Publications by authors named "Rémy Schlichter"

31 Publications

Enhanced analgesic cholinergic tone in the spinal cord in a mouse model of neuropathic pain.

Neurobiol Dis 2021 Apr 18;155:105363. Epub 2021 Apr 18.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France; University of Strasbourg Institute for Advanced Study (USIAS), 67000 Strasbourg, France. Electronic address:

Endogenous acetylcholine (ACh) is an important modulator of nociceptive sensory processing in the spinal cord. An increased level of spinal ACh induces analgesia both in humans and rodents while interfering with cholinergic signaling is allodynic, demonstrating that a basal tone of spinal ACh modulates nociceptive responses in naïve animals. The plasticity undergone by this cholinergic system in chronic pain situation is unknown, and the mere presence of this tone in neuropathic animals is controversial. We have addressed these issues in mice through behavioral experiments, histology, electrophysiology and molecular biology, in the cuff model of peripheral neuropathy. Our behavior experiments demonstrate the persistence, and even increased impact of the analgesic cholinergic tone acting through nicotinic receptors in cuff animals. The neuropathy does not affect the number or membrane properties of dorsal horn cholinergic neurons, nor specifically the frequency of their synaptic inputs. The alterations thus appear to be in the neurons receiving the cholinergic signaling, which is confirmed by the fact that subthreshold doses of acetylcholinesterase (AChE) inhibitors in sham animals become anti-allodynic in cuff mice and by the altered expression of the β2 nicotinic receptor subunit. Our results demonstrate that endogenous cholinergic signaling can be manipulated to relieve mechanical allodynia in animal models of peripheral neuropathy. Until now, AChE inhibitors have mainly been used in the clinics in situations of acute pain (parturition, post-operative). The fact that lower doses (thus with fewer side effects) could be efficient in chronic pain conditions opens new avenues for the treatment of neuropathic pain. SIGNIFICANCE STATEMENT: Chronic pain continues to be the most common cause of disability that impairs the quality of life, accruing enormous and escalating socio-economic costs. A better understanding of the plasticity of spinal neuronal networks, crucially involved in nociceptive processing, could help designing new therapeutic avenues. We here demonstrate that chronic pain modifies the spinal nociceptive network in such a way that it becomes more sensitive to cholinergic modulations. The spinal cholinergic system is responsible for an analgesic tone that can be exacerbated by acetylcholinesterase inhibitors, a property used in the clinic to relief acute pain (child birth, post-op). Our results suggest that lower doses of acetylcholinesterases, with even fewer side effects, could be efficient to relieve chronic pain.
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http://dx.doi.org/10.1016/j.nbd.2021.105363DOI Listing
April 2021

[Nociception pain and autism].

Med Sci (Paris) 2021 Feb 16;37(2):141-151. Epub 2021 Feb 16.

CNRS, 5 rue Blaise-Pascal, 67000 Strasbourg, France.

Autistic subjects frequently display sensory anomalies. Those regarding nociception and its potential outcome, pain, are of crucial interest. Indeed, because of numerous comorbidities, autistic subjects are more often exposed to painful situation. Despite being often considered as less sensitive, experimental studies evaluating this point are failing to reach consensus. Using animal model can help reduce variability and bring, regarding autism, an overview of potential alterations of the nociceptive system at the cellular and molecular level.
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http://dx.doi.org/10.1051/medsci/2020280DOI Listing
February 2021

Involvement of the lateral habenula in fear memory.

Brain Struct Funct 2020 Sep 8;225(7):2029-2044. Epub 2020 Jul 8.

Université de Strasbourg, Centre National de La Recherche Scientifique, Laboratoire de Neurosciences Cognitives Et Adaptatives (LNCA), UMR 7364, 12 rue Goethe, 67000, Strasbourg, France.

Increasing evidence points to the engagement of the lateral habenula (LHb) in the selection of appropriate behavioral responses in aversive situations. However, very few data have been gathered with respect to its role in fear memory formation, especially in learning paradigms in which brain areas involved in cognitive processes like the hippocampus (HPC) and the medial prefrontal cortex (mPFC) are required. A paradigm of this sort is trace fear conditioning, in which an aversive event is preceded by a discrete stimulus, generally a tone, but without the close temporal contiguity allowing for their association based on amygdala-dependent information processing. In a first experiment, we analyzed cellular activations (c-Fos expression) induced by trace fear conditioning in subregions of the habenular complex, HPC, mPFC and amygdala using a factorial analysis to unravel functional networks through correlational analysis of data. This analysis suggested that distinct LHb subregions engaged in different aspects of conditioning, e.g. associative processes and onset of fear responses. In a second experiment, we performed chemogenetic LHb inactivation during the conditioning phase of the trace fear conditioning paradigm and subsequently assessed contextual and tone fear memories. Whereas LHb inactivation did not modify rat's behavior during conditioning, it induced contextual memory deficits and enhanced fear to the tone. These results demonstrate the involvement of the LHb in fear memory. They further suggest that the LHb is engaged in learning about threatening environments through the selection of relevant information predictive of a danger.
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http://dx.doi.org/10.1007/s00429-020-02107-5DOI Listing
September 2020

High-resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline.

