Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation

alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.     

Rat dorsal root ganglia express distinctive forms of the alpha2 calcium channel subunit

Calcium channel alpha2 delta subunit is a glycosylated structural subunit consistent of the alpha2 subunit and the delta peptide. Previous studies have indicated distinctive alpha2 subunit expression in rat spinal cord and dorsal root ganglia (DRG). This study examined whether differential glycosylation underlies the molecular basis of distinct alpha2 delta subunits. The migration patterns of deglycosylated alpha2 subunits from rat spinal cord, DRG, brain and skeletal muscle were compared in Western blots. The data reported indicate that there are two forms of the alpha2 subunit in DRG that are different from the alpha2 subunit in other tissues examined, at least at the glycosylation level. Thus, post-translational modification may be important in tissue specific and functional expression of the alpha2 delta subunit.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

Genetic abnormalities underlying familial epilepsy syndromes

Genetic defects have been recently identified in certain inherited epilepsy syndromes in which the phenotypes are similar to common idiopathic epilepsies. Mutations in the neuronal nicotinic acetylcholine receptor 4 and 2 subunit genes have been detected in families with autosomal dominant nocturnal frontal lobe epilepsy. Both receptors are components of neuronal acetylcholine receptor, a ligand-gated ion channel in the brain. Furthermore, mutations of two K+-channel genes were also identified as the underlying genetic abnormalities of benign familial neonatal convulsions. Mutations in the voltage-gated Na+-channel 1, 2 and 1 and the gamma aminobutyric acid (GABAA) receptor 2 subunit genes were found as a cause of generalized epilepsy with febrile seizures plus, a clinical subset of febrile convulsions. Na+-channels, GABAA receptor and their auxiliaries may be involved in the pathogenesis of this subtype and even in simple febrile convulsions. Mutation of a voltage-gated K+-channel gene can cause partial seizures associated with periodic ataxia type 1 and some forms of juvenile myoclonic epilepsy and idiopathic generalized epilepsy can result from mutations of a Ca2+-channel. This line of evidence suggests the involvement of channels expressed in the brain in the pathogenesis of certain types of epilepsy. Our working hypothesis is to view certain idiopathic epilepsies as disorders of ion channels, i.e. 'channelopathies'. Such hypothesis should provide a new insight to our understanding of the genetic background of epilepsy.     

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.     

Distribution and regulation of L-type calcium channels in deep dorsal horn neurons after sciatic nerve injury in rats

Deep dorsal horn neurons are involved in the processing of nociceptive information in the spinal cord. Previous studies revealed a role of the intrinsic bioelectrical properties (plateau potentials) of deep dorsal horn neuron in neuronal hyperexcitability, indicating their function in pain sensitization. These properties were considered to rely on L-type calcium currents. Two different isotypes of L-type calcium channel alpha 1 subunit have been cloned (Ca(V)1.2 and Ca(V)1.3). Both are known to be expressed in the spinal cord. However, no data were available on their subcellular localization. Moreover, possible changes in Ca(V)1.2 and Ca(V)1.3 expression had never been investigated in nerve injury models. Our study provides evidence for a differential expression of Ca(V)1.2 and Ca(V)1.3 subunits in the somato-dendritic compartment of deep dorsal horn neurons. Ca(V)1.2 immunoreactivity is restricted to the soma and proximal dendrites whereas Ca(V)1.3 immunoreactivity is found in the whole somato-dendritic compartment, up to distal dendritic segments. Moreover, these specific immunoreactive patterns are also found in electrophysiologically identified deep dorsal horn neurons expressing plateau potentials. After nerve injury, namely total axotomy or partial nerve ligation, Ca(V)1.2 and Ca(V)1.3 expression undergo differential changes, showing up- and down-regulation, respectively, both at the protein and at the mRNA levels. Taken together, our data support the role of L-type calcium channels in the control of intrinsic biolectrical regenerative properties. Furthermore, Ca(V)1.2 and Ca(V)1.3 subunits may have distinct and specific roles in sensory processing in the dorsal horn of the spinal cord, the former being most likely involved in long-term changes after nerve injury.     

Immunocytochemical localization of TNF type 1 and type 2 receptors in the rat spinal cord

Tumor necrosis factor-alpha (TNF-alpha) is secreted in numerous pathophysiological situations by a variety of cell types. Tactile hypersensitivity (allodynia) is one component of a constellation of "illness behaviors" triggered by TNF-alpha. TNF-alpha is also implicated in neuropathic pain after peripheral nerve injury and apoptosis after spinal cord injury (SCI). It is possible that SCI, illness- and peripheral injury-induced hypersensitivity may share a similar spinal mediated etiology. These studies identify the locus of type-1 TNF (TNFR1 or p55) and type-2 TNF (TNFR2 or p75) receptors within the spinal cord. At all spinal levels, TNFR1 receptor immunoreactivity (TNFR1-ir) was constitutively expressed on cells and afferent fibers within the dorsal root ganglia, afferent fibers of the dorsal root, dorsal root entry zone (REZ) and within lamina I and II of the dorsal horn. Unilateral dorsal rhizotomy eliminated the characteristic pattern of TNFR1-ir at the rhizotomized REZ. In contrast, TNFR2-ir was consistently absent from dorsal root fibers and the region of the root entry zone. Consistent with our previous report, medullary afferent fibers in the solitary tract and spinal trigeminal tract labelled for TNF1-ir, but did not express TNFR2-ir. The presence TNFR1-ir on dorsal horn afferents, suggests that TNF-alpha may be a mechanism responsible for tactile hypersensitivity during illness. The presence of TNFR1 receptors, and perhaps their long-term activation or plasticity, may also play a critical role in the chronic allodynia and hyperreflexia observed after SCI or peripheral nerve damage.     

Peripheral nerve injury induces reorganization of galanin-containing afferents in the superficial dorsal horn of monkey spinal cord

Peripheral nerve injury-induced structural and chemical modifications of the sensory circuits in the dorsal horn of the spinal cord contribute to the mechanism of neuropathic pain. In contrast to the topographic projection of primary afferents in laminae I-IV in the rat spinal cord, the primary afferents of Macaca mulatta monkeys almost exclusively project into laminae I-II of the spinal cord. After peripheral nerve injury, up-regulation of galanin has been found in sensory neurons in both monkey and rat dorsal root ganglia. However, the nerve injury-induced ultrastructural modification of galanin-containing afferents in the monkey spinal cord remains unknown. Using immunoelectron microscopy, we found that 3 weeks after unilateral sciatic nerve transection, the number of galanin-containing afferents was increased in ipsilateral lamina II of monkey spinal cord. Branching of these galanin-positive afferents was often observed. The afferent terminals contained a large number of synaptic vesicles, peptidergic vesicles and mitochondria, whereas the number of synapses was markedly reduced. Some of the afferents-enriched microtubules were often packed into bundles. Moreover, galanin-labeling could be associated with endosomal structures in many dendrites and axonal terminals of dorsal horn neurons. These results suggest that peripheral nerve injury induces an expansion of the central projection of galanin-containing afferents in lamina II of the monkey spinal cord, not only by increasing galanin levels in primary afferents but also by triggering afferent branching.     

Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.     

Differential expression of gamma-aminobutyric acid--a receptor subunits in rat dorsal and ventral hippocampus

Recent data demonstrate weaker gamma-aminobutyric acid (GABA)-ergic inhibition in ventral (VH) compared with dorsal (DH) hippocampus. Therefore, we examined possible differences regarding the GABAA receptors between VH and DH as follows: 1) the expression of the GABAA receptor subunits (alpha1/2/4/5, beta1/2/3, gamma2, delta) mRNA and protein and 2) the quantitative distribution and kinetic parameters of [3H] muscimol (GABAA receptor agonist) binding. VH compared with DH showed: 1) lower levels for alpha1, beta2, gamma2 but higher levels for alpha2 and beta1 subunits in CA1, CA2, and CA3, the differences being more pronounced in CA1 region; in the CA1 region, the mRNA levels of alpha5 were higher, whereas those of alpha4 subunit were slightly lower; in dentate gyrus, the mRNA levels of alpha4, beta3, and delta subunits were significantly lower, presumably suggesting a lower expression of the alpha4/beta3/delta receptor subtype; and 2) lower levels of [3H]muscimol binding, with the lowest value observed in CA1, apparently resulting from weaker binding affinity, insofar as the KD values were higher in VH, whereas the Bmax values were similar between DH and VH. The differences in the subunit expression and the lower affinity of GABAA receptor binding observed predominantly in the CA1 region of VH suggest that the alpha1/beta2/gamma2 GABAA receptor subtype dominates in DH, and the alpha2/beta1/gamma2 subtype prevails in VH. This could underlie the lower GABAA-mediated inhibition observed in VH and, to some extent, explain 1) the higher liability of VH for epileptic activity and 2) the differential involvement of DH and VH in cognitive and emotional processes.     

Prolonged zymosan-induced inflammatory pain hypersensitivity in mice lacking glycine receptor alpha2

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons under normal conditions and during persistent pain. However, the role of different glycine receptor (GlyR) subtypes in pain processing has only begun to be unraveled. Here, we analysed whether the GlyR alpha2 subunit might be involved in the processing of acute or persistent pain. Real-time RT-PCR and in situ hybridization analyses revealed that GlyR alpha2 mRNA is enriched in the dorsal horn of the mouse spinal cord. Mice lacking GlyR alpha2 (Glra2^−/− mice) demonstrated a normal nociceptive behavior in models of acute pain and after peripheral nerve injury. However, mechanical hyperalgesia induced by peripheral injection of zymosan was significantly prolonged in Glra2^−/− mice as compared to wild-type littermates. We conclude that spinal GlyRs containing the alpha2 subunit exert a previously unrecognized role in the resolution of inflammatory pain.     

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.     

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.     

Distinct spatiotemporal distributions of the N-Methyl-D-aspartate receptor channel subunit mRNAs in the mouse cervical cord

The distribution of five N-methyl-D -aspartate (NMDA) receptor channel subunit mRNAs in the mouse spinal cord from embryonic day 13 (E13) through postnatal day 56 (P56) was semiquantitatively examined at the cervical level via in situ hybridization with subunit-specific oligonucleotide probes. Signals for the ξ1 subunit mRNA were restricted to the most ventral portion of the spinal cord during embryonic stages. They extended to all laminae of the spinal cord except for the lamina 2 (substantia gelatinosa) during postnatal development. A wide expression of the ?2 subunit mRNA was found in the spinal gray matter from E13 through neonatal stages, but the signals became restricted to the lamina 2 by P21. No significant signals for the ?3 subunit mRNA were detected in the spinal cord at any developmental stages. The ?4 subunit mRNA was distributed widely in the spinal cord during embryonic and early postnatal periods but decreased nearly to background levels by P21. In contrast to the differential distribution of the ? subunit mRNAs, the ξ1 subunit mRNA was found ubiquitously at each developmental stage examined. These findings suggest that the molecular organization of the ? subunits may be difference between the dorsal horn and the remaining regions in the mature spinal cord, which provides a molecular basis for functional heterogeneity of the NMDA receptor channel. Moreover, this spatial heterogeneity might be generated through drastic alterations in the subunit composition of the channel complex during spinal cord development. © 1994 Wiley-Liss, Inc.