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

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.journal articleresearch support, non-u.s. gov't2013 Feb 27importe

Etude de la distribution et des proprietes pharmacologiques des recepteurs du GABA sur les afferences primaires du rat

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

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

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

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

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

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

Steroids exert long-term modulatory effects on numerous physiological functions by acting at intracellular/nuclear receptors influencing gene transcription. Steroids and neurosteroids can also rapidly modulate membrane excitability and synaptic transmission by interacting with ion channels, that is, ionotropic neurotransmitter receptors or voltage-dependent Ca2+ or K+ channels. More recently, the cloning of a plasma membrane-located G protein-coupled receptor for progestins in various species has suggested that steroids/neurosteroids could also influence second-messenger pathways by directly interacting with specific membrane receptors. Here we review the experimental evidence implicating steroids/neurosteroids in the modulation of synaptic transmission and the evidence for a role of endogenously produced neurosteroids in such modulatory effects. We present some of our recent results concerning inhibitory synaptic transmission in lamina II of the spinal cord and show that endogenous 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

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

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

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

International audiencePrevious 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

Inflammatory pain upregulates spinal inhibition via endogenous neurosteroid production

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