Purinergic Signal 2019 09 23;15(3):403-420. Epub 2019 Aug 23.

Centre National de la Recherche Scientifique (UPR3212), Institut des Neurosciences Cellulaires et Intégratives, Université de Strasbourg, 67000, Strasbourg, France.

Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200 nM progesterone (HEK293-P2X2-PROG), a potent positive allosteric modulator of homomeric P2X2 receptors, and used to detect low nanomolar concentrations of extracellular ATP. Fura-2-loaded HEK293-P2X2-PROG cells were acutely plated on top of cultured DH glial cells to quantify ATP release from single DH glial cells. Application of the α1 adrenoceptor agonist phenylephrine (PHE, 20 μM) or of a low K (0.2 mM) solution evoked reversible increases in the intracellular calcium concentration ([Ca]) in the biosensor cells. A reversible increase in [Ca] was also detected in half of the biosensor cells following the interruption of general extracellular perfusion. All increases in [Ca] were blocked in the presence of the P2X2 antagonist PPADS or after preloading the glial cells with the calcium chelator BAPTA, indicating that they were due to calcium-dependent ATP release from the glial cells. ATP release induced by PHE was blocked by -L-phenylalanine 2-naphtylamide (GPN) that permeabilizes secretory lysosomes and bafilomycin A1 (Baf A1), an inhibitor of the H-pump of acidic secretory vesicles. By contrast, ATP release induced by application of a low-K solution was abolished by Baf A1 but not by GPN. Finally, spontaneous ATP release observed after interrupting general perfusion was insensitive to both GPN and Baf A1 pretreatment. Our results indicate that ATP is released in a calcium-dependent manner from two distinct vesicular pools and one non-vesicular pool coexisting in DH glial cells and that noradrenaline and PHE selectively target the secretory lysosome pool.
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http://dx.doi.org/10.1007/s11302-019-09673-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737151PMC
September 2019

Neuronal networks and nociceptive processing in the dorsal horn of the spinal cord.

Neuroscience 2016 Dec 3;338:230-247. Epub 2016 Sep 3.

Institut des Neurosciences Cellulaires et Intégratives, UPR3212, Centre National de la Recherche Scientifique, 67084 Strasbourg, France; Université de Strasbourg, 67084 Strasbourg, France. Electronic address:

The dorsal horn (DH) of the spinal cord receives a variety of sensory information arising from the inner and outer environment, as well as modulatory inputs from supraspinal centers. This information is integrated by the DH before being forwarded to brain areas where it may lead to pain perception. Spinal integration of this information relies on the interplay between different DH neurons forming complex and plastic neuronal networks. Elements of these networks are therefore potential targets for new analgesics and pain-relieving strategies. The present review aims at providing an overview of the current knowledge on these networks, with a special emphasis on those involving interlaminar communication in both physiological and pathological conditions.
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http://dx.doi.org/10.1016/j.neuroscience.2016.08.048DOI Listing
December 2016

Loss of inhibitory tone on spinal cord dorsal horn spontaneously and nonspontaneously active neurons in a mouse model of neuropathic pain.

Pain 2016 07;157(7):1432-1442

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, Strasbourg, France.

Plasticity of inhibitory transmission in the spinal dorsal horn (SDH) is believed to be a key mechanism responsible for pain hypersensitivity in neuropathic pain syndromes. We evaluated this plasticity by recording responses to mechanical stimuli in silent neurons (nonspontaneously active [NSA]) and neurons showing ongoing activity (spontaneously active [SA]) in the SDH of control and nerve-injured mice (cuff model). The SA and NSA neurons represented 59% and 41% of recorded neurons, respectively, and were predominantly wide dynamic range (WDR) in naive mice. Nerve-injured mice displayed a marked decrease in the mechanical threshold of the injured paw. After nerve injury, the proportion of SA neurons was increased to 78%, which suggests that some NSA neurons became SA. In addition, the response to touch (but not pinch) was dramatically increased in SA neurons, and high-threshold (nociceptive specific) neurons were no longer observed. Pharmacological blockade of spinal inhibition with a mixture of GABAA and glycine receptor antagonists significantly increased responses to innocuous mechanical stimuli in SA and NSA neurons from sham animals, but had no effect in sciatic nerve-injured animals, revealing a dramatic loss of spinal inhibitory tone in this situation. Moreover, in nerve-injured mice, local spinal administration of acetazolamide, a carbonic anhydrase inhibitor, restored responses to touch similar to those observed in naive or sham mice. These results suggest that a shift in the reversal potential for anions is an important component of the abnormal mechanical responses and of the loss of inhibitory tone recorded in a model of nerve injury-induced neuropathic pain.
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http://dx.doi.org/10.1097/j.pain.0000000000000538DOI Listing
July 2016

Noradrenaline-mediated facilitation of inhibitory synaptic transmission in the dorsal horn of the rat spinal cord involves interlaminar communications.

Eur J Neurosci 2015 Nov 14;42(9):2654-65. Epub 2015 Oct 14.

Centre National de la Recherche Scientifique (CNRS UPR 3212), Institut des Neurosciences Cellulaires et Intégratives, 5 rue Blaise Pascal, 67084, Strasbourg, France.

