In vivo control of NMDA receptor transcript level in motoneurons by viral transduction of a short antisense gene

Glutamate receptors play critical roles in normal and pathological processes. We developed an antisense gene delivery strategy to modulate the NMDA type of glutamate receptor. Using transient transfection in vitro and viral mediated gene transfer in vitro and in vivo, the effect of expression of an antisense gene fragment (60 bp) of the NR1 subunit was tested. Immunoblot analysis showed an antisense-concentration-dependent reduction in the NR1 subunit upon transient co-transfection of a plasmid expressing a sense NR1 gene and a plasmid expressing the antisense fragment into COS-7 cells. After recombination into an adenoviral vector, this antisense fragment reduced the amount of endogenous NR1 protein in PC12 cells. Finally, direct intraparenchymal injection of the viral vector into rat spinal cord resulted in diminished NR1 in motor neurons. Our results demonstrate the efficacy of this approach, which combines antisense with viral gene delivery to control the expression of specific genes in vivo. This approach may also be useful in reducing excitatory neurotransmission in vivo, with implications for the treatment of spinal disorders such as amyotrophic lateral sclerosis or chronic pain.     

An antisense construct reduces N-methyl-D-aspartate receptor 2A expression and receptor-mediated excitotoxicity as determined by a novel flow cytometric approach

The N-methyl-D-aspartate receptor (NMDAR) is a major neurotransmitter receptor in the central nervous system (CNS), with functional roles in learning, memory, and sensation. Several mechanisms potentiate NMDARs, and NMDAR hyperexcitability plays pathophysiological roles in many conditions, such as neurodegenerative disease, ischemia, and chronic conditions arising from spinal cord injury. Previous research suggests that the NR2A subunit of the receptor contributes to NMDAR excitotoxicity in heterologous cells and in neurons in vivo. To investigate the role of NR2A in NMDAR excitotoxicity, we have developed a system based on flow cytometry that allows rapid evaluation of the effect of antisense constructs on protein expression and channel function. The enhanced yellow fluorescent protein (EYFP) was fused to obligatory NMDAR subunits, allowing expression to be monitored in living cultured cells. An NR2A antisense construct, asNR2A, specifically and effectively reduced NR2A-EYFP expression. NR1 and NR2A fusion proteins formed functional, excitotoxic channels upon co-expression. The asNR2A RNA significantly reduced NMDAR excitotoxicity when NR2A levels were limiting for channel formation. Using our assay system, further optimization can be achieved rapidly. The asNR2A construct and the assays developed for this study can be used to provide insights into NMDAR biology and disease.     

Injection of antisense oligos to nNOS into nucleus tractus solitarii increases blood pressure.

We investigated the cardiovascular effects of bilateral microinjection of antisense oligodeoxynucleotides (oligos) into the nucleus tractus solitarii (NTS) to neuronal nitric oxide synthase (nNOS) to suppress the expression of nNOS molecular biologically. In urethane-anesthetized, paralyzed Wistar-Kyoto rats, bilateral microinjection of nNOS antisense oligos (20 pmol in a 50nl volume) into the NTS produced a significant increase in mean arterial blood pressure at 30-60min after injection, compared with rats injected with nNOS sense or scrambled oligos. Immunohistochemical study demonstrated that nNOS immunoreactivity in the rat NTS was suppressed by nNOS antisense oligos. These results indicate that suppression of the nNOS gene using antisense in the NTS increases blood pressure.     

NR2B phosphorylation at tyrosine 1472 in spinal dorsal horn contributed to N-methyl-D-aspartate-induced pain hypersensitivity in mice

