GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.     

Cross-talk between NMDA and GABA<Subscript>A</Subscript> receptors in cultured neurons of the rat inferior colliculus

Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABA_A receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk between the NMDA receptor and the GABA_A receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional interactions between the NMDA receptor and the GABA_A receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L⁻¹ were suppressed by the pre-perfusion of GABA at 100 μmol L⁻¹, indicating cross-inhibition of NMDA receptors by activation of GABA_A receptors. Moreover, we found that the currents induced by GABA at 100 μmol L⁻¹ (I _GABA) were not suppressed by the pre-perfusion of 100 μmol L⁻¹ aspartate, but those induced by GABA at 3 μmol L⁻¹ were suppressed, indicating concentration-dependent cross-inhibition of GABA_A receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca²⁺ channels with CdCl₂ in a solution that contained Ca²⁺, however, CdCl₂ effectively attenuated the inhibition of I _GABA by aspartate when it was perfused in a solution that contained Ba²⁺ instead of Ca²⁺ or a solution that contained Ca²⁺ and 10 mmol L⁻¹ BAPTA, a membrane-permeable Ca²⁺ chelator, suggesting that this inhibition is mediated by Ca²⁺ influx through NMDA receptors, rather than voltage-dependent Ca²⁺ channels. Finally, KN-62, a potent inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I _GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction between NMDA and GABA_A receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.     

Pharmacological characterization of GABA<Subscript>A</Subscript> receptors in taurine-fed mice

Background

Taurine is one of the most abundant free amino acids especially in excitable tissues, with wide physiological actions. Chronic supplementation of taurine in drinking water to mice increases brain excitability mainly through alterations in the inhibitory GABAergic system. These changes include elevated expression level of glutamic acid decarboxylase (GAD) and increased levels of GABA. Additionally we reported that GABA_A receptors were down regulated with chronic administration of taurine. Here, we investigated pharmacologically the functional significance of decreased / or change in subunit composition of the GABA_A receptors by determining the threshold for picrotoxin-induced seizures. Picrotoxin, an antagonist of GABA_A receptors that blocks the channels while in the open state, binds within the pore of the channel between the β2 and β3 subunits. These are the same subunits to which GABA and presumably taurine binds.

Methods

Two-month-old male FVB/NJ mice were subcutaneously injected with picrotoxin (5 mg kg^-1) and observed for a) latency until seizures began, b) duration of seizures, and c) frequency of seizures. For taurine treatment, mice were either fed taurine in drinking water (0.05%) or injected (43 mg/kg) 15 min prior to picrotoxin injection.

Results

We found that taurine-fed mice are resistant to picrotoxin-induced seizures when compared to age-matched controls, as measured by increased latency to seizure, decreased occurrence of seizures and reduced mortality rate. In the picrotoxin-treated animals, latency and duration were significantly shorter than in taurine-treated animas. Injection of taurine 15 min before picrotoxin significantly delayed seizure onset, as did chronic administration of taurine in the diet. Further, taurine treatment significantly increased survival rates compared to the picrotoxin-treated mice.

Conclusions

We suggest that the elevated threshold for picrotoxin-induced seizures in taurine-fed mice is due to the reduced binding sites available for picrotoxin binding due to the reduced expression of the beta subunits of the GABA_A receptor. The delayed effects of picrotoxin after acute taurine injection may indicate that the two molecules are competing for the same binding site on the GABA_A receptor. Thus, taurine-fed mice have a functional alteration in the GABAergic system. These include: increased GAD expression, increased GABA levels, and changes in subunit composition of the GABA_A receptors. Such a finding is relevant in conditions where agonists of GABA_A receptors, such as anesthetics, are administered.

     

D1‐like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation

The proliferation and differentiation of neural progenitor (NP) cells can be regulated by neurotransmitters including GABA and dopamine. The present study aimed to examine how these two neurotransmitter systems interact to affect post‐natal hippocampal NP cell proliferation in vitro. Mouse hippocampal NP cells express functional GABA_A receptors, which upon activation led to an increase in intracellular calcium levels via the opening of L‐type calcium channels. Activation of these GABA_A receptors also caused a significant decrease in proliferation; an effect that required the entry of calcium through L‐type calcium channels. Furthermore, while activation of D₁‐like dopamine receptors had no effect on proliferation, it abrogated the suppressive effects of GABA_A receptor activation on proliferation. The effects of D₁‐like dopamine receptors are associated with a decrease in the ability of GABA_A receptors to increase intracellular calcium levels, and a reduction in the surface expression of GABA_A receptors. In this way, D₁‐like dopamine receptor activation can increase the proliferation of NP cells by preventing GABA_A receptor‐mediated inhibition of proliferation. These results suggest that, in conditions where NP cell proliferation is under the tonic suppression of GABA, agonists which act through D₁‐like dopamine receptors may increase the proliferation of neural progenitors.     