In the dorsal horn of the spinal cord (DH), noradrenaline (NA) is released by axons originating from the locus coeruleus and induces spinal analgesia, the mechanisms of which are poorly understood. Here, the effects of NA on synaptic transmission in the deep laminae (III-V) of the DH were characterized. It was shown that exogenously applied, as well as endogenously released, NA facilitated inhibitory [γ-aminobutyric acid (GABA)ergic and glycinergic] synaptic transmission in laminae III-IV of the DH by activating α1-, α2- and β-adrenoceptors (ARs). In contrast, NA had no effect on excitatory (glutamatergic) synaptic transmission. Physical interruption of communications between deep and more superficial laminae (by a mechanical transection between laminae IV and V) totally blocked the effects of α2-AR agonists and strongly reduced the effects of α1-AR agonists on inhibitory synaptic transmission in laminae III-IV without directly impairing synaptic release of GABA or glycine from neurons. Short-term pretreatment of intact spinal cord slices with the glial cell metabolism inhibitor fluorocitrate or pharmacological blockade of ionotropic glutamate and ATP receptors mimicked the consequences of a mechanical transection between laminae IV and V. Taken together, the current results indicate that the facilitation of inhibitory synaptic transmission in laminae III-IV of the DH by NA requires functional interlaminar connections between deep and more superficial laminae, and might strongly depend on glia to neuron interactions. These interlaminar connections and glia to neuron interactions could represent interesting targets for analgesic strategies.
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http://dx.doi.org/10.1111/ejn.13077DOI Listing
November 2015

Activation of transient receptor potential vanilloid 2-expressing primary afferents stimulates synaptic transmission in the deep dorsal horn of the rat spinal cord and elicits mechanical hyperalgesia.

Eur J Neurosci 2014 Oct 8;40(8):3189-201. Epub 2014 Aug 8.

Institut des Neurosciences Cellulaires et Integratives, UPR 3212 Centre National de la Recherche Scientifique, Strasbourg, France; Universite de Strasbourg, 5 rue Blaise Pascal, F-67084, Strasbourg, France.

Probenecid, an agonist of transient receptor vanilloid (TRPV) type 2, was used to evaluate the effects of TRPV2 activation on excitatory and inhibitory synaptic transmission in the dorsal horn (DH) of the rat spinal cord and on nociceptive reflexes induced by thermal heat and mechanical stimuli. The effects of probenecid were compared with those of capsaicin, a TRPV1 agonist. Calcium imaging experiments on rat dorsal root ganglion (DRG) and DH cultures indicated that functional TRPV2 and TRPV1 were expressed by essentially non-overlapping subpopulations of DRG neurons, but were absent from DH neurons and DH and DRG glial cells. Pretreatment of DRG cultures with small interfering RNAs against TRPV2 suppressed the responses to probenecid. Patch-clamp recordings from spinal cord slices showed that probenecid and capsaicin increased the frequencies of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents in a subset of laminae III-V neurons. In contrast to capsaicin, probenecid failed to stimulate synaptic transmission in lamina II. Intrathecal or intraplantar injections of probenecid induced mechanical hyperalgesia/allodynia without affecting nociceptive heat responses. Capsaicin induced both mechanical hyperalgesia/allodynia and heat hyperalgesia. Activation of TRPV1 or TRPV2 in distinct sets of primary afferents increased the sEPSC frequencies in a largely common population of DH neurons in laminae III-V, and might underlie the development of mechanical hypersensitivity following probenecid or capsaicin treatment. However, only TRPV1-expressing afferents facilitated excitatory and/or inhibitory transmission in a subpopulation of lamina II neurons, and this phenomenon might be correlated with the induction of thermal heat hyperalgesia.
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http://dx.doi.org/10.1111/ejn.12688DOI Listing
October 2014

Antidepressants suppress neuropathic pain by a peripheral β2-adrenoceptor mediated anti-TNFα mechanism.

Neurobiol Dis 2013 Dec 23;60:39-50. Epub 2013 Aug 23.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, 67084 Strasbourg, France; Université de Strasbourg, 67084 Strasbourg, France.

Neuropathic pain is pain arising as a direct consequence of a lesion or disease affecting the somatosensory system. It is usually chronic and challenging to treat. Some antidepressants are first-line pharmacological treatments for neuropathic pain. The noradrenaline that is recruited by the action of the antidepressants on reuptake transporters has been proposed to act through β2-adrenoceptors (β2-ARs) to lead to the observed therapeutic effect. However, the complex downstream mechanism mediating this action remained to be identified. In this study, we demonstrate in a mouse model of neuropathic pain that an antidepressant's effect on neuropathic allodynia involves the peripheral nervous system and the inhibition of cytokine tumor necrosis factor α (TNFα) production. The antiallodynic action of nortriptyline is indeed lost after peripheral sympathectomy, but not after lesion of central descending noradrenergic pathways. More particularly, we report that antidepressant-recruited noradrenaline acts, within dorsal root ganglia, on β2-ARs expressed by non-neuronal satellite cells. This stimulation of β2-ARs decreases the neuropathy-induced production of membrane-bound TNFα, resulting in relief of neuropathic allodynia. This indirect anti-TNFα action was observed with the tricyclic antidepressant nortriptyline, the selective serotonin and noradrenaline reuptake inhibitor venlafaxine and the β2-AR agonist terbutaline. Our data revealed an original therapeutic mechanism that may open novel research avenues for the management of painful peripheral neuropathies.
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http://dx.doi.org/10.1016/j.nbd.2013.08.012DOI Listing
December 2013

A novel population of cholinergic neurons in the macaque spinal dorsal horn of potential clinical relevance for pain therapy.