Calcium influx via N-methyl-D-aspartate (NMDA)-subtype glutamate receptors (NMDARs) regulates the intracellular trafficking of NMDARs, leading to long-lasting modification of NMDAR-mediated synaptic transmission that is involved in development, learning, and synaptic plasticity. The present study investigated the contribution of such NMDAR-dependent synaptic trafficking in spinal dorsal horn to the induction of pain hypersensitivity. Our data showed that direct activation of NMDARs by intrathecal NMDA application elicited pronounced mechanical allodynia in intact mice, which was concurrent with a specific increase in the abundance of NMDAR subunits NR1 and NR2B at the postsynaptic density (PSD)-enriched fraction. Selective inhibition of NR2B-containing NMDARs (NR2BR) by ifenprodil dose dependently attenuated the mechanical allodynia in NMDA-injected mice, suggesting the importance of NR2BR synaptic accumulation in NMDA-induced pain sensitization. The NR2BR redistribution at synapses after NMDA challenge was associated with a significant increase in NR2B phosphorylation at Tyr1472, a catalytic site by Src family protein tyrosine kinases (SFKs) that has been shown to prevent NR2B endocytosis. Intrathecal injection of a specific SFKs inhibitor, PP2, to block NR2B tyrosine phosphorylation eliminated NMDA-induced NR2BR synaptic expression and also attenuated the mechanical allodynia. These data suggested that activation of spinal NMDARs was able to accumulate NR2BR at synapses via SFK signaling, which might exaggerate NMDAR-dependent nociceptive transmission and contribute to NMDA-induced nociceptive behavioral hyperresponsiveness.     

Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons

The N‐methyl‐D ‐aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord and the generation of pain hypersensitivity. NMDAR function is modulated by post‐translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity in the spinal cord. We now show that a noxious heat stimulus applied to the rat hindpaw induces phosphorylation of the NMDAR NR1 subunit at a protein kinase C (PKC)‐dependent site, serine‐896, in superficial dorsal horn neurons. Phosphorylation of NR1 serine‐896 is essentially absent in the superficial dorsal horn laminae of naïve rats, but there is rapid (< 2 min) induction following a noxious but not innocuous heat stimulus. The number of pNR1‐immunoreactive neuronal profiles in the superficial dorsal horn peaks 30 min after noxious heat stimulation and persists for up to 1 h. pNR1serine896 induction occurs in the endoplasmic reticulum, suggesting that it contributes to trafficking of the receptor from intracellular stores to the membrane. The phosphorylation of the subunit is attenuated by intrathecal injection of the NMDAR antagonist, MK801, suggesting that the NMDAR is involved via a feed‐forward mechanism in its own phosphorylation. The pNR1serine896‐positive neurons are highly co‐localized with PKCdelta and only rarely with PKCgamma. These data provide evidence for an activity‐dependent NMDAR phosphorylation at the PKC‐dependent site, serine‐896, in spinal cord dorsal horn neurons initiated by peripheral noxious stimuli.     

Dynamic of migration of HSV‐1 from a medullary pronociceptive centre: antinociception by overexpression of the preproenkephalin transgene

Herpes Simplex Virus type 1 (HSV‐1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV‐1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV‐1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of β‐galactosidase (β‐gal) was detected at 2, 4, 7, 10 and 14 days. Numerous β‐gal‐immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV‐1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time‐points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti‐hyperalgesic effects persisted during 4 days, decreasing after that time‐point. The present study demonstrates that selective migration of HSV‐1 should be considered in gene therapy strategies based on HSV‐1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.     

Hypoalgesia induced by the local injection of carbachol into the nucleus raphe magnus

The medial region of the caudal medulla which contains the nucleus raphe magnus and magnocellular reticular formation has been demonstrated to modulate pain perception. Recent reports from this laboratory have shown that neurons in this region are under tonic inhibitory control by noradrenergic neurons. The excitability of neurons in the raphe magnus and adjacent reticular formation may also be controlled by cholinergic neurons since there is evidence that cholinergic terminals are located in the medial region of the caudal medulla. The present study was designed to examine this possibility by microinjecting carbachol, a cholinergic agonist, into the region of the nucleus raphe magnus. The results indicate that the injection of carbachol into the caudal brainstem produces dose-dependent hypoalgesia, i.e. decreased pain sensitivity. This hypoalgesia appears to be mediated by cholinergic muscarinic receptors since it was reversed by the muscarinic antagonist atropine. The cholinergic innervation of the raphe magnus does not appear to be important in the maintenance of nociceptive threshold since injection of atropine alone did not alter pain responses.     

