Downregulation of spinal astrocytic connexin43 leads to upregulation of interleukin‐6 and cyclooxygenase‐2 and mechanical hypersensitivity in mice

Connexin43 (Cx43), involved in intercellular signaling, is expressed in spinal dorsal horn astrocytes and crucial in the maintenance of neuropathic pain. Downregulation of spinal astrocytic Cx43 in mice enhances glutamatergic neurotransmission by decreasing glutamate transporter GLT‐1 expression, resulting in cutaneous hypersensitivity. Decreased expression of astrocytic Cx43 could lead to altered expression of other nociceptive molecules. Transfection of Cx43‐targeting siRNA in cultured spinal astrocytes increased expression of the pronociceptive cytokine interleukin‐6 (IL‐6) and the prostaglandin synthesizing enzyme cyclooxygenase‐2 (COX‐2). Increased expression of IL‐6 and COX‐2 was due to decreased Cx43 expression rather than due to diminished Cx43 channel function. In mice, downregulation of spinal Cx43 expression by intrathecal treatment with Cx43‐targeting siRNA increased IL‐6 and COX‐2 expression and induced hind paw mechanical hypersensitivity. Cx43 siRNA‐induced mechanical hypersensitivity was attenuated by intrathecal treatment with anti‐IL‐6 neutralizing antibody and intraperitoneal treatment of selective COX‐2 inhibitor celecoxib, demonstrating that these molecules play a role in nociceptive processing following Cx43 downregulation. Restoring spinal Cx43 by intrathecal injection of an adenovirus vector expressing Cx43 in mice with a partial sciatic nerve ligation reduced spinal IL‐6 and COX‐2 expression. Suppression of glycogen synthase kinase‐3β (GSK‐3β), a serine/threonine protein kinase, prevented upregulation of IL‐6 and COX‐2 expression induced by Cx43 downregulation in both cultured astrocytes and in mouse spinal dorsal horn. Inhibition of spinal GSK‐3β also ameliorated Cx43 siRNA‐induced mechanical hypersensitivity. The current findings indicate that downregulation of spinal astrocytic Cx43 leads to changes in spinal expression of pronociceptive molecules underlying the maintenance of pain following nerve injury.     

Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice

Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3 days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7 days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca²⁺ channel subunit α₂δ₁ blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been implicated in the induction of central sensitization, particularly through its actions on astrocytes, in the spinal cord following peripheral injury. Intrathecal injection of TNF in naïve mice induced the downregulation of both Cx43 and GLT-1 in spinal dorsal horn, as well as hind paw mechanical hypersensitivity, as observed in PSNL mice. Conversely, intrathecal treatment of PSNL mice with the TNF inhibitor etanercept prevented not only mechanical hypersensitivity but also the downregulation of Cx43 and GLT-1 expression in astrocytes. The current findings indicate that spinal astrocytic Cx43 are essential for the maintenance of neuropathic pain following peripheral nerve injury and suggest modulation of Cx43 as a novel target for developing analgesics for neuropathic pain.     

Spinal p38 mitogen-activated protein kinase mediates allodynia induced by first-degree burn in the rat

Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been implicated in the development and maintenance of pain states. In this study, we tested whether p38 MAPK is involved in the response to first-degree burn of the hind paw. This injury induces central sensitization leading to tactile allodynia and is mediated by activation of Ca(2+) permeable AMPA/kainate receptors through PKC and PKA. We demonstrate that p38 MAPK is rapidly and robustly activated in the superficial spinal dorsal horn after mild thermal injury to the hind paw. Activated p38 MAPK was localized primarily to microglia and to a lesser extent in oligodendrocytes and lamina II neurons. Astrocytes were not involved in the p38 MAPK response. Intrathecal pretreatment of pharmacological inhibitors of p38 MAPK (SB203580, SD-282) dose-dependently blocked development of tactile allodynia, a characteristic of the first-degree burn model. The effects of the inhibitors on tactile allodynia were lost when they were administered after injury. These studies identify p38 MAPK as a major mediator of tactile allodynia, most likely activated downstream of AMPA/kainate receptors.     