J Neurosci 2013 Feb;33(9):3727-37

Institut des Neurosciences Cellulaires et Intégratives, Unité Propre de Recherche 3212, Centre National de la Recherche Scientifique, Department Nociception et Douleur, Strasbourg Cedex 67084, France.

Endogenous acetylcholine (ACh) is a well-known modulator of nociceptive transmission in the spinal cord of rodents. It arises mainly from a sparse population of cholinergic interneurons located in the dorsal horn of the spinal cord. This population was thought to be absent from the spinal cord of monkey, what might suggest that spinal ACh would not be a relevant clinical target for pain therapy. In humans, however, pain responses can be modulated by spinal ACh, as evidenced by the increasingly used analgesic procedure (for postoperative and labor patients) consisting of the epidural injection of the acetylcholinesterase inhibitor neostigmine. The source and target of this ACh remain yet to be elucidated. In this study, we used an immunolabeling for choline acetyltransferase to demonstrate, for the first time, the presence of a plexus of cholinergic fibers in laminae II-III of the dorsal horn of the macaque monkey. Moreover, we show the presence of numerous cholinergic cell bodies within the same laminae and compared their density and morphological properties with those previously described in rodents. An electron microscopy analysis demonstrates that cholinergic boutons are presynaptic to dorsal horn neurons as well as to the terminals of sensory primary afferents, suggesting that they are likely to modulate incoming somatosensory information. Our data suggest that this newly identified dorsal horn cholinergic system in monkeys is the source of the ACh involved in the analgesic effects of epidural neostigmine and could be more specifically targeted for novel therapeutic strategies for pain management in humans.
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http://dx.doi.org/10.1523/JNEUROSCI.3954-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619320PMC
February 2013

Interactions between superficial and deep dorsal horn spinal cord neurons in the processing of nociceptive information.

Eur J Neurosci 2012 Dec 12;36(11):3500-8. Epub 2012 Sep 12.

Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, UPR 3212 Centre National de la Recherche Scientifique, Université de Strasbourg, 21 rue René Descartes, F-67084 Strasbourg, France.

In acute rat spinal cord slices, the application of capsaicin (5 μm, 90 s), an agonist of transient receptor potential vanilloid 1 receptors expressed by a subset of nociceptors that project to laminae I-II of the spinal cord dorsal horn, induced an increase in the frequency of spontaneous excitatory and spontaneous inhibitory postsynaptic currents in about half of the neurons in laminae II, III-IV and V. In the presence of tetrodotoxin, which blocks action potential generation and polysynaptic transmission, capsaicin increased the frequency of miniature excitatory postsynaptic currents in only 30% of lamina II neurons and had no effect on the frequency of miniature excitatory postsynaptic currents in laminae III-V or on the frequency of miniature inhibitory postsynaptic currents in laminae II-V. When the communication between lamina V and more superficial laminae was interrupted by performing a mechanical section between laminae IV and V, capsaicin induced an increase in spontaneous excitatory postsynaptic current frequency in laminae II-IV and an increase in spontaneous inhibitory postsynaptic current frequency in lamina II that were similar to those observed in intact slices. However, in laminae III-IV of transected slices, the increase in spontaneous inhibitory postsynaptic current frequency was virtually abolished. Our results indicate that nociceptive information conveyed by transient receptor potential vanilloid 1-expressing nociceptors is transmitted from lamina II to deeper laminae essentially by an excitatory pathway and that deep laminae exert a 'feedback' control over neurons in laminae III-IV by increasing inhibitory synaptic transmission in these laminae. Moreover, we provide evidence that laminae III-IV might play an important role in the processing of nociceptive information in the dorsal horn.
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http://dx.doi.org/10.1111/j.1460-9568.2012.08273.xDOI Listing
December 2012

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn.

Eur J Neurosci 2011 Oct 21;34(8):1230-40. Epub 2011 Sep 21.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Université de Strasbourg, 21 rue René Descartes, Strasbourg, France.

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.
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http://dx.doi.org/10.1111/j.1460-9568.2011.07846.xDOI Listing
October 2011

Nociceptive thresholds are controlled through spinal β2-subunit-containing nicotinic acetylcholine receptors.

Pain 2011 Sep 26;152(9):2131-2137. Epub 2011 Jul 26.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Strasbourg, France Faculté des Sciences de la Vie, Université de Strasbourg, Strasbourg, France Division of Molecular Psychiatry, Abraham Ribicoff Research Facilities, Connecticut Mental Health Center, Yale University School of Medicine, New Haven, CT, USA.

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.
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http://dx.doi.org/10.1016/j.pain.2011.05.022DOI Listing
September 2011

Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord.

J Comp Neurol 2011 Nov;519(16):3139-58

Institut des Neurosciences Cellulaires et Intégratives, UPR3212 CNRS, Dept. Nociception et Douleur, 67084 Strasbourg, France.

Endogenous acetylcholine is an important modulator of sensory processing, especially at the spinal level, where nociceptive (pain-related) stimuli enter the central nervous system and are integrated before being relayed to the brain. To decipher the organization of the local cholinergic circuitry in the spinal dorsal horn, we used transgenic mice expressing enchanced green fluorescent protein specifically in cholinergic neurons (ChAT::EGFP) and characterized the morphology, neurochemistry, and firing properties of the sparse population of cholinergic interneurons in this area. Three-dimensional reconstruction of lamina III ChAT::EGFP neurons based either on their intrinsic fluorescence or on intracellular labeling in live tissue demonstrated that these neurons have long and thin processes that grow preferentially in the dorsal direction. Their dendrites and axon are highly elongated in the rostrocaudal direction, beyond the limits of a single spinal segment. These unique morphological features suggest that dorsal horn cholinergic interneurons are the main contributors to the plexus of cholinergic processes located in lamina IIi, just dorsal to their cell bodies. In addition, immunostainings demonstrated that dorsal horn cholinergic interneurons in the mouse are γ-aminobutyric acidergic and express nitric oxide synthase, as in rats. Finally, electrophysiological recordings from these neurons in spinal cord slices demonstrate that two-thirds of them have a repetitive spiking pattern with frequent rebound spikes following hyperpolarization. Altogether our results indicate that, although they are rare, the morphological and functional features of cholinergic neurons enable them to collect segmental information in superficial layers of the dorsal horn and to modulate it over several segments.
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http://dx.doi.org/10.1002/cne.22668DOI Listing
November 2011

Selective potentiation of homomeric P2X2 ionotropic ATP receptors by a fast non-genomic action of progesterone.

Neuropharmacology 2010 Mar 16;58(3):569-77. Epub 2009 Dec 16.

Institut des Neurosciences Cellulaires et Intégratives, UPR 3212 Centre National de la Recherche Scientifique, Université de Strasbourg, Département Nociception et Douleur, 21 rue René Descartes, F-67084 Strasbourg cedex, France.

P2X receptors are ligand-gated ion channels activated by ATP that are widely expressed in the organism and regulate many physiological functions. We have studied the effect of progesterone (PROG) on native P2X receptors present in rat dorsal root ganglion (DRG) neurons and on recombinant P2X receptors expressed in HEK293 cells or Xenopus laevis oocytes. The effects of PROG were observed and already maximal during the first coapplication with ATP and did not need any preincubation of the cells with PROG, indicating a fast mechanism of action. In DRG neurons, PROG rapidly and reversibly potentiated submaximal but not saturating plateau-like currents evoked by ATP, but had no effect on the currents activated by alpha,beta-methylene ATP, an agonist of homomeric or heteromeric receptors containing P2X1 or P2X3 subunits. In cells expressing homomeric P2X2 receptors, responses to submaximal ATP, were systematically potentiated by PROG in a dose-dependent manner with a threshold between 1 and 10 nM. PROG had no effect on ATP currents carried by homomeric P2X1, P2X3, and P2X4 receptors or by heteromeric P2X1/5 and P2X2/3 receptors. We conclude that PROG selectively potentiates homomeric P2X2 receptors and, in contrast with dehydroepiandrosterone (DHEA), discriminates between homomeric and heteromeric P2X2-containing receptors. This might have important physiological implications since the P2X2 subunit is the most widely distributed P2X subunit in the organism. Moreover, DHEA and PROG might be useful tools to clarify the distribution and the role of native homo- and heteromeric P2X2 receptors.
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http://dx.doi.org/10.1016/j.neuropharm.2009.12.002DOI Listing
March 2010

Cord blood-derived neurons are originated from CD133+/CD34 stem/progenitor cells in a cell-to-cell contact dependent manner.

Stem Cells Dev 2008 Oct;17(5):1005-16

Neurogenèse et Processus Intégratifs dans l'Olfaction, Laboratoire de Neurosciences, Université de Franche-Comté, Besançon, France.

Previous studies described that neurons could be generated in vitro from human umbilical cord blood cells. However, there are few data concerning their origin. Notably, cells generating neurons are not well characterized. The present study deals with the origin of cord blood cells generating neurons and mechanisms allowing the neuronal differentiation. We studied neuronal markers of both total fractions of cord blood and stem/progenitor cord blood cells before and after selections and cultures. We also compared neuronal commitment of cord blood cells to that observed for the neuronal cell line SK-N-BE(2). Before cultures, neuronal markers are found within the total fraction of cord blood cells. In CD133+ stem/progenitor cell fraction only immature neuronal markers are detected. However, CD133+ cells are unable to give rise to neurons in cultures, whereas this is achieved when total fraction of cord blood cells is used. In fact, mature functional neurons can be generated from CD133+ cells only in cell-to-cell close contact with either CD133- fraction or a neurogenic epithelium. Furthermore, since CD133+ fraction is heterogenous, we used several selections to precisely identify the phenotype of cord blood-derived neuronal stem/progenitor cells. Results reveal that only CD34- cells from CD133+ fraction possess neuronal potential. These data show the phenotype of cord blood neuronal stem/progenitor cells and the crucial role of direct cell-to-cell contact to achieve their commitment. Identifying the neuron supporting factors may be beneficial to the use of cord blood neuronal stem/progenitor cells for regenerative medicine.
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http://dx.doi.org/10.1089/scd.2007.0248DOI Listing
October 2008

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

Mol Pain 2008 May 29;4:19. Epub 2008 May 29.

Department Nociception and Pain, Institut des Neurosciences Cellulaires et Intégratives, Unité Mixte de Recherche 7168, Centre National de la Recherche Scientifique/Université Louis Pasteur, Strasbourg, France.

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 Adelta 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.
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http://dx.doi.org/10.1186/1744-8069-4-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430948PMC
May 2008

Regional differences in the decay kinetics of GABA(A) receptor-mediated miniature IPSCs in the dorsal horn of the rat spinal cord are determined by mitochondrial transport of cholesterol.

J Neurosci 2008 Mar;28(13):3427-37

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Université Louis Pasteur, F-67084 Strasbourg, France.

We examined the possibility of a differential spatial control in the endogenous production of 3alpha5alpha-reduced steroids and its consequences on GABA(A) receptor-mediated miniature IPSCs (mIPSCs) in laminas II and III-IV of the rat spinal cord dorsal horn (DH). Early in postnatal development [younger than postnatal day 8 (P8)], mIPSCs displayed slow decay kinetics in laminas II and III-IV resulting from a continuous local production of 3alpha5alpha-reduced steroids. This was mediated by the tonic activity of the translocator protein of 18 kDa (TSPO), which controls neurosteroid synthesis by regulating the transport of cholesterol across the mitochondrial membrane system. TSPO activity disappeared in laminas III-IV after P8 and was functionally downregulated in lamina II after P15, resulting in a marked reduction of mIPSC duration in these laminas. TSPO-mediated synthesis of 3alpha5alpha-reduced steroids was spatially restricted, because, at P9-P15, when their production was maximal in lamina II, no sign of spillover to laminas III-IV was apparent. Interestingly, after P8, the enzymes necessary for the synthesis of 3alpha5alpha-reduced steroids remained functional in laminas III-IV and could produce such steroids from various precursors or after a single subcutaneous injection of progesterone. Moreover, induction of an acute peripheral inflammation by intraplantar injection of carrageenan, restored a maximal TSPO-mediated neurosteroidogenesis in laminas III-IV. Our results indicate that the decay kinetics of GABA(A) receptor-mediated mIPSCs in the DH of the spinal cord are primarily controlled by 3alpha5alpha-reduced steroids, which can be produced from circulating steroid precursors and/or in a spatially restricted manner by the modulation of the activity of TSPO.
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http://dx.doi.org/10.1523/JNEUROSCI.5076-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670604PMC
March 2008

Differential contribution of GABAergic and glycinergic components to inhibitory synaptic transmission in lamina II and laminae III-IV of the young rat spinal cord.

Eur J Neurosci 2007 Nov;26(10):2940-9

Université Louis Pasteur, Institut des Neurosciences Cellulaires et Intégratives (INCI), Centre National de la Recherche Scientifique (CNRS), UMR7168, F-67084 Strasbourg, France.

Using whole-cell patch-clamp recordings from spinal cord slices of young (10-15 days old) rats, we have characterized and compared the properties of inhibitory synaptic transmission in lamina II and laminae III-IV of the dorsal horn, which are involved in the processing of nociceptive and non-nociceptive sensory information, respectively. All (100%) of laminae III-IV neurons, but only 55% of lamina II neurons, received both gamma-aminobutyric acid (GABA)ergic and glycinergic inputs. The remaining 45% of lamina II neurons received only GABAergic synapses. Neurons receiving only glycinergic synapses were never observed. Among the 55% of lamina II neurons receiving both GABAergic and glycinergic inputs, all displayed a small proportion (approximately 10%) of mixed miniature inhibitory postsynaptic currents (mIPSCs), indicating the presence of a functional GABA/glycine co-transmission at a subset of synapses. Such a co-transmission was never observed in laminae III-IV neurons. The presence of mixed mIPSCs and the differences in decay kinetics of GABAA-type receptor mIPSCs between lamina II and laminae III-IV were due to the endogenous tonic production of 3alpha5alpha-reduced steroids (3alpha5alpha-RS) in lamina II. Stimulation of the local production of 3alpha5alpha-RS was possible in laminae III-IV after incubation of slices with progesterone, subcutaneous injection of progesterone or induction of a peripheral inflammation. This led to the prolongation of GABAergic mIPSCs, but failed to induce the appearance of mixed mIPSCs in laminae III-IV. Our results indicate that, compared with lamina II, inhibitory synaptic transmission in laminae III-IV is characterized by a dominant role of glycinergic inhibition and the absence of a functional GABA/glycine co-transmission.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05919.xDOI Listing
November 2007

PKC activation sets an upper limit to the functional plasticity of GABAergic transmission induced by endogenous neurosteroids.

Eur J Neurosci 2007 Sep;26(5):1173-82

Institut des Neurosciences Cellulaires et Intégratives, Department of Nociception and Pain, Unité Mixte de Recherche 7168 Centre National de la Recherche Scientifique/Université Louis Pasteur, Strasbourg, France.

