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Our knowledge about the detailed wiring of neuronal circuits in the spinal dorsal horn (DH), where initial sensory processing takes place, is still very sparse. While a substantial amount of data is available on the somatodendritic morphology of DH neurons, the laminar and segmental distribution patterns and consequential function of individual axons are much less characterized. In the present study, we fully reconstructed the axonal and dendritic processes of 10 projection neurons (PNs) and 15 interneurons (INs) in lamina I of the rat, to reveal quantitative differences in their distribution. We also performed whole-cell patch-clamp recordings to test the predicted function of certain axon collaterals. In line with our earlier qualitative description, we found that lamina I INs in the lateral aspect of the superficial DH send axon collaterals toward the medial part and occupy mostly laminae I-III, providing anatomical basis for a lateromedial flow of information within the DH. Local axon collaterals of PNs were more extensively distributed including dorsal commissural axon collaterals that might refer to those reported earlier linking the lateral aspect of the left and right DHs. PN collaterals dominated the dorsolateral funiculus and laminae IV-VI, suggesting propriospinal and ventral connections. Indeed, patch-clamp recordings confirmed the existence of a dorsoventral excitatory drive upon activation of neurokinin-1 receptors that, although being expressed in various lamina I neurons, are specifically enriched in PNs. In summary, lamina I PNs and INs have almost identical dendritic input fields, while their segmental axon collateral distribution patterns are distinct. INs, whose somata reside in lamina I, establish local connections, may show asymmetry, and contribute to bridging the medial and lateral halves of the DH. PNs, on the other hand, preferably relay their integrated dendritic input to deeper laminae of the spinal gray matter where it might be linked to other ascending pathways or the premotor network, resulting in a putative direct contribution to the nociceptive withdrawal reflex.

International audience; Background: Early life stress is known to affect the development of the nervous system and its function at a later age. It increases the risk to develop psychiatric disorders as well as chronic pain and its associated affective comorbidities across the lifespan. GABAergic inhibition is important for the regulation of central function and related behaviors, including nociception, anxiety or social interactions, and requires low intracellular chloride levels. Of particular interest, the oxytocinergic (OTergic) system exerts potent anxiolytic, analgesic and pro-social properties and is known to be involved in the regulation of chloride homeostasis and to be impaired following early life stress.Methods: We used behavioral measures to evaluate anxiety, social interactions and pain responses in a rat model of neonatal maternal separation (NMS). Using quantitative PCR, we investigated whether NMS was associated with alterations in the expression of chloride transporters in the cerebrum and spinal cord. Finally, we evaluated the contribution of OTergic signaling and neuro-inflammatory processes in the observed phenotype.Results: NMS animals displayed a long-lasting upregulation of chloride importer Na-K-Cl cotransporter type 1 (NKCC1) expression in the cerebrum and spinal cord. Neonatal administration of the NKCC1 inhibitor bumetanide or oxytocin successfully normalized the anxiety-like symptoms and the lack of social preference observed in NMS animals. Phenotypic alterations were associated with a pro-inflammatory state which could contribute to NKCC1 upregulation.Conclusions: This work suggests that an impaired chloride homeostasis, linked to oxytocin signaling dysfunction and to neuro-inflammatory processes, could contribute to the sensori-affective phenotype following NMS.

Inflammatory processes are critical promoting factors of chronic pain states, mostly by inducing peripheral and central sensitization of the nociceptive system. These processes are associated with a massive increase in glutamatergic transmission, sometimes facilitated by spinal disinhibition. In this study, we used etifoxine, a non-benzodiazepine anxiolytic known to amplify inhibition mediated by gamma-aminobutyric acid type A (GABAA) receptors in pain processing regions, either directly (through allosteric modulation) or indirectly (through the synthesis of endogenous neurosteroids). We used different models of local inflammation to evaluate the possible direct action of etifoxine on analgesia and edema. Pain symptom and edema measurements were performed after intraplantar carrageenan injection or after topical ear inflammation. We found that etifoxine treatment was associated with reduced plantar surface temperature 24 h after intraplantar carrageenan injection. In this model, etifoxine also alleviated thermal hot and mechanical hyperalgesia. A similar finding was observed while analyzing pain symptoms in the late phase of the formalin test. In a model of ear inflammation, etifoxine appeared to have a moderate anti-edemic effect after topical application. This slight action of etifoxine on the limitation of inflammatory processes could be mediated in part by cyclo-oxygenase 1 activity inhibition. Etifoxine appears as a promising therapeutic tool contributing to the limitation of inflammatory pain symptoms. Since etifoxine is already prescribed as an anxiolytic in several countries, it could be a good candidate for the prevention of inflammatory-driven edema and hyperalgesia, although the precise mechanism of action relative to its anti-inflammatory potential remains to be elucidated.