Protein kinase C gamma interneurons in the rat medullary dorsal horn: Distribution and synaptic inputs to these neurons,and subcellular localization of the enzyme

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (II_i) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina II_i PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A‐ and C‐fibers low threshold mechanoreceptors (LTMRs) terminate in lamina II_i raise the general issue of synaptic inputs to lamina II_i PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ‐immunoreactivity is mostly restricted to interneurons in lamina II_i of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ‐immunoreactive interneurons are asymmetric (likely excitatory). PKCγ‐immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3‐immunoreactive bouton, specific to C‐LTMRs, on PKCγ‐immunoreactive interneurons. PKCγ‐immunoreactive interneurons contain GABA_Aergic and glycinergic receptors. At the subcellular level, PKCγ‐immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ‐immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia. J. Comp. Neurol. 522:393–413, 2014. © 2013 Wiley Periodicals, Inc.     

Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild-type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72-78). With the goal of understanding the origin of this increased activity in Cx36-/- mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477-485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36-/- mice. Both correlated and asynchronous action potentials in P10 Cx36-/- RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36-containing gap junctions in suppressing RGC firing between waves during postnatal retinal development.     

Peripheral neurobiologic mechanisms of antiallodynic effect of warm water immersion therapy on persistent inflammatory pain

Water immersion is widely used in physiotherapy and might relieve pain, probably by activating several distinct somatosensory modalities, including tactile, pressure, and thermal sensations. However, the endogenous mechanisms behind this effect remain poorly understood. This study examined whether warm water immersion therapy (WWIT) produces an antiallodynic effect in a model of localized inflammation and whether peripheral opioid, cannabinoid, and adenosine receptors are involved in this effect. Mice were injected with complete Freund's adjuvant (CFA; intraplantar; i.pl.). The withdrawal frequency to mechanical stimuli (von Frey test) was used to determine 1) the effect of WWIT against CFA‐induced allodynia and 2) the effect of i.pl. preadministration of naloxone (a nonselective opioid receptor antagonist; 5 µg/paw), caffeine (a nonselective adenosine receptor antagonist; 150 nmol/paw), 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX; a selective adenosine A₁ receptor antagonist; 10 nmol/paw), and AM630 (a selective cannabinoid receptor type 2 antagonist; 4 µg/paw) on the antiallodynic effect of WWIT against CFA‐induced allodynia. Moreover, the influence of WWIT on paw inflammatory edema was measured with a digital micrometer. WWIT produced a significant time‐dependent reduction of paw inflammatory allodynia but did not influence paw edema induced by CFA. Naloxone, caffeine, DPCPX, and AM630 injected in the right, but not in the left, hind paw significantly reversed the antiallodynic effect of WWIT. This is the first study to demonstrate the involvement of peripheral receptors in the antiallodynic effect of WWIT in a murine model of persistent inflammatory pain. © 2014 Wiley Periodicals, Inc.     

Spinal N-acetyl-alpha-linked acidic dipeptidase (NAALADase) inhibition attenuates mechanical allodynia induced by paw carrageenan injection in the rat

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) hydrolyzes N-acetyl-aspartyl-glutamate (NAAG) to liberate N-acetyl-aspartate and glutamate. NAAG is a putative neurotransmitter and acts as a mixed agonist/antagonist on N-methyl-D-aspartate (NMDA) receptors and acts as an agonist on the metabotropic glutamate receptor 3 (mGluR3). In the present study, we examined the role of spinal NAALADase in the maintenance of mechanical allodynia induced by carrageenan injection, skin incision and mild thermal injury using 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a specific NAALADase inhibitor, in rats. Mechanical allodynia was induced by injection of 2 mg carrageenan into the paw (carrageenan model), by creating a 1-cm longitudinal skin incision of the plantar aspect of the foot (post-operative model), or by application of thermal stimulation (52.5 degrees C) for 45 s to the hind paw (mild thermal injury model). 2-PMPA was administered intrathecally at the time when the maximum mechanical allodynia occurred. Mechanical allodynia was assessed by the measurement of mechanical threshold using von Frey filaments. The mechanical threshold was measured 5, 15, 30, 60 and 90 min after the drug administration. In the carrageenan model, 100 microg of 2-PMPA attenuated the level of mechanical allodynia. 2-PMPA had no effect on the level of mechanical allodynia in both the post-operative pain model and the mild thermal injury model. These data suggested that the inhibition of spinal NAALADase alleviated mechanical allodynia induced by paw carrageenan injection.     

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36‐containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase‐positive and 5‐hydroxytryptamine‐positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens‐1 (ZO‐1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36‐containing gap junctions that also incorporate the scaffolding protein ZO‐1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36‐containing gap junctions, which may now be tested by the use of Cx36 null mice.     

Re‐evaluation of connexins associated with motoneurons in rodent spinal cord,sexually dimorphic motor nuclei and trigeminal motor nucleus

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) are a common feature in mammalian brain circuitry, but less is known about their deployment in spinal cord. It has been reported based on connexin mRNA and/or protein detection that developing and/or mature motoneurons express a variety of connexins, including Cx26, Cx32, Cx36 and Cx43 in trigeminal motoneurons, Cx36, Cx37, Cx40, Cx43 and Cx45 in spinal motoneurons, and Cx32 in sexually dimorphic motoneurons. We re‐examined the localization of these connexins during postnatal development and in adult rat and mouse using immunofluorescence labeling for each connexin. We found Cx26 in association only with leptomeninges in the trigeminal motor nucleus (Mo5), Cx32 only with oligodendrocytes and myelinated fibers among motoneurons in this nucleus and in the spinal cord, and Cx37, Cx40 and Cx45 only with blood vessels in the ventral horn of spinal cord, including those among motoneurons. By freeze‐fracture replica immunolabeling, > 100 astrocyte gap junctions but no neuronal gap junctions were found based on immunogold labeling for Cx43, whereas 16 neuronal gap junctions at postnatal day (P)4, P7 and P18 were detected based on Cx36 labeling. Punctate labeling for Cx36 was localized to the somatic and dendritic surfaces of peripherin‐positive motoneurons in the Mo5, motoneurons throughout the spinal cord, and sexually dimorphic motoneurons at lower lumbar levels. In studies of electrical synapses and electrical transmission between developing and between adult motoneurons, our results serve to focus attention on mediation of this transmission by gap junctions composed of Cx36.     

Expression of connexin30.2 in interneurons of the central nervous system in the mouse

Electrical synapses, particularly gap junctions composed of connexin (Cx) 36, have been suggested to synchronize neuronal network oscillations. Recently, we generated Cx30.2-deficient mice which express beta-galactosidase under control of Cx30.2 gene regulatory elements. In the central nervous system beta-galactosidase activity representing Cx30.2 expression was restricted to NeuN-positive cells, thus identifying Cx30.2 as new neuronal connexin. In the hippocampus, co-immunofluorescence analyses revealed beta-galactosidase/Cx30.2 expression in GABAergic inhibitory interneurons such as parvalbumin- and somatostatin-positive basket, axo-axonic, bistratified or oriens lacunosum-moleculare cells. approximately 94% of the Cx30.2 expressing parvalbumin-positive interneurons also expressed Cx36. Performing field potential recordings from hippocampal slices we found no differences in basal excitation and excitation-inhibition balance between Cx30.2+/+ and Cx30.2LacZ/LacZ)mice. Furthermore, frequency and power of gap junction dependent gamma and ripples oscillations were similar in these animals. This suggests that the lack of Cx30.2 in interneurons can be largely compensated by other connexins, most likely Cx36.     

Connexin 36 Mediates Orofacial Pain Hypersensitivity Through GluK2 and TRPA1

Trigeminal neuralgia is a debilitating condition, and the pain easily spreads to other parts of the face. Here, we established a mouse model of partial transection of the infraorbital nerve (pT-ION) and found that the Connexin 36 (Cx36) inhibitor mefloquine caused greater alleviation of pT-ION-induced cold allodynia compared to the reduction of mechanical allodynia. Mefloquine reversed the pT-ION-induced upregulation of Cx36, glutamate receptor ionotropic kainate 2 (GluK2), transient receptor potential ankyrin 1 (TRPA1), and phosphorylated extracellular signal regulated kinase (p-ERK) in the trigeminal ganglion. Cold allodynia but not mechanical allodynia induced by pT-ION or by virus-mediated overexpression of Cx36 in the trigeminal ganglion was reversed by the GluK2 antagonist NS102, and knocking down Cx36 expression in Nav1.8-expressing nociceptors by injecting virus into the orofacial skin area of Nav1.8-Cre mice attenuated cold allodynia but not mechanical allodynia. In conclusion, we show that Cx36 contributes greatly to the development of orofacial pain hypersensitivity through GluK2, TRPA1, and p-ERK signaling.Electronic supplementary materialThe online version of this article (10.1007/s12264-020-00594-4) contains supplementary material, which is available to authorized users.     