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.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05746.xDOI Listing
September 2007

Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model.

Eur J Neurosci 2007 Jul 26;26(1):113-25. Epub 2007 Jun 26.

Institut des Neurosciences Cellulaires et Intégratives (INCI), UMR7168 Centre National de la Recherche Scientifique, Université Louis Pasteur, F-67084 Strasbourg, France.

The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05649.xDOI Listing
July 2007

Corelease of GABA/glycine in lamina-X of the spinal cord of neonatal rats.

Neuroreport 2007 Jul;18(10):1025-9

Department of Physiology, University of Basel, Pharmazentrum, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.

Spinal-cord slices from neonatal rats were used to record lamina-X neurons using the patch-clamp technique under whole cell recording configuration. Lamina-X surrounds the central canal of the spinal cord and contains sympathetic preganglionic neurons of the central autonomic nucleus. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and kynurenic acid to block action potential-dependent transmitter release and glutamatergic transmissions, respectively. We recorded mixed gamma-amino-n-butyric acid/glycine miniature synaptic currents suggesting that gamma-amino-n-butyric acid and glycine can be coreleased from the same single synaptic vesicles, and that this corelease can be detected by the postsynaptic cell. In addition, acetylcholine can induce the release of gamma-amino-n-butyric acid/glycine by acting presynaptically at nicotinic receptors located on the gamma-amino-n-butyric acid ergic/glycinergic terminals.
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http://dx.doi.org/10.1097/WNR.0b013e3281667c0cDOI Listing
July 2007

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord.

Neuropharmacology 2006 Jul 5;51(1):77-89. Epub 2006 May 5.

Department of Physiology, University of Basel, Pharmazentrum, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9-15days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100microM), a selective agonist at GABA(A) receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA(A) receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100microM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100microM cadmium added to the bath solution. Choline 10mM, an agonist at alpha7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10microM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1microM of dihydro-beta-erythroidine (DHbetaE), an antagonist of non-alpha7 nAChRs but was insensitive to alpha7 nAChRs antagonists (strychnine, alpha-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10microM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non alpha7 nAChRs.
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http://dx.doi.org/10.1016/j.neuropharm.2006.03.007DOI Listing
July 2006

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

J Mol Neurosci 2006 ;28(1):33-51

Institut des Neurosciences Cellulaires et Intégratives-Centre National de la Recherche Scientifique (CNRS), Université Louis Pasteur, 67084 Strasbourg Cedex, France.

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.
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http://dx.doi.org/10.1385/jmn:28:1:33DOI Listing
June 2006

Inflammatory pain upregulates spinal inhibition via endogenous neurosteroid production.

J Neurosci 2005 Dec;25(50):11768-76

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique/Université Louis Pasteur, F-67084 Strasbourg, France.

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 5alpha-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.
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http://dx.doi.org/10.1523/JNEUROSCI.3841-05.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726017PMC
December 2005

The rat spinal cord slice: Its use in generating pharmacological evidence for cholinergic transmission using the alpha7 subtype of nicotinic receptors in the central autonomic nucleus.

J Pharmacol Toxicol Methods 2005 May-Jun;51(3):243-52

Laboratoire de Neurophysiologie Cellulaire et Intégrée, UMR 7519 CNRS/ULP, 21 rue R.Descartes, 67084 Strasbourg Cedex, France.

Lamina X surrounds the central canal of the spinal cord and is an important site for the convergence of somatic and visceral afferent inputs relaying nociceptive information. Lamina X contains sympathetic preganglionic neurons (SPN) in the so-called central autonomic nucleus which may participate to viscero-autonomic reflexes. Here, we describe a transversal slice preparation of postnatal rat thoracolumbar spinal cord which allows the detailed characterization of the morphology, electrophysiological properties, synaptic activities and receptor pharmacology of neurons surrounding the central canal. By means of the patch clamp technique, in its whole cell configuration, and by the use of various pharmacological tools, we show here that lamina X neurons of the central autonomic nucleus express functional alpha7 nicotinic receptors which are located postsynaptically on SPNs where they are involved in a fast cholinergic transmission. Thus, this in vitro preparation is useful to study the mechanisms and the pharmacology of viscero-autonomic reflexes.
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http://dx.doi.org/10.1016/j.vascn.2004.08.013DOI Listing
August 2005

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

J Physiol 2004 Aug 2;559(Pt 1):169-86. Epub 2004 Jul 2.

Laboratoire de Neurophysiologie Cellulaire et Intégrée, UMR 7519 CNRS/ULP, 21 rue R. Descartes, 67084 Strasbourg Cedex, France.

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 microm 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 micromd-2-amino-5-phosphonovalerate (APV), an antagonist of the NMDA receptor, and by 30 microm dichlorokynurenic acid (DCKA), an antagonist of the glycine site of the NMDA receptor. Glycine (1-5 microm) and d-serine (10 microm) 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.
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http://dx.doi.org/10.1113/jphysiol.2004.068858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1665078PMC
August 2004

Production of 5alpha-reduced neurosteroids is developmentally regulated and shapes GABA(A) miniature IPSCs in lamina II of the spinal cord.

