of GABA_A receptors have been proposed to be important for the acquisition and/or maintenance of the epileptic state. Using the kainic acid model of status epilepticus, we have studied the effects of repetitive neonatal episodes of status epilepticus on the expression of cation chloride cotransporter KCC2 in the neonatal hippocampus. In contrast to adults, seizures increased KCC2 mRNA expression in the CA3 region of the neonatal hippocampus. The contrasting patterns of regulation of KCC2 by seizures in mature and immature neurons may be one of the age-related factors that protect the neonatal brain against the development of epilepsy.     

Selective Allocation of GABAA Receptors Containing the α1 Subunit to Neurochemically Distinct Subpopulations of Rat Hippocampal Interneurons

The identification of a large variety of GABA_A receptor subunits by molecular cloning suggests the existence of multiple receptor subtypes differing in localization and functional properties. In the present study we analysed immunohistochemically the cellular distribution of GABA_A receptors containing the α1 subunit in the rat hippocampus with a subunit-specific antiserum. Prominent staining of numerous interneurons was evident in Ammon's horn and the dentate gyrus, which contrasted with moderate and diffuse immunoreactivity in the dendritic layers of pyramidal and granule cells. Double immunofluorescence staining with antibodies to GABA revealed that a subset of GABAergic neurons in the hippocampus were immunoreactive for the α1 subunit. To determine whether these cells represent distinct subpopulations of interneurons, we analysed the co-localization of the GABA_A receptor α1 subunit with selective markers of hippocampal interneurons (selected calcium-binding proteins and neuropeptides). In both Ammon's horn and the dentate gyrus, all parvalbumin-positive neurons and 50% of calretinin-positive neurons were double-labelled, whereas interneurons containing calbindin-D_28k were devoid of α1 subunit staining. Similarly, most neurons positive for neuropeptide Y and a subset of somatostatin-positive cells were double-labelled, in contrast to cholecystokinin- and vasoactive intestinal peptide-containing cells, which lacked the α1 subunit staining. These results demonstrate cell-specific expression of GABA_A receptors containing the α1 subunit among subsets of hippocampal interneurons, pointing to a pronounced functional specialization of these cells. Furthermore, the prominent expression of GABA_A receptors by interneurons suggests that disinhibition may be of major functional relevance in regulating the balance between excitation and inhibition in hippocampal circuits.     

Localization of the mRNAs for Two Dopamine D2 Receptor Isoforms in the Rat Brain

Abstract: Two molecular forms of the dopamine D., receptor were generated by alternative RNA splicing. To investigate the relative distributions of the two mRNAs encoding the D2 receptor isoforms, D2(415) and D2(444), we performed in situ hybridization histochemistry in the rat brain with the two oligonucleotide probes. An insert probe complementary to an additional fragment of the D, receptor mRNA cloned from the rat brain, and a spanning probe complementary to its contiguous sequence were used. These 48 base probes were 3'-end labeled with [35S]dATP. The brains were dissected from male SD rats and frozen in dry ice and acetone. Cryostat sections (16 um) were collected on gelatin coated slides and stored at – 20^oC. In situ hybridization studies were conducted with a probe concentration of 1 × 10e dpm/100 ^1 of buffer per brain slice at 37^oC for 18–20 h in a humid chamber. The slides were washed, dried and exposed to tritium sensitive film for one week. The autoradiograph showed that both mRNA were present at high levels in the corpus striatum, accumbens nucleus and substantia nigra (pars compacta). Identical patterns of labeling were obtained in the rat brain using both the insert and spanning probes, although the optical densities detected with the insert probe were higher than those with the spanning probe in the corpus striatum. This suggests that both D2 receptor mRNAs are expressed similarly in each region of the rat brain and D2(14) expressed dominantly in the corpus striatum.     

