Rémy Schlichter

Coexistence of GABAA and GABAB receptors on Aδ and C primary afferents

1 Intracellular recordings from adult rat dorsal root ganglion neurones were performed in vitro and the coexistence of two γ‐aminobutyric acid (GABA) receptors on the membrane of identified Aδ and C primary afferents was demonstrated. 2 Transient applications of GABA (10−6 − 10−2m) evoked dose‐dependent depolarizations and increased membrane conductance. The responses were mimicked by muscimol, isoguvacine, THIP and 3 amino propane sulphonic acid (3 APS); they were blocked by bicuculline and picrotoxin. Pentobarbitone induced an increase of GABA‐induced depolarizations. 3 Perfusion of tetraethylammonium (TEA, 7.5 mm) and intracellular injection of Cs+ ions unmasked the Ca2+ component of action potentials, which appeared as long‐lasting plateau depolarizations. Such action potentials were shortened in the presence of methoxyverapamil (D600, 5 × 10−6‐ 10−5m) and in a medium without Ca+ ions. 4 Prolonged (5–10 min) perfusion of GABA (10−9‐10−5m) shortened the Ca2+ component of action potentials. This effect was mimicked by baclofen (10−7‐5 × 10−6m) and muscimol (5 × 10−7‐10−5m) and was not affected by bicuculline perfusion (5 × 10−6‐10−5m). Isoguvacine (2.5 × 10−5m) did not affect action potential duration. 5 It is concluded that two GABA receptors coexist on the membrane of slow conducting primary afferents: the bicuculline‐sensitive GABAA receptor mediates depolarizations and the bicuculline‐insensitive GABAB receptor shortens the calcium component of action potentials.

Inflammatory Pain Upregulates Spinal Inhibition via Endogenous Neurosteroid Production

Inhibitory synaptic transmission in the dorsal horn (DH) of the spinal cord plays an important role in the modulation of nociceptive messages because pharmacological blockade of spinal GABAA receptors leads to thermal and mechanical pain symptoms. Here, we show that during the development of thermal hyperalgesia and mechanical allodynia associated with inflammatory pain, synaptic inhibition mediated by GABAA receptors in lamina II of the DH was in fact markedly increased. This phenomenon was accompanied by an upregulation of the endogenous production of 5α-reduced neurosteroids, which, at the spinal level, led to a prolongation of GABAA receptor-mediated synaptic currents and to the appearance of a mixed GABA/glycine cotransmission. This increased inhibition was correlated with a selective limitation of the inflammation-induced thermal hyperalgesia, whereas mechanical allodynia remained unaffected. Our results show that peripheral inflammation activates an endogenous neurosteroid-based antinociceptive control, which discriminates between thermal and mechanical hyperalgesia.

Production of 5α-Reduced Neurosteroids Is Developmentally Regulated and Shapes GABAA Miniature IPSCs in Lamina II of the Spinal Cord

In lamina II of the spinal dorsal horn, synaptic inhibition mediated by ionotropic GABAA and glycine receptors contributes to the integration of peripheral nociceptive messages. Whole-cell patch-clamp recordings were performed from lamina II neurons in spinal cord slices to study the properties of miniature IPSCs (mIPSCs) mediated by activation of GABAA and glycine receptors in immature (<30 d) and adult rats. Blockade of neurosteroidogenesis by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195), an inhibitor of the peripheral benzodiazepine receptor (PBR), or finasteride, which blocks 5α-reductase, accelerated the decay kinetics of GABAA receptor-mediated mIPSCs in immature, but not in adult animals. Glycine receptor-mediated mIPSCs remained unaffected under these conditions. These results suggest the presence of a tonic production of 5α-reduced neurosteroids in young rats that confers slow decay kinetics to GABAA mIPSCs. At all of the ages, selective stimulation of PBR by diazepam in the presence of flumazenil prolonged GABAA mIPSCs in a PK11195- and finasteride-sensitive manner. This condition also increased the proportion of mixed GABAA/glycine mIPSCs in the immature animals and led to the reappearance of mixed GABAA/glycine mIPSCs in the adult. Our results might point to an original mechanism by which the strength of synaptic inhibition can be adjusted locally in the CNS during development and under physiological and/or pathological conditions by controlling the synthesis of endogenous 5α-reduced neurosteroids.

Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABA(A) receptor function. Etifoxine displaced [(35)S]TBPS (t-butylbicyclophosphorothionate) from GABA(A) receptors of rat cortical membranes with an IC(50) of 6.7+/-0.8 microM and [(3)H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC(50) of 27.3+/-1.0 microM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABA(A) receptor-mediated membrane currents elicited by submaximal (5-10 microM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 microM which reflected the post-synaptic potentiation of a small ( approximately 20 pA) tonic and bicuculline-sensitive GABA(A) receptor-gated Cl(-) current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 microM), an antagonist of central-type benzodiazepine sites present at GABA(A) receptors, but were partly inhibited by PK11195 (10 microM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABA(A) receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

Modulation of GABAA receptor-mediated IPSCs by neuroactive steroids in a rat hypothalamo-hypophyseal coculture model.