Peripheral inflammation activated focal adhesion kinase signaling in spinal dorsal horn of mice

Focal adhesion kinase (FAK) is one of the nonreceptor protein tyrosine kinases critical for the dynamic regulation of cell adhesion structures. Recent studies have demonstrated that FAK is also localized at excitatory glutamatergic synapses and is involved in long‐term modification of synaptic strength. However, whether FAK is engaged in nociceptive processing in the spinal dorsal horn remains unresolved. The current study shows that intraplantar injection of complete Freund's adjuvant (CFA) in mice significantly increases FAK autophosphorylation at Tyr397, indicating a close correlation of FAK activation with inflammatory pain. FAK activation depended on the activity of N‐methyl‐D‐aspartate‐subtype glutamate receptor (NMDAR) and metabotropic glutamate receptor (mGluR) because pharmacological inhibition of NMDAR or group I mGluR totally abolished FAK phosphorylation induced by CFA. The active FAK operated to stimulate extracellular signal‐regulated kinase1/2 (ERK1/2), which boosted the protein expression of GluN2B subunit‐containing NMDAR at the synaptosomal membrane fraction. Inhibition of FAK activity by spinal expression of a kinase‐dead FAK(Y397F) mutant repressed ERK1/2 hyperactivity and reduced the synaptic concentration of NMDAR in CFA‐injected mice. Electrophysiological recording demonstrated that intracellular loading of specific anti‐FAK antibody significantly reduced the amplitudes of NMDAR‐mediated excitatory postsynaptic currents on lamina II neurons from inflamed mice but not from naive mice. Behavioral tests showed that spinal expression of FAK(Y397F) generated a long‐lasting alleviation of CFA‐induced mechanical allodynia and thermal hyperalgesia. These data indicate that FAK might exaggerate NMDAR‐mediated synaptic transmission in the spinal dorsal horn to sensitize nociceptive behaviors. © 2015 Wiley Periodicals, Inc.     

Amitriptyline attenuates astrocyte activation and morphine tolerance in rats: role of the PSD-95/NR1/nNOS/PKCγ signaling pathway

The tricyclic antidepressant amitriptyline binds with high affinity to N-methyl-d-aspartate receptors (NMDARs) and inhibits NMDAR-mediated events. Activation of the postsynaptic density protein-95 (PSD-95)/NMDAR-mediated downstream signaling cascade, including neuronal nitric oxide synthase (nNOS) and protein kinase gamma (PKCγ), has been shown to be involved in morphine tolerance. The present study examined the potential effect of amitriptyline on chronic morphine infusion-induced spinal PSD-95/NMDAR/nNOS/PKCγ signaling in morphine tolerance. Male Wistar rats were implanted with an intrathecal catheter and received an intrathecal infusion of saline or amitriptyline (15 μg/h), morphine+saline (tolerance induction, 15 μg/h), or morphine+amitriptyline for 5 days. Co-administration of amitriptyline with morphine not only preserved the antinociceptive effect of morphine, but also attenuated astrocyte activation in the rat spinal cord dorsal horn. On day 5 after drug infusion, increased expression and phosphorylation of spinal membrane NMDAR NR1 subunit and expression of PSD-95 were observed following chronic morphine infusion and these effects were attenuated by amitriptyline co-infusion. Upregulation of NMDAR-induced intracellular nNOS expression was also inhibited by amitriptyline co-infusion in chronic morphine-infused rats. Furthermore, amitriptyline co-infusion significantly inhibited morphine-induced PKCγ expression in both the cytosol and membrane of spinal neurons. These findings suggest that the attenuation of morphine tolerance caused by amitriptyline is due to downregulation of NMDAR NR1 subunit expression in the synaptosomal membrane accompanied by decreased expression of the scaffolding protein PSD-95. The effects of amitriptyline in attenuating astrocyte activation and reversing tolerance to morphine may be due, at least in part, to inhibition of the PSD-95/NMDAR NR1/nNOS/PKCγ signaling cascade.     

Morphine or U-50,488 suppresses Fos protein-like immunoreactivity in the spinal cord and nucleus tractus solitarii evoked by a noxious visceral stimulus in the rat.