Effects of the Anticonvulsant Losigamone and Its Isomers on the GABAA Receptor System

Summary: We conducted in vitro studies to clarify the possible involvement of GABA, receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+losigamone) and AO-294 (-losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, ≤100 μM losigamone did not affect the specific binding of [³H]GABA, [³H]flunitrazepam, or [³⁵S]t-butyl-bicyclo-phosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10-8-10-5 M, stimulated ³⁶Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10^-5 M potentiated the effect of a suboptimal concentration of exogenous GABA M on 3³⁶Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS- induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating ³⁶Cl influx in the spinal cord neurons by a GABA-sensitive mechanism and at a side distant from the GABA channel.     

The Convergence of Axon Terminals from the Mediodorsal Thalamic Nucleus and Ventral Tegmental Area on Pyramidal Cells in Layer V of the Rat Prelimbic Cortex

We investigated the ultrastructural basis of the synaptic convergence of afferent fibres from the mediodorsal thalamic nucleus (MD) and the ventral tegmental area (VTA) on the prefrontal cortical neurons of the rat by examining the synaptic relationships between thalamocortical or tegmentocortical terminals labelled with anterograde markers [lesion-induced degeneration or transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA—HRP)] and randomly selected unlabelled apical dendrites of layer V pyramidal cells in the prelimbic cortex. WGA—HRP-labelled terminals from the VTA ranged in diameter from 0.7 to 2.8 μm and established synaptic contacts with large dendritic profiles, i.e. proximal segments of apical dendritic shafts and spines from layer V pyramidal cells. Symmetrical synapses, i.e. inhibitory synapses, were more often seen than asymmetrical ones. Degenerating terminals from the MD formed asymmetrical synapses on dendritic spines or occasionally on small dendritic shafts of apical dendrites from layer V pyramidal cells, which received tegmentocortical synapses, mostly within layer III. Thalamocortical synapses were more distally distributed over common apical dendrites than tegmentocortical synapses, although some of them overlapped. The numerical density of direct synaptic inputs from the MD and VTA was low. These results suggest that fibres from the VTA exert their inhibitory effects directly on pyramidal cells in layer V via synaptic junctions with apical dendrites of these pyramidal cells, and that the tegmentocortical fibres are in an ideal anatomical position to modulate the reverberatory circuits between the MD and the prelimbic cortex.     

Development of Action Potential-dependent and Independent Spontaneous GABAA Receptor-mediated Currents in Granule Cells of Postnatal Rat Cerebellum

The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl^-conditions. Between postnatal days 7 and 14 (P7–14), bicuculline-and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on tonic current of-12 ± 1.8 pA at -70 mV, that was accompanied by noise with variance of 17 ± 3 pA². Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by?60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (?35%). Between P40 and P53, slPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of-17 pA and 22 pA² at-70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABA_A receptor-mediated slPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABA_A receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities.     

Expression of NMDA Receptor mRNAs in Rat Motoneurons is Down-regulated after Axotomy

The cytotoxic effects of glutamate via the N -methyl-D-aspartate (NMDA) receptor have been suggested to take part in the events leading to death of motoneurons after neonatal axotomy. By the use of in situ hybridization and immunohistochemistry we have investigated motoneuron mRNA expression of the NMDA receptor subunits NR1, NR2B and NR2D and of the NR1 subunit protein in two lesion models leading to partial motoneuron death: sciatic nerve transection early postnatally in the rat and ventral root avulsion in the adult rat. The results were compared with a lesion model with no subsequent death of motoneurons, i.e. sciatic nerve transection in the adult rat. All lesions were followed by down-regulation of the mRNAs for all studied subunits in severed motoneuron populations; down-regulation was detectable already at early stages postoperatively before any significant death had taken place. The strongest down-regulation was in fact seen in the lesion with the largest loss of motoneurons (ventral root avulsion). The reduction in the expression of NR1 mRNA was paralleled by a decrease in NR1 subunit protein. We conclude that down-regulation of NMDA receptor subunit expression is part of the acute response to axonal injury in motoneurons, whether or not neuronal death follows, and that the susceptibility of lesioned motoneurons to excitotoxic effects should be highest early after axonal injury.     