International audience; Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

For a long time, the capacity of the newborn infant to feel pain was denied. Today it is clear that the nociceptive system, even if still immature, is functional enough in the newborn infant to elicit pain responses. Unfortunately, pain is often present in the neonatal period, in particular in the case of premature infants which are subjected to a high number of painful procedures during care. These are accompanied by a variety of environmental stressors, which could impact the maturation of the nociceptive system. Therefore, the question of the long-term consequences of early life stress is a critical question. Early stressful experience, both painful and non-painful, can imprint the nociceptive system and induce long-term alteration in brain function and nociceptive behavior, often leading to an increase sensitivity and higher susceptibility to chronic pain. Different animal models have been developed to understand the mechanisms underlying the long-term effects of different early life stressful procedures, including pain and maternal separation. This review will focus on the clinical and preclinical data about early life stress and its consequence on the nociceptive system.

More than 450 million people worldwide suffer from diabetes, or 1 in 11 people. Chronic hyperglycemia degrades patients’ quality of life and the development of neuropathic pain contributes to the burden of this disease. In this study, we used the mouse model of streptozocin-induced diabetic type 1 neuropathy to assess the analgesic potential of etifoxine. Etifoxine is a prescribed anxiolytic that increases GABAAA receptor function through a direct positive allosteric modulation effect and, indirectly, by stimulating the production of endogenous GABAA receptor positive modulators such as allopregnanolone-type neurosteroids. We show that a post-symptomatic or preventive treatment strongly and durably reduces mechanical hyperalgesia and anxiety in diabetic neuropathic mice. This analgesic and neuroprotective effect on painful symptoms and emotional comorbidities is promising and should now be clinically evaluated.

International audience; Neuropathic pain is frequently associated with anxiety and major depressive disorders, which considerably impact the overall patient experience. Favoring GABAergic inhibition through the pain matrix has emerged as a promising strategy to restore proper processing of nociceptive and affective information in neuropathic pain states. In this context, the non-benzodiazepine anxiolytic etifoxine (EFX), known to amplify GABAergic inhibition through positive modulation of GABAA receptors and neurosteroidogenesis, presents several advantages. Therefore, we sought to investigate the preclinical therapeutic potential of EFX on the somatosensory and affective components of neuropathic pain. Here, we used a murine model in which neuropathic pain was induced by the implantation of a compressive cuff around the sciatic nerve (mononeuropathy). We showed that the intraperitoneal EFX treatment for five consecutive days (50 mg/kg) relieved mechanical allodynia in a sustained manner. Besides its effect on evoked mechanical hypersensitivity, EFX also alleviated aversiveness of ongoing pain as well as anxiodepressive-like consequences of neuropathic pain following cuff-induced mononeuropathy. This effect was also seen 12 weeks after induction of the neuropathy when allodynia was no longer present. Analgesic and neuroprotective actions of EFX were also seen by the absence of neuropathic pain symptoms if a second sciatic nerve constriction injury was applied to the contralateral hindpaw. Mass spectrometry analysis revealed a normalization of brainstem serotonin levels in EFX-treated animals and an increase in norepinephrine. This study suggests that EFX presents promising therapeutic potential for the relief of both somatosensory and affective consequences of neuropathic pain, a beneficial effect that is likely to involve monoamine descending controls.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

International audience; Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D

International audience; Background and purpose: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels.Experimental approach: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms.Key results: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study.Conclusions and implications: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.

International audience; Oxytocin (OT), known for its neurohormonal effects around birth, has recently been suggested for being a critical determinant in neurodevelopmental disorders. This hypothalamic neuropeptide exerts a potent analgesic effect through an action on the nociceptive system. This endogenous control of pain has an important adaptive value but might be altered by early life stress, possibly contributing to its long-term consequences on pain responses and associated comorbidities. We tested this hypothesis using a rat model of neonatal maternal separation (NMS) known to induce long-term consequences on several brain functions including chronic stress, anxiety, altered social behavior, and visceral hypersensitivity. We found that adult rats with a history of NMS were hypersensitive to noxious mechanical/thermal hot stimuli and to inflammatory pain. We failed to observe OT receptor-mediated stress-induced analgesia and OT antihyperalgesia after carrageenan inflammation. These alterations were partially rescued if NMS pups were treated by intraperitoneal daily injection during NMS with OT or its downstream second messenger allopregnanolone. The involvement of epigenetic changes in these alterations was confirmed since neonatal treatment with the histone deacetylase inhibitor SAHA, not only normalized nociceptive sensitivities but also restored OT receptor-mediated stress-induced analgesia and the endogenous antihyperalgesia in inflamed NMS rats. There is growing evidence in the literature that early life stress might impair the nociceptive system ontogeny and function. This study suggests that these alterations might be restored while stimulating OT receptor signaling or histone deacetylase inhibitors, using molecules that are currently available or part of clinical trials for other pathologies.