Immunohistochemical visualization of Fos protein, the nuclear phosphoprotein product of the early-immediate gene c-fos, permits identification of populations of neurons that are activated in response to a variety of stimuli. This study examined the distribution of Fos-like immunoreactive (FLI) neurons in the spinal cord and the nucleus tractus solitarii (NTS) of the caudal medulla evoked by a noxious visceral stimulus in the unanesthetized rat. It also compared the inhibition of pain behavior and Fos expression by a mu-selective opioid agonist, morphine, and a kappa-selective opioid agonist, U-50,488. Intraperitoneal injection of 3.5% acetic acid in the unanesthetized rat evoked the expression of FLI in a discrete population of spinal cord neurons, the distribution of which closely mirrored the spinal terminations of visceral primary afferents. Specifically, FLI neurons were concentrated in laminae I, IIo, V, VII, and X. Large numbers of Fos-immunoreactive neurons were also present in the NTS of the caudal medulla, most likely as a result of spinosolitary tract and vaginal afferent input. The number of labeled neurons in both the spinal cord and the NTS was significantly correlated with the number of abdominal stretches, a pain behavior measure. Both morphine (1-10 mg/kg s.c.) and U-50,488 (3-30 mg/kg s.c.) produced a dose-dependent inhibition of the pain behavior in these animals and a dose-dependent suppression of the number of FLI neurons in both the spinal cord and in the NTS; complete suppression of FLI neurons was, however, not necessary for the production of antinociception. Furthermore, although equianalgesic doses of morphine and U-50,488 reduced the number of labelled neurons in the spinal cord to a comparable extent, morphine reduced the number of immunoreactive neurons in the NTS to a greater extent than did U-50,488. These results suggest that morphine and U-50,488 have comparable effects on the transmission of visceral nociceptive messages by spinal neurons, but differentially affect the autonomic response to noxious visceral stimuli.     

Masseteric inflammation-induced Fos protein expression in the trigeminal interpolaris/caudalis transition zone: contribution of somatosensory-vagal-adrenal integration.

The effects of vagotomy and adrenalectomy on the expression of Fos protein in brainstem neurons following the inflammation of masseter muscle were examined in order to differentiate the Fos activation related to nociceptive processing in contrast to that due to somatoautonomic processing. The inflammation was induced by a unilateral injection of complete Freund's adjuvant (CFA) into the masseter muscle under methohexital anesthesia after a small skin-cut (S-cut). After the CFA injection, Fos positive neurons were identified in bilateral spinal trigeminal nucleus (VSP), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM) and inferior medial olivary nucleus (IOM). At the level of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition zone, there was a selective induction of Fos-like immunoreactivity (LI) in the VSP and NTS, when compared to control rats (anesthesia with or without S-cut). A major portion of the Fos-LI in the VSP at the level of the caudal Vc was apparently activated by S-cut. Bilateral adrenalectomy or a unilateral vagotomy resulted in a selective reduction of inflammation-induced Fos-LI in the VSP at the Vi/Vc transition zone (P<0.05) and NTS (P<0.05), but had less effect on Fos-LI in the caudal Vc. These results suggest that the inflammation of the masseter muscle, an injury of orofacial deep tissue, results in a widespread change in neuronal activity in the VSP and NTS that depends in part on the integrity of the adrenal cortex and vagus. Thus, in addition to somatotopically organized nociceptive responses, orofacial deep tissue injury also is coupled to somatovisceral and somatoautonomic processing that contribute to central neural activation.     

NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation

In the present study, we examined the distribution of neurons in the parabrachial nucleus (PB), the Kölliker–Fuse nucleus (KF), the spinal trigeminal nucleus caudalis (Sp5C), the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM), which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-d-aspartate (NMDA) receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases (p≤0.001) of Fos-immunoreactive (-ir) neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Bötzinger/pre-Bötzinger complex, and in the ventrolateral and commissural NTS the increases were only moderately significant (p≤0.05). With respect to the numbers of NR1-/Fos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C (p≤0.001) and the midlevel KF (p≤0.01) and moderately significant (p≤0.05) in the external lateral PB, Bötzinger/pre-Bötzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses.     