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species‐specific expression,compensation in Cx36 null mice and co‐localization in neurons vs. glia

Electrical synapses formed by connexin36 (Cx36)‐containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter‐based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36‐puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild‐type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36‐puncta co‐localized with Cx45‐puncta, which in turn was associated with the scaffolding protein zonula occludens‐1. In rats, Cx45‐puncta were also co‐localized with Cx36‐puncta and additionally occurred along Bergmann glial processes adjacent to Cx43‐puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.     

Red nucleus glutamate facilitates neuropathic allodynia induced by spared nerve injury through non‐NMDA and metabotropic glutamate receptors

Previous studies have demonstrated that glutamate plays an important role in the development of pathological pain. This study investigates the expression changes of glutamate and the roles of different types of glutamate receptors in the red nucleus (RN) in the development of neuropathic allodynia induced by spared nerve injury (SNI). Immunohistochemistry indicated that glutamate was constitutively expressed in the RN of normal rats. After SNI, the expression levels of glutamate were significantly increased in the RN at 1 week and reached the highest level at 2 weeks postinjury compared with sham‐operated and normal rats. The RN glutamate was colocalized with neurons, oligodendrocytes, and astrocytes but not microglia under physiological and neuropathic pain conditions. To elucidate further the roles of the RN glutamate and different types of glutamate receptors in the development of neuropathic allodynia, antagonists to N‐methyl‐D‐aspartate (NMDA), non‐NMDA, or metabotropic glutamate receptors (mGluRs) were microinjected into the RN contralateral to the nerve‐injury side of rats with SNI, and the paw withdrawal threshold (PWT) was dynamically assessed with von Frey filaments. Microinjection of the NMDA receptor antagonist MK‐801 into the RN did not show any effect on SNI‐induced mechanical allodynia. However, microinjection of the non‐NMDA receptor antagonist 6,7‐dinitroquinoxaline‐2,3(1H,4H)‐dione or the mGluR antagonist (±)‐α‐methyl‐(4‐carboxyphenyl) glycine into the RN significantly increased the PWT and alleviated SNI‐induced mechanical allodynia. These findings suggest that RN glutamate is involved in regulating neuropathic pain and facilitates the development of SNI‐induced neuropathic allodynia. The algesic effect of glutamate is transmitted by the non‐NMDA glutamate receptor and mGluRs. © 2015 Wiley Periodicals, Inc.     

Connexin36 in gap junctions forming electrical synapses between motoneurons in sexually dimorphic motor nuclei in spinal cord of rat and mouse

Pools of motoneurons in the lumbar spinal cord innervate the sexually dimorphic perineal musculature, and are themselves sexually dimorphic, showing differences in number and size between male and female rodents. In two of these pools, the dorsomedial nucleus (DMN) and the dorsolateral nucleus (DLN), dimorphic motoneurons are intermixed with non‐dimorphic neurons innervating anal and external urethral sphincter muscles. As motoneurons in these nuclei are reportedly linked by gap junctions, we examined immunofluorescence labeling for the gap junction‐forming protein connexin36 (Cx36) in male and female mice and rats. Fluorescent Cx36‐labeled puncta occurred in distinctly greater amounts in the DMN and DLN of male rodents than in other spinal cord regions. These puncta were localized to motoneuron somata, proximal dendrites, and neuronal appositions, and were distributed either as isolated or large patches of puncta. In both rats and mice, Cx36‐labeled puncta were associated with nearly all (> 94%) DMN and DLN motoneurons. The density of Cx36‐labeled puncta increased dramatically from postnatal days 9 to 15, unlike the developmental decreases in these puncta observed in other central nervous system regions. In females, Cx36 labeling of puncta in the DLN was similar to that in males, but was sparse in the DMN. In enhanced green fluorescent protein (EGFP)–Cx36 transgenic mice, motoneurons in the DMN and DLN were intensely labeled for the EGFP reporter in males, but less so in females. The results indicate the presence of Cx36‐containing gap junctions in the sexually dimorphic DMN and DLN of both male and female rodents, suggesting coupling of not only sexually dimorphic but also non‐dimorphic motoneurons in these nuclei.     