Brain regional distribution of GABAA receptors exhibiting atypical GABA agonism: Roles of receptor subunits

The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA_A receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[³⁵S]thionate ([³⁵S]TBPS) binding to GABA_A receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA_A receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [³⁵S]TBPS binding. THIP displaced more [³⁵S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain.Our results suggest that native GABA_A receptors in brain sections showing reduced displacing capacity of [³⁵S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA_A receptors.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

Neuronal transmembrane chloride electrochemical gradient: A key player in GABA<Subscript>A</Subscript> receptor activation physiological effect

Summary.  It has long been accepted that GABA is the main inhibitory neurotransmitter in the mammalian brain, acting via GABA_A or GABA_B receptors. However, new evidences have shown that it may work as an excitatory transmitter, especially in the brain of newly-born animals and acting via GABA_A receptors. The difference in the end results of GABA_A receptors activation in the two cases is not due to the receptor associated channels, which in both cases are chloride channels. The different physiological effect in the two cases is due to different electrochemical gradients for chloride. When GABA acting via GABA_A receptors is inhibitory, either there is no transmembrane electrochemical gradient for chloride or there is one forcing such negative ions into the nerve cell, once chloride channels are open. Viceversa, GABA is excitatory when the electrochemical gradient is such to make chloride ions flow outside the cell, upon opening of the GABA activated chloride channels. In this review this concept is discussed in details and evidence in the scientific literature for the existence of different types of chloride pumps (either internalizing or extruding chloride) is compiled. Received August 5, 2002 Accepted October 30, 2002 Published online March 17, 2003 Acknowledgement The author thanks Dr. Simona Scarrone, Genova, for helping him with the schemes in Fig. 1. Author's address: Dr. Aroldo Cupello, Istituto di Bioimmagini e Fisiologia Molecolare, Via De Toni 5, I-16132 Genova, Italy, Fax: 39-010354180, E-mail: dcupel@neurologia.unige.it     

GABA Transporter-1 Deficiency Confers Schizophrenia-Like Behavioral Phenotypes

The mechanism underlying the pathogenesis of schizophrenia remains poorly understood. The hyper-dopamine and hypo-NMDA receptor hypotheses have been the most enduring ideas. Recently, emerging evidence implicates alterations of the major inhibitory system, GABAergic neurotransmission in the schizophrenic patients. However, the pathophysiological role of GABAergic system in schizophrenia still remains dubious. In this study, we took advantage of GABA transporter 1 (GAT1) knockout (KO) mouse, a unique animal model with elevated ambient GABA, to study the schizophrenia-related behavioral abnormalities. We found that GAT1 KO mice displayed multiple behavioral abnormalities related to schizophrenic positive, negative and cognitive symptoms. Moreover, GAT1 deficiency did not change the striatal dopamine levels, but significantly enhanced the tonic GABA currents in prefrontal cortex. The GABA_A receptor antagonist picrotoxin could effectively ameliorate several behavioral defects of GAT1 KO mice. These results identified a novel function of GAT1, and indicated that the elevated ambient GABA contributed critically to the pathogenesis of schizophrenia. Furthermore, several commonly used antipsychotic drugs were effective in treating the locomotor hyperactivity in GAT1 KO mice, suggesting the utility of GAT1 KO mice as an alternative animal model for studying schizophrenia pathogenesis and developing new antipsychotic drugs.     

Decreased GABA<Subscript>A</Subscript> Receptors Functional Regulation in the Cerebral Cortex and Brainstem of Hypoxic Neonatal Rats: Effect of Glucose and Oxygen Supplementation

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABA_A in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABA_Aα1 and GABA_Aγ5. In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABA_A receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABA_A receptors, andGABA_Aα1 and GABA_Aγ5 gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABA_A receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

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

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

GABAergic Modulation of Striatal Cholinergic Interneurons: An In Vivo Microdialysis Study

Abstract: Striatal cholinergic interneurons have been shown to receive input from Striatal γ-aminobutyric acid (GABA)-containing cell elements. GABA is known to act on two different types of receptors, the GABA_A and the GABA₆ receptor. Using in vivo microdialysis, we have studied the effect of intrastriatal application of the GABA_A-selective compounds muscimol and bicuculline and the GA- BA_B-selective compounds baclofen and 2-hydroxysaclofen, agonists and antagonists, respectively, at GABA receptors, on the output of Striatal acetylcholine (ACh). Intrastriatal infusion of 1 and 10 μmol/L concentrations of the GABA_A antagonist bicuculline resulted in a significant increase in Striatal ACh output, whereas infusion of 1 and 10 /μmol/L concentrations of the GABA_A agonist muscimol significantly decreased the output of Striatal ACh. Both compounds were ineffective in changing the output of Striatal ACh at lower concentrations. Infusion of concentrations up to 100 μmol/L of the GABA_B-selective antagonist 2-hydroxy-saclofen failed to affect Striatal ACh output, whereas infusion of 10 and 100 μmol/L baclofen, but not 0.1 and 1 μmol/L baclofen, significantly decreased the output of Striatal ACh. Thus, agonist-stimulation of GABAA and GABA_B receptors decreases the output of striatal ACh in a dose-dependent fashion, whereas the GABAergic system appears to inhibit tonically the output of striatal ACh via GABA_A receptors, but not via GABA_B receptors. We hypothesize that although GABA_A mediated regulation of striatal ACh occurs via GABA receptors on the cholinergic neuron, the GABA_B mediated effects may be explained by presynaptic inhibition of the glutamatergic input of the striatal cholinergic neuron.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