Originally confined to the initiation of parturition and milk ejection after birth, the hypothalamic nonapeptide oxytocin (OT) is now recognized as a critical determinant of social behavior and emotional processing. It accounts for the modulation of sensory processing and pain perception as OT displays a potent analgesic effect mediated by OT receptors (OTRs) expressed in the peripheral and central nervous systems. In our chapter, we will first systemically analyze known efferent and afferent OT neuron projections, which form the anatomical basis for OT modulation of somatosensory and pain processing. Next, we will focus on the synergy of distinct types of OT neurons (e.g., magno- and parvocellular OT neurons) which efficiently control acute inflammatory pain perception. Finally, we will describe how OT signaling mechanisms in the spinal cord control nociception, as well as how OT is able to modulate emotional pain processing within the central amygdala. In the conclusions at the end of the chapter, we will formulate perspectives in the study of OT effects on pain anticipation and pain memory, as well as propose some reasons for the application of exogenous OT for the treatment of certain types of pain in human patients.

Recent studies describe sex and gender as critical factors conditioning the experience of pain and the strategies to respond to it. It is now clear that men and women have different physiological and behavioral responses to pain. Some pathological pain states are also highly sex-specific. This clinical observation has been often verified with animal studies which helped to decipher the mechanisms underlying the observed female hyper-reactivity and hyper-sensitivity to pain states. The role of gonadal hormones in the modulation of pain responses has been a straightforward hypothesis but, if pertinent in many cases, cannot fully account for this complex sensation, which includes an important cognitive component. Clinical and fundamental data are reviewed here with a special emphasis on possible developmental processes giving rise to sex-differences in pain processing.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Background Growing evidence in the literature shows that oxytocin (OT) has a strong spinal anti-nociceptive action. Oxytocinergic axons originating from a subpopulation of paraventricular hypothalamic neurons establish synaptic contacts with lamina II interneurons but little is known about the functional role of OT with respect to neuronal firing and excitability. Results Using the patch-clamp technique, we have recorded lamina II interneurons in acute transverse lumbar spinal cord slices of rats (15 to 30 days old) and analyzed the OT effects on action potential firing ability. In the current clamp mode, we found that bath application of a selective OT-receptor agonist (TGOT) reduced firing in the majority of lamina II interneurons exhibiting a bursting firing profile, but never in those exhibiting a single spike discharge upon depolarization. Interestingly, OT-induced reduction in spike frequency and increase of firing threshold were often observed, leading to a conversion of the firing profile from repetitive and delayed profiles into phasic ones and sometimes further into single spike profile. The observed effects following OT-receptor activation were completely abolished when the OT-receptor agonist was co-applied with a selective OT-receptor antagonist. In current and voltage clamp modes, we show that these changes in firing are strongly controlled by voltage-gated potassium currents. More precisely, transient IA currents and delayed-rectifier currents were reduced in amplitude and transient IA current was predominantly inactivated after OT bath application. Conclusion This effect of OT on the firing profile of lamina II neurons is in good agreement with the antinociceptive and analgesic properties of OT described in vivo.

International audience; Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non-genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3alpha neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3beta isomer was inefficient. More interestingly, 3alpha5beta neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3alpha5beta neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3alpha5beta neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

International audience; Because of its severity, chronicity, resistance to usual therapy and its consequences on quality of life, neuropathic pain represents a real clinical challenge. Fundamental research on this pathology uses metabolic, pharmacological or traumatic models in rodents that reproduce the characteristic human pain symptoms. In 1996, Mosconi and Kruger morphologically described a model of peripheral neuropathy in which a cuff of polyethylene tubing was placed around the sciatic nerve in rats. In the present study, we evaluated the behavioral consequences of this neuropathic pain model in C57Bl/6J mice which is the main genetic background used for studies in transgenic mice. A short cuff of polyethylene tubing was unilaterally placed around the main branch of the sciatic nerve. It induced an ipsilateral heat thermal hyperalgesia lasting around 3 weeks, and a sustained ipsilateral mechanical allodynia lasting at least 2 months. We showed that this neuropathic pain model is insensitive to ketoprofen, a non-steroidal anti-inflammatory drug. Morphine treatment acutely suppressed the mechanical allodynia, but tolerance to this effect rapidly developed. The analysis of video recordings revealed that most aspects of spontaneous behavior remained unaffected on the long term, excepted for a decrease in the time spent at social interaction for the neuropathic mice. Using the elevated plus-maze and the marble-burying test, we also showed that neuropathic mice develop an anxiety phenotype. Our data indicate that sciatic nerve cuffing in mice is a pertinent model for the study of nociceptive and emotional consequences of sustained neuropathic pain.

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic–pituitary–adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3a5a-reduced steroids, which modulate GABAA receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3a5a-reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non-genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C-fiber-mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

Etifoxine (EFX) is a non-benzodiazepine anxiolytic which potentiate GABAA receptor (GABAAR) function directly or indirectly via the production of 3α-reduced neurosteroids. The later effect is now recognized to account for the long-term reduction of pain symptoms in various neuropathic and inflammatory pain models. In the present study, we characterized the acute antinociceptive properties of EFX during spinal pain processing in naive and monoarthritic rats using in vivo electrophysiology. The topical application of EFX on lumbar spinal cord segment, at concentrations higher than 30 μM, reduced the excitability of wide dynamic range neurons receiving non-nociceptive and nociceptive inputs. Windup discharge resulting from the repetitive stimulation of the peripheral receptive field, and recognized as a short-term plastic process seen in central nociceptive sensitization, was significantly inhibited by EFX at these concentrations. In good agreement, mechanical nociceptive thresholds were also significantly increased following an acute intrathecal injection of EFX. The acute modulatory properties of EFX on spinal pain processing were never seen in the simultaneous presence of bicuculline. This result further confirmed EFX antinociception to result from the potentiation of spinal GABAA receptor function.

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.

International audience; Inflammatory and degenerative diseases of the joint are major causes of chronic pain. Long-lasting pain symptoms are thought to result from a central sensitization of nociceptive circuits. These processes include activation of microglia and spinal disinhibition. Using a monoarthritic rat model of pain, we tried to potentiate neural inhibition by using etifoxine (EFX), a nonbenzodiazepine anxiolytic that acts as an allosteric-positive modulator of gamma-aminobutyric acid type A (GABAA) receptor function. Interestingly, EFX also can bind to the mitochondrial translocator protein (TSPO) complex and stimulate the synthesis of 3α-reduced neurosteroids, the most potent positive allosteric modulator of GABAA receptor function. Here we show that a curative and a preventive treatment with 50mg/kg of EFX efficiently reduced neuropathic pain symptoms. In the spinal cord, EFX analgesia was accompanied by a reduction in microglial activation and in the levels of proinflammatory mediators. Using electrophysiological tools, we found that EFX treatment not only amplified spinal GABAergic inhibition, but also prevented prostaglandin E2-induced glycinergic disinhibition and restored a "normal" spinal pain processing. Because EFX is already distributed in several countries under the trade name of Stresam for its anxiolytic actions in humans, new clinical trials are now required to further extend its therapeutic indications as pain killer.

As a first step towards elucidating mechanisms involved in neuroendocrine synaptogenesis, we developed a model of co-culture based on hypothalamic-intermediate pituitary interactions. Dissociated hypothalamic neurons from fetal rats at embryonic day 15 were cultured in a defined medium together with melanotrope cells of the pituitary intermediate lobe from neonatal rats. In these co-cultures, establishment of synaptic contacts between GABAergic or dopaminergic neurons and an endocrine target cell, the melanotrope cell, was studied by morphofunctional approaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and AE-melanocyte-stimulating hormone, we demonstrated morphological contacts between GABAergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synapsin I, we showed a modification of synapsin I immunoreactivity from diVuse to punctate distribution correlated with the establishment of contacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrophysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic currents, which occurred either spontaneously and/or could be evoked chemically by 50 mM KCl or 100 µM kainate. The proportion of the melanotrope cells receiving functional synaptic inputs increased until 10 days in culture, a stage at which virtually all melanotrope cells in contact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approaches. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd.

Morphine, codeine, morphine-6-glucuronide, and morphine-3-glucuronide are synthesized de novo in mammalian cells and in the central nervous system. Knowledge on endogenous morphine-like compound distribution in the adult mouse brain has been recently improved, and new hypotheses have been suggested about the potential implications in brain physiology. Endogenous morphine-like compounds have been shown to be synthesized in the spinal cord, but their localization is unknown. Here we describe the distribution of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the adult mouse spinal cord using a well-validated antibody. By using different microscopy approaches, we found the presence of morphine, codeine, or morphine glucuronides in γ-aminobutyric acid (GABA)-ergic neurons and astrocytes of the spinal cord. Whereas GABAergic neurons containing endogenous morphine-like compounds were located primarily in the ventral horn, astrocytes that were labeled for morphine-like compounds were found throughout the gray matter and the white matter. Our study demonstrates the possibility that endogenous morphine-like compounds in the central nervous system have other functions beyond their analgesic functions. J. Comp. Neurol. 520:1547–1561, 2012. © 2011 Wiley Periodicals, Inc.

The properties of neuronal-type nicotinic acetylcholine receptors (nAChRs) present on the neuroendocrine cells of the porcine pars intermedia of the pituitary were studied in intact single cell using measurements of the free intracellular Ca2+ concentration ([Ca]i) with the calcium-sensitive dye fura 2. Local application of an extracellular solution containing 50 mM K+ or of the selective nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) depolarised the cells and induced an elevation in [Ca]i. The effect of DMPP on [Ca]i was dose dependent (EC50 = 6 microM), reversibly blocked by d-tubocurarine and strictly dependent on the concentration of extracellular Ca2+. The calcium channel blocker Cd2+ (100 microM) reversibly blocked 80% of the response induced by 50 mM K+, whereas it reduced the DMPP response by only 50%. In the absence of extracellular Na+, DMPP no longer depolarised the cells but still increased [Ca]i. The rise in [Ca]i under these conditions represented 41% of the control response, i.e. in the presence of external Na+. Thus activation of nAChRs induced an elevation in [Ca]i which was in part independent of cell depolarisation. This was confirmed by recording simultaneously, under whole-cell voltage-clamp, a rise in [Ca]i associated with the inward nicotinic current. During prolonged application of the agonist (50 s), the amplitude of the nicotinic current decayed rapidly to a very low plateau level reflecting nAChR desensitisation. However, photometric experiments performed on intact non-dialysed cells revealed the presence of a slowly decaying phase in [Ca]i throughout the application of DMPP. This suggests the persistence of a substantial Ca2+ influx during prolonged exposure to the agonist. Taken together, our results show that stimulation of nAChRs induces an influx of Ca2+ which elevates [Ca]i. This phenomenon is due to activation of voltage-dependent Ca2+ channels and to Ca2+ entry through the nAChR.

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions. J. Comp. Neurol. 519:2390–2416, 2011. © 2011 Wiley-Liss, Inc.

Evaluation of pain is a critical issue in human pathologies but also in animal experimentation. In human studies there is growing evidence that cardiovascular outputs such as heart rate variability (HRV) might be of interest to detect and measure pain expression. Indeed, systems controlling cardiovascular function are closely coupled to the perception of pain. To demonstrate the interest of HRV, we have combined radiotelemetry and remote-controlled nociceptive tests in rats submitted to various situations of acute and persistent inflammatory pain. We found the Poincare plot descriptor SD1 and pNN18 to represent robust indicators of pain, especially in the case of persistent inflammatory states. Further studies will be performed in order to understand by which mechanisms pain-related increases in HRV are produced and if these descriptors can be used for other persistent pain states.

Although cholinergic drugs are known to modulate nociception, the role of endogenous acetylcholine in nociceptive processing remains unclear. In the current study, we evaluated the role of cholinergic transmission through spinal β(2)-subunit-containing nicotinic acetylcholine receptors in the control of nociceptive thresholds. We show that mechanical and thermal nociceptive thresholds are significantly lowered in β(2)(∗)-knockout (KO) mice. Using nicotinic antagonists in these mice, we demonstrate that β(2)(∗)-nAChRs are responsible for tonic inhibitory control of mechanical thresholds at the spinal level. We further hypothesized that tonic β(2)(∗)-nAChR control of mechanical nociceptive thresholds might implicate GABAergic transmission since spinal nAChR stimulation can enhance inhibitory transmission. Indeed, the GABA(A) receptor antagonist bicuculline decreased the mechanical threshold in wild-type but not β(2)(∗)-KO mice, and the agonist muscimol restored basal mechanical threshold in β(2)(∗)-KO mice. Thus, β(2)(∗)-nAChRs appeared to be necessary for GABAergic control of nociceptive information. As a consequence of this defective inhibitory control, β(2)(∗)-KO mice were also hyperresponsive to capsaicin-induced C-fiber stimulation. Our results indicate that β(2)(∗)-nAChRs are implicated in the recruitment of inhibitory control of nociception, as shown by delayed recovery from capsaicin-induced allodynia, potentiated nociceptive response to inflammation and neuropathy, and by the loss of high-frequency transcutaneous electrical nerve stimulation (TENS)-induced analgesia in β(2)(∗)-KO mice. As high-frequency TENS induces analgesia through Aβ-fiber recruitment, these data suggest that β(2)(∗)-nAChRs may be critical for the gate control of nociceptive information by non-nociceptive sensory inputs. In conclusion, acetylcholine signaling through β(2)(∗)-nAChRs seems to be essential for setting nociceptive thresholds by controlling GABAergic inhibition in the spinal cord.

Background-: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. Results-: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. Conclusions-: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

Effective relief of acute and long-term postoperative pain is of utmost importance to patients undergoing surgery. Here, we worked on a controlled procedure of abdominal surgery in the rat inducing persistent postoperative pain symptoms for up to 10 days and tested the efficacy of perioperative care with the local anesthetic ropivacaine. Laparotomy was likewise used to implant radiotelemetric probes by which electrocardiogram, body temperature, and locomotor activity were recorded in freely moving animals. We showed that postoperative pain symptoms (mechanical allodynia) measured in periphery of the scar were associated over time with persistent tachycardia, elevated heart rate variability, and loss of mobility. Furthermore, a single subcutaneous infiltration of the local anesthetic ropivacaine in the periphery of the abdominal incision was sufficient to prevent the appearance of allodynia and the associated cardiac and motor signs of pain, monitored by radio- telemetry. These beneficial effects were observed when the infiltration was performed in the perio- perative period, but not later. This study on freely moving animals exhibiting long-lasting postoperative pain symptoms and altered autonomic/motor function illustrates well the importance of the timing of preemptive analgesia care with long-acting local anesthetics. Moreover, it empha- sizes the utility of monitoring heart rate variability to quantify spontaneous expression of long- lasting postoperative pain. Perspective: Speeding the recovery time after surgery using perioperative ropivacaine care is of significant clinical relevance because it might limit the risk of chronic pain and postoperative compli- cations. In humans, chronobiological analysis of heart rate variability could also help quantify spon- taneous pain expression with minimal emotional bias. a 2011 by the American Pain Society

In animal studies, thermal sensitivity is mostly evaluated on the basis of nociceptive reaction latencies in response to a given thermal aversive stimulus. However, these techniques may be inappropriate to differentiate allodynia from hyperalgesia or to provide information differentiating the activation of nociceptor subtypes. The recent development of dynamic hot and cold plates, allowing computer-controlled ramps of temperature, may be useful for such measures. In this study, we characterized their interest for studying thermal nociception in freely moving mice and rats. We showed that escape behavior (jumps) was the most appropriate parameter in C57Bl/6J mice, whereas nociceptive response was estimated by using the sum of paw lickings and withdrawals in Sprague-Dawley rats. We then demonstrated that this procedure allows the detection of both thermal allodynia and hyperalgesia after peripheral pain sensitization with capsaicin in mice and in rats. In a condition of carrageenan-induced paw inflammation, we observed the previously described thermal hyperalgesia, but we also revealed that rats exhibit a clear thermal allodynia to a cold or a hot stimulus. These results demonstrate the interest of the dynamic hot and cold plate to study thermal nociception, and more particularly to study both thermal allodynia and hyperalgesia within a single paradigm in awake and freely moving rodents.Despite its clinical relevance, thermal allodynia is rarely studied by researchers working on animal models. As shown after stimulation of capsaicin-sensitive fibers or during inflammatory pain, the dynamic hot and cold plate validated in the present study provides a useful tool to distinguish between thermal allodynia and thermal hyperalgesia in rodents.

The central processing of peripheral nociceptive messages is highly controlled by the activity of local inhibitory networks in the spinal cord and supraspinal centers. Recently, it has been shown that endogenous 3alpha-reduced neurosteroids (3alphaNS) exert a significant spinal antinociception by potentiating GABA(A) receptor function. Because endogenous 3alphaNS can be produced in many relay structures of the nociceptive system, we tested the potential analgesic efficacy of promoting the production of neurosteroids by using etifoxine (ETX, 50mg/kg i.p.). This prescribed non-benzodiazepine anxiolytic was shown previously to stimulate neurosteroidogenesis in its early step after binding to the mitochondrial translocator protein complex (TSPO). Using an animal model of generalized neuropathic pain resulting from a 2-week treatment with the antitumoral agent vincristine sulfate (VCR, 0.1mg/kg i.p.), we show that injections of ETX (50mg/kg i.p.) given every day reduced the VCR-induced mechanical and thermal pain symptoms but also prevented their appearance, if used in prophylaxia 1 week before VCR. Both the curative and preventive effects of ETX on pain symptoms were mediated by the production of 3alphaNS as demonstrated in animals treated with the enzymatic inhibitor provera (6-medroxyprogesterone acetate; 20mg/kg s.c.). Altogether, this study shows for the first time that promoting 3alphaNS could be a possible therapeutic strategy to treat neuropathic pain symptoms. Since ETX is already available as an anxiolytic, its use in humans, provided that its analgesic properties are confirmed, could be rapidly considered.

Background: Recent 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. Results: We 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. Conclusion: Our 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.

The activity of GABAergic inhibitory interneurones located in lamina II of the spinal cord is of fundamental importance for the processing of peripheral nociceptive messages. We have recently shown that 3alpha-hydroxy ring A-reduced pregnane neurosteroids [3alpha5alpha-neurosteroids (3alpha5alphaNS)], potent allosteric modulators of GABA(A) receptors (GABA(A)Rs), are synthesized in the spinal cord and limit thermal hyperalgesia during inflammatory pain. Because changes in the expression of calcium-dependent protein kinases [protein kinase C (PKC)] are observed during pathological pain in the spinal cord, we examined the possible interactions between PKC and 3alpha5alphaNS at synaptic GABA(A)Rs. Using patch-clamp recordings of lamina II interneurones in the spinal cord of 15-20-day-old rats, we showed that synaptic inhibition mediated by GABA(A)Rs and its modulation by 3alpha5alphaNS in lamina II of the spinal cord largely depend on activation of PKC. Our experimental results suggested that activation of PKC locks synaptic GABA(A)Rs in a functional state precluding further positive allosteric modulation by endogenous and exogenous 3alpha5alphaNS. This effect was fully prevented by coadministration of chelerythrin, an inhibitor of PKC. Furthermore, application of chelerythrin alone rendered synaptic GABA(A)Rs hypersensitive to endogenously produced or exogenously applied 3alpha5alphaNS. These findings confirmed that there was a significant production of endogenous 3alpha5alphaNS in lamina II of the spinal cord but also indicated that PKC-dependent phosphorylation processes were tonically activated to control GABA(A)R-mediated inhibition under resting conditions. We therefore can conclude that PKC activation sets an upper limit to the functional plasticity of GABAergic transmission induced by endogenous neurosteroids.

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 5alpha-reduced neurosteroids are produced locally in lamina II and modulate synaptic gamma-aminobutyric acid A(GABAA) receptor function during development, as well as during inflammatory pain. The production of 5alpha-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 GABAergic 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.

Devices designed for mechanical pain threshold studies are often difficult to implement. The purpose of this study was to investigate a simple tool based on calibrated forceps to induce quantifiable mechanical stimulation in the rat on a linear scale. The most suitable protocol was tested by determining the effects of 3 repetitive measurements on both hind paws, respectively, during long-term (9 days), mid-term (1 day), and short-term (2 hours). Only threshold increase related to weight gain over long-term was observed, suggesting that moderate rat training can be used. The capacity of the device to reveal hyperalgesia was tested in a model of carrageenan-induced inflammation in the hind paw. The hyperalgesia was maximal 6 hours after carrageenan injection and progressively decreased. Similar, although more variable, responses were observed with von Frey filaments. Morphine-induced analgesia resulted in a dose-dependent increase of paw threshold. Tolerance to morphine administrated on a once daily schedule (10 mg/kg) during 5 days was revealed by a significant decrease in analgesia by day 3. Taken together, these results demonstrated accuracy of this device for easy, fast, and reproducible measure of mechanical pain threshold on rat limbs. Moreover, it allows the performance of rat testing with minimal constraint, which reduces data variability. Perspective The calibrated forceps is an easy to use device well-suited to rapidly test mechanical pain threshold with accuracy. It is well-designed for preclinical behavioral screening of noxious or analgesic properties of molecules.

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 GABAAreceptors 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 GABAAreceptors 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 GABAAreceptor-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.

Prostaglandin E 2 (PGE 2 ) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PGE 2 -induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE 2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE 2 injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy.

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 concentrations1 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.

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAAreceptor subunits. Yet, the effect of phosphorylation on GABAAreceptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAAreceptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAAreceptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipiddependent protein kinase (PKC) activation on the function of synaptic GABAAreceptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAAreceptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAAreceptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the α-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 μm) and SR95531 (5 μm), two competitive antagonists of the GABAAreceptor. At a holding potential of −60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10–100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast (≤1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant, 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mminduced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8–10 GABAAchannels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

Glutamate is the major excitatory neurotransmitter in the central nervous system, yet little is known about its actions on endocrine cells. We have investigated the membrane effects of glutamate in cultured neonatal rat pituitary intermediate lobe (IL) cells using the whole-cell configuration of the patch-clamp technique. In a standard Na(+)-based extracellular solution, glutamate failed to induce a detectable membrane current at a holding potential (HP) of -60 mV (n = 40). However, when cyclothiazide (50 microM), a benzothiazide that blocks desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole-propanoic acid (AMPA)-type receptors, was added to the extracellular solution, glutamate (0.5-1 mM) induced an inward current at a HP of -60 mV in 65% of the cells tested (n = 72). This response was usually small in amplitude (mean amplitude: 28.6 +/- 37.5 pA, n = 47). The glutamate-induced current reversed polarity close to 0 mV and was reversibly blocked when extracellular Na+ was replaced by the impermeant cation N-methyl-D-glucamine, suggesting that this current was a nonselective cation current. The response to glutamate (1 mM) was reproduced by AMPA (50 microM), kainate (200 microM), and quisqualate (200 microM). N-Methyl-D-aspartate (NMDA, 100 microM) in the presence of 10 microM glycine did not induce any membrane current in cells responding to glutamate (n = 8). The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) reversibly inhibited the response to glutamate (0.5 mM) by 85 +/- 14% (n = 7), whereas D(-)-2-amino-5-phosphonopentanoic acid (20 microM), an antagonist of NMDA receptors, had no effect on the glutamate-induced current (n = 3). Moreover, we show that although the amplitude of the glutamate currents was small, the latter induced large (30-mV) membrane depolarizations and triggered the firing of action potentials. Taken together, our results indicate that neonatal rat IL cells possess AMPA-type glutamate receptors that could possibly underlie a fast excitatory glutamatergic synaptic input to these cells.

Whole-cell patch-clamp recordings were made from CA1 pyramidal and dentate gyrus granule cells (GCs) in hippocampal slices to assess the effects of withdrawal from chronic flurazepam (FRZ) treatment on the function of synaptic GABAAreceptors. In slices from control rats, acute perfusion of FRZ (30 μm) increased the monoexponential decay time constant of miniature IPSCs (mIPSCs) in CA1 and GCs (from 3.4 ± 0.6 to 7.6 ± 2.1 msec and from 4.2 ± 0.6 to 7.1 ± 1.8 msec, respectively) but did not change their mean conductance, 10–90% rise time, or frequency of occurrence. Withdrawal (2–5 d) from chronicin vivoFRZ treatment (40–110 mg/kg per day, per os) resulted in a dramatic loss of mIPSCs in CA1 neurons. On day 5 of withdrawal, no mIPSCs could be recorded in 40% of CA1 pyramidal cells. In the remaining 60% of the neurons, mIPSCs had a reduced mean conductance (from 0.78 ± 0.12 nS in vehicle-treated controls to 0.31 ± 0.05 nS) and a diminished frequency of occurrence (from 20.7 ± 7.9 to 4.1 ± 0.6 Hz). We have estimated that >80% of GABAAsynapses on CA1 pyramidal cells had become silent, whereas at still-active synapses the number of functional GABAAreceptor channels decreased by 60%. This reduction rapidly reverted to control levels on day 6 of withdrawal. FRZ withdrawal did not alter mIPSC properties in GCs. Our results are consistent with the hypothesis that chronic benzodiazepine treatment leads to a reduced number of functional synaptic GABAAreceptors in a region-specific manner that may stem from differences in the subunit composition of synaptic GABAAreceptors.