1. We have used the whole‐cell configuration of the patch‐clamp technique to investigate the effects of neuroactive steroids on GABAA receptor‐mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre‐ and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor‐mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half‐maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha‐pregnan‐3 alpha‐ol‐20‐one) reversibly potentiated IGABA in a dose‐dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/‐ 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta‐isomer of allopregnanolone, epipregnanolone (5 beta‐pregnan‐3 beta‐ol‐20‐one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5‐pregnen‐3 alpha‐ol‐20‐one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of ‐80 and ‐60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre‐ and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition

BackgroundRecent evidence suggests that oxytocin (OT), secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN) neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception.ResultsWe show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II). This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages.ConclusionOur results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.

Role of glial and neuronal glycine transporters in the control of glycinergic and glutamatergic synaptic transmission in lamina X of the rat spinal cord

Using whole cell voltage clamp recordings from lamina X neurones in rat spinal cord slices, we investigated the effect of glycine transporter (GlyT) antagonists on both glycinergic inhibitory postsynaptic current (IPSCs) and glutamatergic excitatory postsynaptic current (EPSCs). We used ORG 24598 and ORG 25543, selective antagonists of the glial GlyT (GlyT1) and neuronal GlyT (GlyT2), respectively. In rats (P12–P16) and in the presence of kynurenic acid, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and bicuculline, ORG 24598 and ORG 25543 applied individually at a concentration of 10 μm induced a mean inward current of −10/−50 pA at −60 mV and increased significantly the decay time constants of miniature (mIPSCs), spontaneous (sIPSCs) and electrically evoked glycinergic (eIPSCs) inhibitory postsynaptic currents. ORG 25543, but not ORG 24598, decreased the frequency of mIPSCs and sIPSCs. Replacing extracellular sodium with N‐methyl‐d‐glucamine or superfusing the slice with micromolar concentrations of glycine also increased the decay time constant of glycinergic IPSCs. By contrast, the decay time constant, amplitude and frequency of miniature GABAergic IPSCs recorded in the presence of strychnine were not affected by ORG 24598 and ORG 25543. In the presence of strychnine, bicuculline and CNQX, we recorded electrically evoked NMDA receptor‐mediated EPSCs (eEPSCs). eEPSCs were suppressed by 30 μmd‐2‐amino‐5‐phosphonovalerate (APV), an antagonist of the NMDA receptor, and by 30 μm dichlorokynurenic acid (DCKA), an antagonist of the glycine site of the NMDA receptor. Glycine (1–5 μm) and d‐serine (10 μm) increased the amplitude of eEPSCs whereas l‐serine had no effect. ORG 24598 and ORG 25543 increased significantly the amplitude of NMDA receptor‐mediated eEPSCs without affecting the amplitude of non‐NMDA receptor‐mediated eEPSCs. We conclude that blocking glial and/or neuronal glycine transporters increased the level of glycine in spinal cord slices, which in turn prolonged the duration of glycinergic synaptic current and potentiated the NMDA‐mediated synaptic response.

Fast nongenomic effects of steroids on synaptic transmission and role of endogenous neurosteroids in spinal pain pathways.

Steroids exert long-term modulatory effects on numerous physiological functions by acting at intracellular/nuclear receptors influencing gene transcription. Steroids and neurosteroids can also rapidly modulate membrane excitability and synaptic transmission by interacting with ion channels, that is, ionotropic neurotransmitter receptors or voltage-dependent Ca2+ or K+ channels. More recently, the cloning of a plasma membrane-located G protein-coupled receptor for progestins, in various species has suggested that steroids/neurosteroids could also influence second-messenger pathways by directly interacting with specific membrane receptors. Here we review the experimental evidence implicating steroids/neurosteroids in the modulation of synaptic transmission and the evidence for a role of endogenously produced neurosteroids in such modulatory effects. We present some of our recent results concerning inhibitory synaptic transmission in lamina II of the spinal cord and show that endogenous 5α-reduced neurosteroids are produced locally in lamina II and modulate synaptic γ-aminobutyric acid A (GABAA) receptor function during development, as well as during inflammatory pain. The production of 5α-reduced neurosteroids is controlled by the endogenous activation of the peripheral benzodiazepine receptor (PBR), which initiates the first step of neurosteroidogenesis by stimulating the translocation, of cholesterol across the inner mitochondrial membrane. Tonic neurosteroidogenesis observed in immature animals was decreased during postnatal, development, resulting in an acceleration of GABAA receptor-mediated miniature inhibitory postsynaptic current (mIPSC) kinetics observed in the adult. Stimulation of the PBR resulted in a prolongation of GABA ergic mIPSCs at all ages and was observed during inflammatory pain. Neurosteroidogenesis might play an, important role in the control of nociception at least at the spinal cord level.