Phosphatidylinositol 4,5-Bisphosphate Induced Flunitrazepam Sensitive-GABA<Subscript>A</Subscript> Receptor Increase in Synaptosomes from Chick Forebrain

The flunitrazepam sensitive-GABA_A receptor density was increased by cytochalasins C and D at 37°C suggesting that microfilament depolymerization induces exposure to the radioligand of a GABA_A receptor in synaptosomes (Pharm Biochem Behav 72 (2002) 497). Similarly, phosphatidylinositol-4,5-bisphosphate (1–5 μM), but not a mixture of phospholipids, induced an increase of GABA_A receptors in synaptosomes. Furthermore, N-ethyl maleimide, an inactivator of the sensitive fusion protein, which interacts with GABA_A receptor, abolished the receptor increase induced by phosphatidylinositol-4,5-bisphosphate. Together, the results suggest that phosphatidylinositol-4,5-bisphosphate, acts via microfilament depolymerization increasing the binding of the radioligand to receptors possibly by modulation of their interaction with proteins involved in trafficking and docking mechanisms.     

Inhibitory effects of insulin on GABAA currents modulated by the GABAA alpha subunit

Abstract

Insulin, when co-applied with GABA, can cause an inhibition of the induced current at GABA_A receptors. This study investigated that inhibitory effect of insulin at a variety of receptor isoforms, concentrating on α1, α2 and α4 containing receptors. Various isoforms were expressed in Xenopus oocytes and currents determined using two-electrode voltage clamp. Submaximal GABA currents at all isoforms studied were inhibited by nanomolar concentrations of insulin. At α2 and α4 containing forms, insulin could inhibit maximal GABA currents. The ability to inhibit maximal currents, and the general potency and effects at submaximal currents paralleled the number of potential MAPK sites on the α subunits. The differences in insulin inhibition of GABA currents at different α containing GABA_A receptors could be important in autocrine and paracrine control of hormone secretion in the pancreas, and in control of reward and food intake circuits of the brain.     

γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABA_B receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABA_A receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABA_A receptors, where GHB acts as an agonist with an EC₅₀ of 140 nM. We investigated three neuronal cell types that express a tonic GABA_A receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABA_A receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABA_B receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.     

Dopamine Preferentially Inhibits NMDA Receptor-Mediated EPSCs by Acting on Presynaptic D1 Receptors in Nucleus Accumbens during Postnatal Development

Nucleus accumbens (nAcb), a major site of action of drugs of abuse and dopamine (DA) signalling in MSNs (medium spiny neurons), is critically involved in mediating behavioural responses of drug addiction. Most studies have evaluated the effects of DA on MSN firing properties but thus far, the effects of DA on a cellular circuit involving glutamatergic afferents to the nAcb have remained rather elusive. In this study we attempted to characterize the effects of dopamine (DA) on evoked glutamatergic excitatory postsynaptic currents (EPSCs) in nAcb medium spiny (MS) neurons in 1 to 21 day-old rat pups. The EPSCs evoked by local nAcb stimuli displayed both AMPA/KA and NMDA receptor-mediated components. The addition of DA to the superfusing medium produced a marked decrease of both components of the EPSCs that did not change during the postnatal period studied. Pharmacologically isolated AMPA/KA receptor-mediated response was inhibited on average by 40% whereas the isolated NMDA receptor-mediated EPSC was decreased by 90%. The effect of DA on evoked EPSCs were mimicked by the D₁-like receptor agonist SKF 38393 and antagonized by the D₁-like receptor antagonist SCH 23390 whereas D₂-like receptor agonist or antagonist respectively failed to mimic or to block the action of DA. DA did not change the membrane input conductance of MS neurons or the characteristics of EPSCs produced by the local administration of glutamate in the presence of tetrodotoxin. In contrast, DA altered the paired-pulse ratio of evoked EPSCs. The present results show that the activation D₁-like dopaminergic receptors modulate glutamatergic neurotransmission by preferentially inhibiting NMDA receptor-mediated EPSC through presynaptic mechanisms.     

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

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

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja