Ipsilateral Superficial Dorsal Horn Research Articles

Dependence of c-fos Expression on Amplitude of High-Frequency Spinal Cord Stimulation in a Rodent Model

Clinical high-frequency spinal cord stimulation (hfSCS) (>250 Hz) applied at subperception amplitudes reduces leg and low back pain. This study investigates, via labeling for c-fos-a marker of neural activation, whether 500 Hz hfSCS applied at amplitudes above and below the dorsal column (DC) compound action potential (CAP) threshold excites dorsal horn neurons. DC CAP thresholds in rats were determined by applying single biphasic pulses of SCS to T12 -T13 segments using pulse widths of 40 or 200 μsec via a ball electrode placed over the left DC and increasing amplitude until a short latency CAP was observed on the L5 DC and sciatic nerve. The result of this comparison allowed us to substitute sciatic nerve CAP for DC CAP. SCS at T12 -T13 was applied continuously for two hours using: sham or hfSCS at 500 Hz SCS, 40 μsec pulse width, and 50, 70, 90, or 140% CAP threshold. Spinal cord slices from T11 -L1 were immunolabeled for c-fos, and the number of c-fos-positive cells was quantified. 500 Hz hfSCS applied at 90 and 140% CAP threshold produced substantial (≥6 c-fos + neurons on average per slice per segment) c-fos expression in more segments between T11 and L1 than did sham stimulation (p < 0.025, 90% CAP; p < 0.001, 140% CAP, Fisher's Exact Tests) and resulted in more c-fos-positive neurons on average per slice per segment ipsilateral to than contralateral to the SCS electrode at 70, 90, and 140% CAP threshold (p < 0.01, Wilcoxon Signed Rank Tests). The finding of enhanced c-fos expression in the ipsilateral superficial dorsal horn provides evidence for activation/modulation of neuronal circuitry associated with subperception hfSCS.

Lumbar Spinal Cord Astrocytic Hyperactivation is Associated with Thrombospondin‐4 Upregulation during Persistent Low Back Pain

Low back pain (LBP) is a leading cause of global disability. Due to a lack of understanding of its underlying mechanisms, this condition is poorly managed and necessitates novel approaches to improve treatment options. Here we used a preclinical rat LBP model induced by nerve growth factor (NGF) injections into the lumbar paraspinal muscles to study the neurobiological changes in the lumbar spinal cord that contribute to LBP. NGF injections induce behavioral changes, including hypersensitivity to a noxious mechanical stimulus (i.e., mechanical hyperalgesia) in the low back (LBP). NGF‐induced LBP was associated with spinal cord dorsal horn pain neuron hyperexcitability, a pro‐nociceptive process that contributes to central sensitization, a state implicated in persistent pain. We previously reported that the maintenance of NGF‐induced LBP was associated with astrocytic hyperactivation (i.e., increased glial fibrillary acidic protein, GFAP, with somatic &amp; process hypertrophy) in the superficial dorsal horn (SDH), a location for pain processing and modulation. Our findings suggested a role for neuroimmune activation, a glial‐cell dependent process that promotes inflammation and central sensitization. Although glial hyperactivation was previously implicated in LBP patients, the mechanisms by which spinal astrocytes contribute to LBP are unclear. In this study we examined the potential role of the astrocyte‐dependent glycoprotein, thrombospondin‐4 (TSP‐4). TSP‐4 is predominantly expressed and released by spinal cord astrocytes and promotes inflammation and synaptic plasticity. Although unreported in LBP, spinal astrocytic TSP‐4 upregulation was implicated in neuropathic pain, in part, by promoting excitatory synaptogenesis. We hypothesized that persistent LBP and spinal astrocytic hyperactivation at day 14 (D14) induced by NGF injections would be associated with spinal cord dorsal horn TSP‐4 upregulation. To test our hypothesis, male rats received two injections, 5 days apart, of NGF (0.8μM/50μl) or vehicle into the left paraspinal muscles (5th lumbar vertebral level). At D14, lumbar spinal cord dorsal horns (L1–6) were harvested and evaluated for TSP‐4 mRNA (DNA microarray; n=3/group) and the location and cellular expression of TSP‐4 protein (n=4/group) via immunofluorescence with confocal microscopy. Compared to vehicle, NGF injected rats had significant ipsilateral low back mechanical hyperalgesia that persisted through D14. NGF rats also had upregulation of ipsilateral dorsal horn TSP‐4 mRNA expression (1.5‐fold increase) compared to controls. TSP‐4 immunofluorescence was significantly increased (2 fold) in the ipsilateral SDH in NGF rats. High magnification confocal z‐stacks demonstrated TSP‐4 immunolabeling was located in GFAP positive astrocytes. These findings demonstrate that spinal astrocytic TSP‐4 upregulation occurs during the maintenance of persistent LBP. Further work is required to determine the mechanisms that promote spinal TSP‐4 upregulation and its role in LBP. Our findings could help to reveal the central mechanisms contributing to LBP and to develop novel LBP treatments.Support or Funding InformationSLU Center for Neuroscience SCFN‐SGP‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone Acetylation in Microglia Contributes to Exercise-Induced Hypoalgesia in Neuropathic Pain Model Mice.