Primary afferent terminals that express presynaptic NR1 in rats are mainly from myelinated,mechanosensitive fibers

Presynaptic N-methyl-D-aspartate (NMDA) receptors in terminals of primary afferents to spinal cord of rats were first reported by Liu et al. (1994; Proc. Natl. Acad. Sci. USA 91:8383-8387) and were proposed to modulate nociceptive input (Liu et al. [1997] Nature 386:721-724). We previously demonstrated kainate and AMPA receptors in numerous primary afferent terminals in the spinal cord fixed with diluted paraformaldehyde and no glutaraldehyde. Therefore, we reinvestigated the occurrence of presynaptic NMDAR1 (NR1) with this fixation protocol. With confocal microscopy, numerous immunofluorescent puncta were double-stained for NR1 and the presynaptic marker synaptophysin throughout the spinal gray. NR1-immunostained puncta costained more frequently with a tracer that labels myelinated afferents (cholera toxin subunit B; CTB) than with a tracer that labels non-peptidergic unmyelinated afferents (Griffonia simplicifolia isolectin B4; IB4). Virtually no double staining was found for NR1 and calcitonin gene-related peptide (CGRP), which labels somatic peptidergic primary afferents. In the gracile nucleus, virtually all puncta labeled for CTB appeared immunopositive for NR1. At the electron microscopic level, most immunopositive terminals in spinal cord and gracile nucleus displayed morphological characteristics of endings of myelinated primary afferents. NR1 was presynaptic in 60-65% of all synapses in which it was expressed pre- or postsynaptically, or both, in spinal laminae I-IV. Estimates for the gracile nucleus were higher (80%). No presynaptic NR1 was found in the ventroposterior thalamus. Because of the relative sparsity of presynaptic NR1 in terminals in laminae I and IIo and in terminals of peptidergic unmyelinated afferents, it is suggested that presynaptic NMDA receptors play a more significant role in modulation of mechanosensitive, innocuous input than in nociception.     

Effects of protein phosphatase inhibitors on the phosphorylation of spinal cord N-methyl-D-aspartate receptors following electroacupuncture stimulation in rats

The present study investigated the role of inhibitor of protein phosphatases 1 and 2A on the modulation of the phosphorylation of the spinal N-methyl-D-aspartate receptor (NMDAR) NR1 and NR2B subunits following electroacupuncture (EA) stimulation in rats. Bilateral 2Hz EA stimulations with 1.0 mA were delivered at those acupoints corresponding to Zusanli and Sanyinjiao to men via needles for 30 min. EA analgesia was slightly reduced by the intrathecal injection of calyculin A during EA stimulation. At 60 min after the termination of EA stimulation, the levels of c-fos, serine phosphorylation of NR1 and NR2B by Western analysis had increased in the L(4-5) segments of the spinal cord after EA treatment. These expressions were enhanced by the intrathecal injection of calyculin A and immunohistochemical analyses confirmed the significant increase of these proteins. As for the regional reaction of NMDAR subunits, a mean integrated optical density of phosphorylated NR1 and NR2B subunits was potentiated by calyculin A injections in the superficial laminae and neck region and superficial laminae and nucleus proprius, respectively. It can be concluded that protein phosphatase may play an important role in EA analgesia by modulating the phosphorylation state of spinal NMDAR subunits.     

Dexmedetomidine prevents remifentanil-induced postoperative hyperalgesia and decreases spinal tyrosine phosphorylation of N-methyl-d-aspartate receptor 2B subunit

Numerous studies have demonstrated that prolonged opioid exposure can enhance pain sensitivity that presents as opioid-induced hyperalgesia (OIH). Activation of spinal α2-adrenergic receptor may play an important role in the development of OIH. Dexmedetomidine is an α2-adrenergic agonist that has been shown to synergize with opioids. The aim of this study was to investigate the antihyperalgesia effects of dexmedetomidine on remifentanil-induced postinfusion hyperalgesia in a rat model of incision pain. We also evaluated whether the antihyperalgesic effects of dexmedetomidine were associated with suppression of NMDAR excitability, as measured by a reduction in spinal cord NR2B phosphorylation. Dexmedetomidine (12.5 μg/kg, 25 μg/kg, 50 μg/kg) was administered subcutaneously 30 min before plantar incision. Pretreatment with dexmedetomidine significantly decreased remifentanil-induced hyperalgesia, as indicated by increased paw withdrawal latencies and thresholds to thermal and mechanical stimulation respectively. Correlated with the pain behavior changes, Western blotting experiments also revealed that dexmedetomidine could decrease NR2B subunit phosphorylation (Tyr1472 site) in the dorsal horn, which was upregulated after remifentanil infusion. These results suggest that dexmedetomidine can efficiently alleviate OIH and it may be an effective novel option for the treatment of OIH. Our data also provide evidence that dexmedetomidine's anti-hyperalgesic effect may depend on its ability to modulate spinal cord NMDAR activation via suppression of NR2B phosphorylation.     