Cerebrospinal fluid‐contacting neurons in the rat spinal cord, a γ‐aminobutyric acidergic system expressing the P2X2 subunit of purinergic receptors, PSA‐NCAM, and GAP‐43 immunoreactivities: Light and electron microscopic study

Cerebrospinal fluid‐contacting neurons (CSFcNs) occur in various brain regions of lower vertebrates. In mammals, they are restricted to medullospinal areas, and little is known about their projection sites. In the present work, we investigated some morphofunctional characteristics of such neurons in the rat spinal cord by light and electron microscopic immunocytochemistry. CSFcNs expressing the P2X2 subunit of purinergic receptors were present throughout the spinal cord, though more numerous at lower thoracolumbar and sacral levels. These neurons coexpressed GAD and the polysialylated neural cell adhesion molecule (PSA‐NCAM), a marker of cellular plasticity. From low thoracic levels downward, tiny amyelinic axons (less than 200 nm in diameter) were tightly packed in bundles, which ran along the ependyma and extended ventrally, eventually concentrating against the walls of the ventral median fissure. In addition to P2X2, GAD, γ‐aminobutyric acid (GABA), and PSA, these axons expressed GAP‐43 immunoreactivity. Moreover, they were labelled along their entire lengths with antibodies against synaptotagmin and synaptophysin, but these failed to reveal intraspinal terminal fields. Taken together, our observations indicate the presence in the rat spinal cord of a highly plastic system of GABAergic CSFcNs that express the P2X2 subunit of purinergic receptors. The function of this original system remains open to question. In these neurons, the P2X2 receptors may confer a sensitivity to ATP either present in the CSF or released by nearby neurons of the central autonomic area. J. Comp. Neurol. 457:159–174, 2003.

Modulation of GABA‐gated chloride currents by intracellular Ca2+ in cultured porcine melanotrophs.

1. The modulatory role of intracellular Ca2+ concentration ([Ca2+]i) on gamma‐aminobutyric acid type A (GABAA) receptor‐gated Cl‐ currents was investigated in dialysed and intact cells of cultured porcine pituitary intermediate lobe (IL) cells using the patch‐clamp technique. In order to isolate Ca2+ and Cl‐ currents all other membrane currents were blocked pharmacologically. Isoguvacine, a specific GABAA receptor agonist, was used to activate selectively GABAA receptor‐mediated whole‐cell and single‐channel Cl‐ currents. 2. In the whole‐cell recording (WCR) configuration inward Ca2+ currents triggered before and/or during the application of isoguvacine (100 microM), did not inhibit the GABAA receptor‐mediated response. This lack of effect of calcium currents was obtained in all situations tested, i.e. when the intracellular Ca2+ concentration was only weakly buffered (0.5 mM‐EGTA in the pipette solution), not buffered at all (no EGTA added to the pipette solution) or when the resting [Ca2+]i was buffered at 10(‐7) M (pCa 7) with internal EGTA. 3. At pCa 7, simultaneous application of isoguvacine (100 microM) and caffeine (10 mM) resulted in a 47 +/‐ 15% reduction of the whole‐cell GABAA response. In the same conditions, a ten times lower concentration of caffeine (1 mM), induced a transient increase of the GABAA response which turned into a steady‐state inhibition during the subsequent applications. 4. At pCa 7, when isoguvacine (100 microM) was applied together with 3Me‐His2‐TRH (50 nM), a potent analogue of the calcium‐recruiting thyrotrophin‐releasing hormone, the GABAA receptor‐gated Cl‐ current was increased by 40 +/‐ 8%. In the absence of the Ca2+ chelator EGTA in the pipette solution, either potentiating or inhibitory effects of 3Me‐His2‐TRH on the GABAA response were observed. 5. If a high concentration (18 mM) of the calcium chelator EGTA was included in the pipette solution, caffeine and 3Me‐His2‐TRH had markedly lower effects on the GABAA response than those observed at pCa 7, suggesting that the effect of both substances was mediated by an increase in [Ca2+]i. 6. In the absence of extracellular Ca2+, the effects of caffeine and 3Me‐His2‐TRH were not significantly different from those obtained in the presence of Ca2+ (5 mM), suggesting that Ca2+ influx was not the major route for increasing [Ca2+]i. 7. In the cell‐attached (CA) configuration, the presence of isoguvacine (3‐5 microM) in the pipette solution triggered the opening of channels displaying multiple current levels.(ABSTRACT TRUNCATED AT 400 WORDS)