Physical exercise can attenuate neuropathic pain (NPP), but the exact mechanism underlying exercise-induced hypoalgesia (EIH) remains unclear. Recent studies have shown that histone hyperacetylation via pharmacological inhibition of histone deacetylases in the spinal cord attenuates NPP, and that histone acetylation may lead to the production of analgesic factors including interleukin 10. We intended to clarify whether histone acetylation in microglia in the spinal dorsal horn contributes to EIH in NPP model mice. C57BL/6J mice underwent partial sciatic nerve ligation (PSL) and PSL- and sham-runner mice ran on a treadmill at a speed of 7m/min for 60min/d, 5days per week, from 2days after the surgery. PSL-sedentary mice developed mechanical allodynia and heat hyperalgesia, but such behaviors were significantly attenuated in PSL-runner mice. In immunofluorescence analysis, PSL surgery markedly increased the number of histone deacetylase 1-positive/CD11b-positive microglia in the ipsilateral superficial dorsal horn, and they were significantly decreased by treadmill-running. Moreover, the number of microglia with nuclear expression of acetylated H3K9 in the ipsilateral superficial dorsal horn was maintained at low levels in PSL-sedentary mice, but running exercise significantly increased them. Therefore, we conclude that the epigenetic modification that causes hyperacetylation of H3K9 in activated microglia may play a role in producing EIH. This article presents the importance of epigenetic modification in microglia in producing EIH. The current research is not only helpful for developing novel nonpharmacological therapy for NPP, but will also enhance our understanding of the mechanisms and availability of exercise in our daily life.

Axotomy of tributaries of the pelvic and pudendal nerves induces changes in the neurochemistry of mouse dorsal root ganglion neurons and the spinal cord

Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4-S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6-S1 axotomy induced dramatic de novo expression of ATF3 in many L6-S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6-S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6-S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6-S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity.

Persistent at-level thermal hyperalgesia and tactile allodynia accompany chronic neuronal and astrocyte activation in superficial dorsal horn following mouse cervical contusion spinal cord injury.