Intraspinal amino acid neurotransmitter activities are involved in the generation of rhythmic sympathetic nerve discharge in newborn rat spinal cord

Endogenous neurotransmitter activities underlying the sympathetic nerve discharge (SND) generated by newborn rat spinal cord in vitro were investigated using glutamatergic, glycinergic, and GABAergic antagonists. Under control conditions, the SND power spectrum had two major frequency components: synchronous bursting SND (bSND) with power dominant at < 0.1 Hz and quasiperiodic SND (qSND) oscillating at 1-2 Hz. Using high Mg2+ solution (12-24 mM) to block Ca2+-dependent synaptic transmission reversibly abolished SND. An interruption of glutamatergic neurotransmission by CNQX (non-NMDA receptor blocker) or L-AP4 (reducing the synaptic release of glutamate) failed to affect qSND, but consistently reduced bSND. Application of kynurenate, a broad-spectrum ionotropic glutamate receptor blocker, only caused an unstable SND but did not reduce SND. In contrast, strychnine (Stry, glycine receptor antagonist) consistently reduced qSND in a dose-dependent manner. Bicuculline (Bic, GABA(A) receptor antagonist) induced a synchronous bSND of irregular rhythm, which could be further regularized by adding Stry. Bic-induced bSND was reversibly abolished by CNQX or L-AP4. In conclusion, intraspinal glycinergic, GABAergic, and glutamatergic activities are involved in the generation of the spinal cord-derived SND in newborn rats. Intraspinal GABAergic interneurons may tonically inhibit the glutamatergic bursting neurons that generate a synchronous bSND. Activities of these glutamatergic bursting neurons may also be modulated by intraspinal glycinergic interneurons.     

A brain slice model for in vitro analyses of astrocytic gap junction and connexin43 regulation: actions of ischemia, glutamate and elevated potassium

Brain slices prepared from adult rats and maintained for up to 3 h in vitro were used to investigate the effects of pharmacological treatments on the phosphorylation state, immunolabelling characteristics and ultrastructural localization of astrocytic gap junctions and connexin43 (Cx43). Slices deprived of glucose/oxygen to mimic ischemia or those exposed to 1 mM glutamate for 1 h exhibited Cx43 dephosphorylation, epitope masking and gap junction internalization as revealed by Western blotting and Cx43 immunolocalization with various antibodies. Treatment with 15 mM K+ caused Cx43 dephosphorylation without junction internalization. The effects of glutamate and K+ were completely blocked by the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 2-amino-5-phosphonovalerate (APV), which acts largely on neuronal NMDA receptors, suggesting neuronal mediation of glial gap junction responses to these treatments. Astrocytes contained a dephosphorylated form of Cx43 with a typical migration profile at 41 kDa as well as novel, apparently dephosphorylated or partially phosphorylated, forms migrating at 43 kDa. These results indicate that slices prepared from adult brain can serve as a convenient model to investigate the molecular basis and receptor-mediated mechanisms underlying astrocytic Cx43 responses that have been observed in vivo following cerebral ischemia or neural activation. These processes can be related in part to neuronal regulation of astrocytic gap junctional coupling state, which is also amenable to analysis in brain slices.     

Nociceptive response to innocuous mechanical stimulation is mediated via myelinated afferents and NK-1 receptor activation in a rat model of neuropathic pain