Masseteric inflammation-induced Fos protein expression in the trigeminal interpolaris/caudalis transition zone: contribution of somatosensory–vagal–adrenal integration

The effects of vagotomy and adrenalectomy on the expression of Fos protein in brainstem neurons following the inflammation of masseter muscle were examined in order to differentiate the Fos activation related to nociceptive processing in contrast to that due to somatoautonomic processing. The inflammation was induced by a unilateral injection of complete Freund's adjuvant (CFA) into the masseter muscle under methohexital anesthesia after a small skin-cut (S-cut). After the CFA injection, Fos positive neurons were identified in bilateral spinal trigeminal nucleus (VSP), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM) and inferior medial olivary nucleus (IOM). At the level of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition zone, there was a selective induction of Fos-like immunoreactivity (LI) in the VSP and NTS, when compared to control rats (anesthesia with or without S-cut). A major portion of the Fos-LI in the VSP at the level of the caudal Vc was apparently activated by S-cut. Bilateral adrenalectomy or a unilateral vagotomy resulted in a selective reduction of inflammation-induced Fos-LI in the VSP at the Vi/Vc transition zone (P<0.05) and NTS (P<0.05), but had less effect on Fos-LI in the caudal Vc. These results suggest that the inflammation of the masseter muscle, an injury of orofacial deep tissue, results in a widespread change in neuronal activity in the VSP and NTS that depends in part on the integrity of the adrenal cortex and vagus. Thus, in addition to somatotopically organized nociceptive responses, orofacial deep tissue injury also is coupled to somatovisceral and somatoautonomic processing that contribute to central neural activation.     

A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells

To develop effective gene therapy techniques that target populations of neurons in the spinal cord, suitable vectors must be developed that will undergo efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte et al, 1998a) has demonstrated that a replication defective herpes simplex virus vector 14Hdelta3vhsZ, that has been substantially detoxified, is retrogradely transported from peripheral sites and can infect large numbers of the targeted spinal neurons. We plan to develop targeted gene therapy approaches designed to modulate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spinal cord neurons as a means of ameliorating a form of episodic high blood pressure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus vector does not appear to alter aspects of the cytoskeletal architecture important to the proper distribution of the NMDA receptor. In turn, the distribution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfected NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expression. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. These results indicate that the 14Hdelta3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

Antisense knockdown of spinal-mGluR1 reduces the sustained phase of formalin-induced nociceptive responses

To examine the role of mGluR1 (a subunit of the group I metabotropic glutamate receptor) in the nociceptive responses of rats following a subcutaneous injection of formalin into the plantar surface of the hind paw, we delivered antisense oligonucleotides (ODNs) against mGluR1 into the rat lumbar spinal cord (L3–L5) intrathecally using an HVJ–liposome-mediated gene transfer method. Rats treated with a single injection of mGluR1 antisense ODNs into the intrathecal space of the lumbar spinal cord showed a marked reduction of the early-sustained phase of formalin-induced nociceptive responses, but not of their acute phase. The reduction of nociceptive behavioral responses became apparent at day 2 after the antisense treatment and lasted for 2 days. This corresponded to a long-lasting down-regulation (46%) of mGluR1 expression in the lumbar cord. This down-regulated mGluR1 was observed at day 2 and persisted until day 4 after the intrathecal infusion of mGluR1 antisense ODN. In contrast, rats treated with mGluR1 sense or mismatch ODNs showed none of these changes. These results suggest that mGluR1 may play a crucial role in the sustained nociception of formalin-induced behavioral responses.