Altered Gene Expression of the N-Methyl-d-Aspartate Receptor Channel Subunits in Purkinje Cells of the Staggerer Mutant Mouse

The gene expression of five NMDA receptor channel subunits, the ɛ1 ɛ2, epsiv;3, ɛ4 and ζ1 subunits, was examined in cerebellar Purkinje cells of the staggerer mouse at postnatal day 21. In the midline region of the staggerer cerebellum, signals for the ɛ1, ɛ4, and ζ1 subunit mRNAs were distributed in Purkinje cells, which have a large cell body aligned in a monolayer between the granular and molecular layers. In addition to the midline region, labelled neurons in the intermediate cerebellar region were, though at lower levels, aligned almost in a monolayer between the granular and molecular layers. In the hemisphere, most labelled neurons occurred in various locations in the granular layer and the cerebellar medulla. These regions, populated with Purkinje cells expressing the ɛl, ɛ4, and ζ1 subunit mRNAs, were separated from each other by narrow gap regions that contained neurons without any detectable NMDA receptor channel subunit mRNAs. These results suggest that there is discrete mediolateral heterogeneity in staggerer Purkinje cell populations, in terms of expression properties of the NMDA receptor channel subunits. When compared with wild-type Purkinje cells that express the ζ1 subunit alone, additional expression of the E subunits presumably explains the persistence of NMDA responses in adult staggerer Purkinje cells (Dupont et al Neuroscience, 12, 613-619, 1984).     

Radioautographic Evidence that the GABAA Receptor Antagonist SR 95531 is a Substrate Inhibitor of MAO-A in the Rat and Human Locus Coeruleus

The locus coeruleus (LC), a major noradrenergic nucleus in the brain, probably has a functional role in the regulation of anxiety states as well as vigilance, attention, learning and memory. LC neurons are under the inhibitory control of γ-aminobutyric acid (GABA) via ionotropic GABA_A receptors. However, to date, little is known of the receptor binding characteristics of these neurons. In the present investigation we therefore examined by receptor radioautography the localization of the binding sites for different components of the GABA_A receptor complex in the rat and human LC. Both rat and human LC neurons have a high density of binding sites for the pyridazinyl-GABA derivative [³H]SR 95531 (gabazine, a GABA_A receptor antagonist for low affinity GABA recognition sites). However, at the concentrations used, no binding sites in the LC were detectable for the benzodiazepine receptor antagonist [³H]flumazenil, the GABA_A receptor agonist (for high affinity sites) [³H]muscimol or the ionophore ligand [³⁵S]t -butyl bicyclophosphorothionate (TBPS). Unexpectedly, the pharmacological specificity of [³H]SR 95531 binding to the LC differed markedly from that to most brain regions (IC₅₀ values for GABA and RU 5135 respectively in the LC were > 10^-2 and 10^-3 M; and, for example, in the dentate gyrus the most labelled structure after the LC, 8 × 10^-7 and 1.8 × 10^-9 M). These differences prompted the further characterization of [³H]SR 95531 binding in the LC, revealing a significant affinity for monoamine oxidase type A (MAO-A), which is highly concentrated in this nucleus. In a competition binding study, a reduction of up to 25% of the [³H]SR 95531 binding was observed with MAO-A but not MAO-B inhibitors, at concentrations which produce maximum but selective enzyme inhibition. Correspondingly, 2 h after the oral administration of supramaximal doses of the MAO-A inhibitors moclobemide and Ro 41 -1049 (but not the MAO-B inhibitor lazabemide) the in vitro binding of [³H]SR 95531 was markedly reduced (by 77 and 82% of controls respectively). Moreover, enzyme radioautography with [³H]Ro 41 -1049 revealed that SR 95531 has a significant affinity for MAO-A (ICgo values were 10^-5 and 4x 10^-6 M in the LC and dentate gyrus respectively) but not for MAO-B ([³H]lazabemide binding). Altogether, these findings suggest that the high-affinity binding of [³H]SR 95531 to the LC mainly reflects its affinity for MAO-A, which questions its utility as a selective ligand for low-affinity GABA recognition sites in the CNS. It remains to be seen whether the dual pharmacological profile of SR 95531 (desinhibition of LC neurons and facilitation of noradrenergic transmission) can be exploited as a principle in the development of new anxiolytics or antidepressants.