J Neurosci 2004 Jan;24(4):907-15

Laboratoire de Neurophysiologie Cellulaire et Intégrée, Université Louis Pasteur/Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7519, F-67084 Strasbourg, France.

In lamina II of the spinal dorsal horn, synaptic inhibition mediated by ionotropic GABA(A) 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 GABA(A) 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 5alpha-reductase, accelerated the decay kinetics of GABA(A) 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 5alpha-reduced neurosteroids in young rats that confers slow decay kinetics to GABA(A) mIPSCs. At all of the ages, selective stimulation of PBR by diazepam in the presence of flumazenil prolonged GABA(A) mIPSCs in a PK11195- and finasteride-sensitive manner. This condition also increased the proportion of mixed GABA(A)/glycine mIPSCs in the immature animals and led to the reappearance of mixed GABA(A)/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 5alpha-reduced neurosteroids.
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http://dx.doi.org/10.1523/JNEUROSCI.4642-03.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729827PMC
January 2004

Convergent control of synaptic GABA release from rat dorsal horn neurones by adenosine and GABA autoreceptors.

J Physiol 2003 Sep 4;551(Pt 2):479-89. Epub 2003 Jul 4.

Laboratoire de Neurophysiologie Cellulaire et Intégrée, UMR 7519-CNRS, Université Louis Pasteur, 21 rue René Descartes, 67084 Strasbourg Cedex, France.

Perforated patch clamp recordings were performed on cultured superficial neonatal rat dorsal horn (DH) spinal cord neurones in order to study the presynaptic modulation of GABA release at unitary synaptic connections. Since ATP can be coreleased with GABA at about two-thirds of GABAergic synapses between DH neurones, and can be rapidly metabolized to adenosine in the extracellular space, we investigated the potential role of A1 adenosine receptors and GABAB receptors which might function as inhibitory autoreceptors. Adenosine and GABAB receptor agonists reduced the amplitude of electrically evoked GABAergic inhibitory postsynaptic currents (eIPSCs) as well as the frequency of GABAergic miniature IPSCs, suggesting a presynaptic action of these substances. The actions of adenosine were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The effects of adenosine and GABAB agonists were occlusive, indicating a functional convergence of the signalling pathways engaged by A1 and GABAB receptors. A1 and GABAB antagonists increased the amplitude of eIPSCs in a supra-additive manner, suggesting a tonic activation of these receptors by ambient adenosine and GABA. Moreover, using trains of electrical stimulations, we were able to unravel a phasic (activity-dependent) activation of presynaptic A1 and GABAB autoreceptors only in the case of neurones coreleasing ATP and GABA, despite the presence of functional presynaptic A1 and GABAB receptors on all GABAergic DH neurones. This selective, convergent and activity-dependent inhibition of GABA release by A1 and GABAB autoreceptors might modulate the integrative properties of postsynaptic DH neurones under physiological conditions and/or during the development of pathological pain states.
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http://dx.doi.org/10.1113/jphysiol.2003.047894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2343215PMC
September 2003

Dehydroepiandrosterone potentiates native ionotropic ATP receptors containing the P2X2 subunit in rat sensory neurones.

J Physiol 2003 Oct 4;552(Pt 1):59-71. Epub 2003 Jul 4.

Laboratoire de Neurophysiologie Cellulaire et Intégrée, UMR 7519-CNRS, Université Louis Pasteur, 21 rue René Descartes, 67084 Strasbourg Cedex, France.

We have studied the modulatory effect of dehydroepiandrosterone (DHEA), the most abundant neurosteroid produced by glial cells and neurones, on membrane currents induced by the activation of ionotropic ATP (P2X) receptors in neonatal rat dorsal root ganglion neurones. ATP (1 microM) induced three types of currents/responses termed F (fast and transient), S (slowly desensitizing) and M (mixed, sum of F- and S-type responses). DHEA (10 nM to 100 microM) concentration-dependently increased the amplitude of plateau-like currents of S- and M-type responses evoked by submaximal (1 microM) but not saturating (100 microM or 1 mM) concentrations of ATP. Alphabeta-methylene ATP (alphabetame-ATP, 5 microM) also evoked F-, S- and M-type responses, the plateau phases of which were potentiated by lowering external pH (6.3) and by ivermectin (IVM, 3 microM), indicating the presence heteromeric P2X2-containing receptors and possibly of functional native P2X4/6 receptors. There was a strict correlation between the potentiating effects of low pH and DHEA on alphabetame-ATP responses but not between that of IVM and DHEA, suggesting that DHEA selectively modulated P2X2-containing receptors. DHEA also potentiated putative homomeric P2X2 receptor responses recorded in the continuous presence of 1 microM 2'-(or 3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Our results constitute the first demonstration of a fast potentiation of P2X receptors by a neurosteroid and suggest that DHEA could be an endogenous modulator of P2X2-containing receptors thereby contributing to the facilitation of the detection and/or the transmission of nociceptive messages, particularly under conditions of inflammatory pain where the P2X receptor signalling pathway appears to be upregulated.
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http://dx.doi.org/10.1113/jphysiol.2003.046078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2343311PMC
October 2003