In humans, sensory abnormalities, including neuropathic pain, often result from traumatic spinal cord injury (SCI). SCI can induce cellular changes in the CNS, termed central sensitization, that alter excitability of spinal cord neurons, including those in the dorsal horn involved in pain transmission. Persistently elevated levels of neuronal activity, glial activation, and glutamatergic transmission are thought to contribute to the hyperexcitability of these dorsal horn neurons, which can lead to maladaptive circuitry, aberrant pain processing and, ultimately, chronic neuropathic pain. Here we present a mouse model of SCI-induced neuropathic pain that exhibits a persistent pain phenotype accompanied by chronic neuronal hyperexcitability and glial activation in the spinal cord dorsal horn. We generated a unilateral cervical contusion injury at the C5 or C6 level of the adult mouse spinal cord. Following injury, an increase in the number of neurons expressing ΔFosB (a marker of chronic neuronal activation), persistent astrocyte activation and proliferation (as measured by GFAP and Ki67 expression), and a decrease in the expression of the astrocyte glutamate transporter GLT1 are observed in the ipsilateral superficial dorsal horn of cervical spinal cord. These changes have previously been associated with neuronal hyperexcitability and may contribute to altered pain transmission and chronic neuropathic pain. In our model, they are accompanied by robust at-level hyperaglesia in the ipsilateral forepaw and allodynia in both forepaws that are evident within two weeks following injury and persist for at least six weeks. Furthermore, the pain phenotype occurs in the absence of alterations in forelimb grip strength, suggesting that it represents sensory and not motor abnormalities. Given the importance of transgenic mouse technology, this clinically-relevant model provides a resource that can be used to study the molecular mechanisms contributing to neuropathic pain following SCI and to identify potential therapeutic targets for the treatment of chronic pathological pain.

P.1.c.002 Biological peculiarities of the analgesic drug – nefopam in rats

Previous findings indicate that the brain stem descending system becomes more active in modulating spinal nociceptive processes during the development of persistent pain. The present study further identified the supraspinal sites that mediate enhanced descending modulation of behavior hyperalgesia and dorsal horn hyperexcitability (as measured by Fos-like immunoreactivity) produced by subcutaneous complete Freund's adjuvant (CFA). Selective chemical lesions were produced in the nucleus raphe magnus (NRM), the nuclei reticularis gigantocellularis (NGC), or the locus coeruleus/subcoeruleus (LC/SC). Compared to vehicle-injected animals with injection of vehicle alone, microinjection of a serotoninergic neurotoxin 5,7-dihydroxytryptamine into the NRM significantly increased thermal hyperalgesia and Fos protein expression in lumbar spinal cord after hindpaw inflammation. In contrast, the selective bilateral destruction of the NGC with a soma-selective excitotoxic neurotoxin, ibotenic acid, led to an attenuation of hyperalgesia and a reduction of inflammation-induced spinal Fos expression. Furthermore, if the NGC lesion was extended to involve the NRM, the behavioral hyperalgesia and CFA-induced Fos expression were similar to that in vehicle-injected rats. Bilateral LC/SC lesions were produced by microinjections of a noradrenergic neurotoxin, DSP-4. There was a significant increase in inflammation-induced spinal Fos expression, especially in the ipsilateral superficial dorsal horn following LC/SC lesions. These results demonstrated that multiple specific brain stem sites are involved in descending modulation of inflammatory hyperalgesia. Both NRM and LC/SC descending pathways are major sources of enhanced inhibitory modulation in inflamed animals. The persistent hyperalgesia and neuronal hyperexcitability may be mediated in part by a descending pain facilitatory system involving NGC. Thus, the intensity of perceived pain and hyperalgesia is fine-tuned by descending pathways. The imbalance of these modulating systems may be one mechanism underlying variability in acute and chronic pain conditions.

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the α7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

Effect of genetic knockout or pharmacologic inhibition of neuronal nitric oxide synthase on complete Freund's adjuvant-induced persistent pain