Peripheral nerve injury in humans can produce a persistent pain state characterized by spontaneous pain and painful responses to normally innocuous stimuli (allodynia). Here we attempt to identify some of the neurophysiological and neurochemical mechanisms underlying neuropathic pain using an animal model of peripheral neuropathy induced in male Sprague-Dawley rats by placing a 2-mm polyethylene cuff around the left sciatic nerve according to the method of Mosconi and Kruger. von Frey hair testing confirmed tactile allodynia in all cuff-implanted rats before electrophysiological testing. Rats were anesthetized and spinalized for extracellular recording from single spinal wide dynamic range neurons (L(3-4)). In neuropathic rats (days 11-14 and 42-52 after cuff implantation), ongoing discharge was greater and hind paw receptive field size was expanded compared to control rats. Activation of low-threshold sensory afferents by innocuous mechanical stimulation (0.2 N for 3 s) in the hind paw receptive field evoked the typical brief excitation in control rats. However, in neuropathic rats, innocuous stimulation also induced a nociceptive-like afterdischarge that persisted 2-3 min. This afterdischarge was never observed in control rats, and, in this model, is the distinguishing feature of the spinal neural correlate of tactile allodynia. Electrical stimulation of the sciatic nerve at 4 and at 20 Hz each produced an initial discharge that was identical in control and in neuropathic rats. This stimulation also produced an afterdischarge that was similar at the two frequencies in control rats. However, in neuropathic rats, the afterdischarge produced by 20-Hz stimulation was greater than that produced by 4-Hz stimulation. Given that acutely spinalized rats were studied, only peripheral and/or spinal mechanisms can account for the data obtained; as synaptic responses from C fibers begin to fail above approximately 5-Hz stimulation [Pain 46 (1991) 327], the afterdischarge in response to 20-Hz stimulation suggests a change mainly in myelinated afferents and a predominant role of these fibers in eliciting this afterdischarge. These data are consistent with the suggestion that peripheral neuropathy induces phenotypic changes predominantly in myelinated afferents, the sensory neurons that normally respond to mechanical stimulation. The NK-1 receptor antagonist, CP-99,994 (0.5 mg/kg, i.v.), depressed the innocuous pressure-evoked afterdischarge but not the brief initial discharge of wide dynamic range neurons, and decreased the elevated ongoing rate of discharge in neuropathic rats. These results support the concept that following peripheral neuropathy, myelinated afferents may now synthesize and release substance P. A result of this is that tonic release of substance P from the central terminals of these phenotypically altered neurons would lead to ongoing excitation of NK-1-expressing nociceptive spinal neurons. In addition, these spinal neurons would also exhibit exaggerated responses to innocuous pressure stimulation. The data in this study put forth a possible neurophysiological and neurochemical basis of neuropathic pain and identify substance P and the NK-1 receptor as potential neurochemical targets for its management.     

Effects of intraplantar botulinum toxin‐B on carrageenan‐induced changes in nociception and spinal phosphorylation of GluA1 and Akt

Increasing evidence suggests that botulinum neurotoxins (BoNTs) delivered into the skin and muscle in certain human and animal pain states may exert antinociceptive efficacy though their uptake and transport to central afferent terminals. Cleavage of soluble N‐methylaleimide‐sensitive attachment protein receptor by BoNTs can impede vesicular mediated neurotransmitter release as well as transport/insertion of channel/receptor subunits into plasma membranes, an effect that can reduce activity‐evoked facilitation. Here, we explored the effects of intraplantar botulinum toxin‐ B (BoNT‐B) on peripheral inflammation and spinal nociceptive processing in an inflammatory model of pain. C57BL/6 mice (male) received unilateral intraplantar BoNT (1 U, 30 μL) or saline prior to intraplantar carrageenan (20 μL, 2%) or intrathecal N‐methyl‐D‐aspartate (NMDA), substance P or saline (5 μL). Intraplantar carrageenan resulted in edema and mechanical allodynia in the injected paw and increased phosphorylation of a glutamate subunit (pGluA1ser845) and a serine/threonine‐specific protein kinase (pAktser473) in spinal dorsal horn along with an increased incidence of spinal c‐Fos positive cells. Pre‐treatment with intraplantar BoNT‐B reduced carrageenan evoked: (i) allodynia, but not edema; (ii) pGluA1 and pAkt and (iii) c‐Fos expression. Further, intrathecal NMDA and substance P each increased dorsal horn levels of pGluA1 and pAkt. Intraplantar BoNT‐B inhibited NMDA, but not substance P evoked phosphorylation of GluA1 and Akt. These results suggest that intraplantar toxin is transported centrally to block spinal activation and prevent phosphorylation of a glutamate receptor subunit and a kinase, which otherwise contribute to facilitated states.     

Medullary N-type and P/Q-type calcium channels contribute to neuropathy-induced allodynia

The present study was designed to determine the contribution of N-type, P/Q-type and L-type calcium channels in the rostral ventromedial medulla to tactile allodynia following peripheral nerve injury. L5/L6 spinal nerve ligation in rats produced tactile allodynia, which was dose-dependently inhibited by intrarostral ventromedial medulla microinjection of the N-type calcium channel antagonist omega-conotoxin MVIIA. Similarly, intrarostral ventromedial medulla microinjection of the P/Q-type calcium channel antagonist omega-agatoxin IVA inhibited spinal nerve ligation-induced tactile allodynia, whereas intrarostral ventromedial medulla microinjection of the L-type calcium channel antagonist nimodipine had no effect. These results demonstrate that N-type and P/Q-type calcium channels in the rostral ventromedial medulla contribute to tactile allodynia following peripheral neuropathy, likely via neurotransmitter-mediated activation of descending facilitatory systems from the rostral ventromedial medulla.