Nitric oxide (NO) acts as a neurotransmitter or neuromodulator involving in the modulation of thermal and/or inflammatory hyperalgesia. The neuronal nitric oxide synthase (nNOS) is a key enzyme for NO production in normal neuronal tissues, but its functional role in chronic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to address the role of nNOS in the central mechanism of complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. Targeted disruption of the nNOS gene significantly reduced CFA-induced mechanical pain hypersensitivity during the maintenance (but not the development) of inflammatory pain, while it failed to attenuate either development or maintenance of CFA-induced thermal pain hypersensitivity. Intraperitoneal administration of L-N G-nitro-arginine methyl ester (L-NAME), a non-specific NOS inhibitor, blocked CFA-evoked thermal and mechanical pain hypersensitivity at both development (2 h) and maintenance (24 h) phase in wild type mice, but had no effect in the knockout mice. Furthermore, intrathecal injection of either L-NAME or 7-nitroindazole, a selective nNOS inhibitor, markedly attenuated mechanical pain hypersensitivity at both 2 and 24 h after CFA injection. Finally, spinal cord nNOS (but not endothelial NOS or inducible NOS) expression was up-regulated at 24 h after CFA injection, occurring mainly in the ipsilateral superficial dorsal horn. Together, these data indicate that spinal cord nNOS may be essential for the maintenance of mechanical pain hypersensitivity and that it may also be sufficient for the development of mechanical pain hypersensitivity and for the development and maintenance of thermal pain hypersensitivity after chronic inflammation. Our findings suggest that spinal cord nNOS might play a critical role in central mechanisms of the development and/or maintenance of chronic inflammatory pain.

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP 8-37 markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Origins of GABA B receptor-like immunoreactive terminals in the rat spinal dorsal horn

By means of immunohistochemistry for γ-aminobutyric acid receptor B subtype (GABA BR), the origins of GABA BR-like immunoreactive (GABA BR-LI) terminals in the rat spinal dorsal horn were investigated. After dorsal root rhizotomy and/or spinal cord hemisection, the densities of GABA BR-LI terminals were remarkably depleted in the ipsilateral superficial dorsal horn of relevant segments, whereas GABA BR-LI neurons and sparsely distributed GABA BR-LI terminals remained. After injection of Fluoro-Gold (FG) into the left side of superficial lumbar dorsal horn, FG retrograde-labeled neurons were mainly observed in the ipsilateral rostral ventromedial medulla (RVM) and brainstem raphe nuclei. Some of the FG-labeled neurons, especially in the RVM, exhibited GABA BR-like immunoreactivity. Additionally, immunofluorescence histochemical double-staining revealed that the majority of GABA BR-LI neurons in the periaqueductal gray (PAG), RVM and brainstem raphe nuclei showed 5-hydroxytryptamine (5-HT)-like immunoreactivity. The present study morphologically proves that GABA BR-LI terminals in the spinal dorsal horn originate from peripheral afferents, intrinsic neurons and supraspinal structures; GABA BR and 5-HT co-exist in many neurons in the PAG, RVM and brainstem raphe nuclei. Considering that PAG, RVM, brainstem raphe nuclei and spinal dorsal horn are important structures involved in the pain modulation, we suggest that the descending pain modulation system might be mediated, at least in part, by GABA BR.

C-fos induction in rat superficial dorsal horn following cutaneous application of noxious chemical or mechanical stimuli

The method of c-fos immunodetection was used to map the distributions of neurons in the lumbar spinal dorsal horn that were activated following intracutaneous (i.c.) microinjection, or iontophoretic application, of different irritant chemicals to the lateral hindpaw of rats. Microinjections (1 microl) of histamine, serotonin (5-HT), nicotine, capsaicin, or formalin each elicited similar distributions of Fos-like immunoreactivity (FLI) in laminae I-II of the ipsilateral superficial dorsal horn, with little or no FLI in deeper laminae or contralaterally. In laminae I and II, FLI cell counts were significantly higher following i.c. histamine, 5-HT, capsaicin, formalin, and noxious pinch, compared to i.c. saline controls. Capsaicin-evoked FLI was dose-dependent. Multivariate analysis of variance revealed no significant difference in spatial distributions of FLI induced by any of the chemical or pinch stimuli. Iontophoretic application of histamine, 5-HT, or nicotine also elicited similar distributions of FLI in the superficial dorsal horn, and cell counts of FLI were significantly higher compared to controls receiving iontophoretic vehicle (methyl cellulose). These results indicate either that individual laminae I-II neurons are activated by each of the irritant chemicals, or that neurons selectively responsive to a given irritant are comingled without any apparent laminar segregation.

Laminar-selective noradrenergic and serotoninergic modulation includes spinoparabrachial cells after inflammation.

We evaluated the effects of chemical lesions on hindpaw inflammation-induced Fos protein expression in spinoparabrachial neurons that were retrogradely labeled by Fluoro-Gold. The descending serotoninergic and noradrenergic pathways were destroyed by the selective neurotoxins, 5,7-DHT and DSP-4, respectively. After 5,7-DHT treatment there was a significant increase in double-labeled neurons only in the lateral reticulated neck of the dorsal horn 24h after inflammation compared with vehicle-injected controls. In contrast, the DSP-4 treatment resulted in a more robust increase in double-labeled neurons in the ipsilateral superficial dorsal horn than in the neck of the dorsal horn. These results indicate that after inflammation the enhanced modulation from descending serotoninergic and noradrenergic pathways targets supraspinally projecting neurons to dampen increased ascending nociceptive input. Further, these pathways differentially suppress the responses of spinoparabrachial neurons in the deep and superficial dorsal horn.

Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation

Previous findings indicate that the brain stem descending system becomes more active in modulating spinal nociceptive processes during the development of persistent pain. The present study further identified the supraspinal sites that mediate enhanced descending modulation of behavior hyperalgesia and dorsal horn hyperexcitability (as measured by Fos-like immunoreactivity) produced by subcutaneous complete Freund's adjuvant (CFA). Selective chemical lesions were produced in the nucleus raphe magnus (NRM), the nuclei reticularis gigantocellularis (NGC), or the locus coeruleus/subcoeruleus (LC/SC). Compared to vehicle-injected animals with injection of vehicle alone, microinjection of a serotoninergic neurotoxin 5,7-dihydroxytryptamine into the NRM significantly increased thermal hyperalgesia and Fos protein expression in lumbar spinal cord after hindpaw inflammation. In contrast, the selective bilateral destruction of the NGC with a soma-selective excitotoxic neurotoxin, ibotenic acid, led to an attenuation of hyperalgesia and a reduction of inflammation-induced spinal Fos expression. Furthermore, if the NGC lesion was extended to involve the NRM, the behavioral hyperalgesia and CFA-induced Fos expression were similar to that in vehicle-injected rats. Bilateral LC/SC lesions were produced by microinjections of a noradrenergic neurotoxin, DSP-4. There was a significant increase in inflammation-induced spinal Fos expression, especially in the ipsilateral superficial dorsal horn following LC/SC lesions. These results demonstrated that multiple specific brain stem sites are involved in descending modulation of inflammatory hyperalgesia. Both NRM and LC/SC descending pathways are major sources of enhanced inhibitory modulation in inflamed animals. The persistent hyperalgesia and neuronal hyperexcitability may be mediated in part by a descending pain facilitatory system involving NGC. Thus, the intensity of perceived pain and hyperalgesia is fine-tuned by descending pathways. The imbalance of these modulating systems may be one mechanism underlying variability in acute and chronic pain conditions.

Inflammation-induced up-regulation of protein kinase C γ immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing

Activation of various second messengers contributes to long-term changes in the excitability of dorsal horn neurons and to persistent pain conditions produced by injury. Here, we compared the time-course of decreased mechanical nociceptive thresholds and the density of protein kinase C γ immunoreactivity in the dorsal horn after injections of complete Freund's adjuvant in the plantar surface of the rat hindpaw. Complete Freund's adjuvant significantly increased paw diameter and mechanical sensitivity ipsilateral to the inflammation. The changes peaked one day post-injury, but endured at least two weeks. In these rats, we recorded a 75–100% increase in protein kinase C γ immunoreactivity in the ipsilateral superficial dorsal horn of the L4 and L5 segments at all time-points. Electron microscopy revealed that the up-regulation was associated with a significant translocation of protein kinase C γ immunoreactivity to the plasma membrane. In double-label cytochemical studies, we found that about 20% of the protein kinase C γ-immunoreactive neurons, which are concentrated in inner lamina II, contain glutamate decarboxylase-67 messenger RNA, but none stain for parvalbumin or nitric oxide synthase. These results indicate that persistent changes in protein kinase C γ immunoreactivity parallel the time-course of mechanical allodynia and suggest that protein kinase C γ contributes to the maintenance of the allodynia produced by peripheral inflammation. The minimal expression of protein kinase C γ in presumed inhibitory neurons suggests that protein kinase C γ-mediated regulation of excitatory interneurons underlies the changes in spinal cord activity during persistent nociception.

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons.

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.

Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve.

Following the transection of one pelvic nerve and both hypogastric nerves, the urinary bladder of male Sprague-Dawley rats was injected with pseudorabies virus (PRV; Bartha strain). The central stump of the transected pelvic nerve was labelled with fast blue (FB), and rats were maintained for 2, 2.5, and 3 days following viral infection. Tissue was processed with antisera against PRV and choline acetyltransferase (CAT). In the L6-S1 spinal cord, neurons in the ipsilateral intermediolateral area (IML) were labelled after 2 days. After 2.5 days, labelled neurons were also found in the dorsal gray commissure (DGC), the ipsilateral superficial dorsal horn, and the contralateral IML area. After 3 days, many labelled neurons appeared in the superficial dorsal horns and, bilaterally, in the L6-S1 dorsal root ganglia. In both IMLs, two groups of PRV-labelled neurons were found: 1) CAT-positive preganglionic cells and 2) smaller, CAT-negative cells located slightly dorsal to the preganglionic neurons. No other doubly stained neurons were found in the spinal cord. Contralateral DRG neurons stained for either PRV or FB or both. Ipsilateral DRG neurons stained only for PRV. PRV-immunoreactive (IR) neurons appeared in the brainstem only after 3 days. These were located primarily in the pontine micturition centers (equal numbers), the ventral locus coeruleus, and the raphe and lateral reticular areas.

The transcription factor CREB is not phosphorylated at serine 133 in axotomized neurons: implications for the expression of AP-1 proteins

The present study has investigated whether nerve fiber transection alters the phosphorylation of serine at position 133 (Ser 133) of the transcription factor CREB (phosphoCREB). Activation of CREB by phosphorylation has a major function in the control of gene transcription. PhosphoCREB was visualized by antisera that specificly react with an epitope comprising the phosphorylated Ser 133 of CREB as well as of CREM and ATF1 proteins. In untreated rats, nuclear immunoreactivity (IR) of phosphoCREB was consistently visible, e.g. in the cortex, thalamic and hypothalamic compartments and central termination areas of primary somatosensory afferents. Transection of peripheral (sciatic nerve), cranial (hypoglossal and facial nerve) and central (medial forebrain bundle and mammillo-thalamic tract) nerve fibers did not increase phosphoCREB-IR in the axotomized neurons between 5 min and 30 days post-axotomy. In contrast, phosphoCREB-IR appeared after 24 h in glial cells adjacent to the axotomized motoneurons and persisted up to 4 weeks. This increase in glial phosphoCREB-IR was paralleled by enhanced expression of the CREB protein itself. Between 20 min and 24 h following sciatic nerve transection, the number of phosphoCREB labeled nuclei also increased in neurons of the ipsilateral superficial dorsal horn of lumbar L3–L5 spinal cord segments. These data suggest that phosphorylation of Ser 133 in CREB/CREM/ATF1 proteins is not involved in the transcriptional control of early-response genes such as c- jun in axotomized neurons, following nerve transection. This is in contrast to the reported phosphorylation of CREB and its trans-acting effects on immediate-early genes such as c- fos after transynaptic neuronal excitation.