Multireceptive Dorsal Horn Neurons Research Articles

Upregulation of Sodium Channel Nav1.3 and Functional Involvement in Neuronal Hyperexcitability Associated with Central Neuropathic Pain after Spinal Cord Injury

Spinal cord injury (SCI) can result in hyperexcitability of dorsal horn neurons and central neuropathic pain. We hypothesized that these phenomena are consequences, in part, of dysregulated expression of voltage-gated sodium channels. Because the rapidly repriming TTX-sensitive sodium channel Nav1.3 has been implicated in peripheral neuropathic pain, we investigated its role in central neuropathic pain after SCI. In this study, adult male Sprague Dawley rats underwent T9 spinal contusion injury. Four weeks after injury when extracellular recordings demonstrated hyperexcitability of L3-L5 dorsal horn multireceptive nociceptive neurons, and when pain-related behaviors were evident, quantitative RT-PCR, in situ hybridization, and immunocytochemistry revealed an upregulation of Nav1.3 in dorsal horn nociceptive neurons. Intrathecal administration of antisense oligodeoxynucleotides (ODNs) targeting Nav1.3 resulted in decreased expression of Nav1.3 mRNA and protein, reduced hyperexcitability of multireceptive dorsal horn neurons, and attenuated mechanical allodynia and thermal hyperalgesia after SCI. Expression of Nav1.3 protein and hyperexcitability in dorsal horn neurons as well as pain-related behaviors returned after cessation of antisense delivery. Responses to normally noxious stimuli and motor function were unchanged in SCI animals administered Nav1.3 antisense, and administration of mismatch ODNs had no effect. These results demonstrate for the first time that Nav1.3 is upregulated in second-order dorsal horn sensory neurons after nervous system injury, showing that SCI can trigger changes in sodium channel expression, and suggest a functional link between Nav1.3 expression and neuronal hyperexcitability associated with central neuropathic pain.

Serotonergic neural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons after spinal hemisection in rat

Hemisection of the rat spinal cord at thoracic level 13 provides a model of spinal cord injury that is characterized by chronic pain attributable to hyperexcitability of dorsal horn neurons. Presuming that this hyperexcitability can be explained in part by interruption of descending inhibitory modulation by serotonin, we hypothesized that intrathecal transplantation of RN46A-B14 serotonergic precursor cells, which secrete serotonin and brain-derived neurotrophic factor, would reduce this hyperexcitability by normalizing the responses of low-threshold mechanoreceptive, nociceptive-specific, and multireceptive dorsal horn neurons. Three groups ( n=45 total) of 30-day-old male Sprague–Dawley rats underwent thoracic level 13 spinal hemisection, after which four weeks were allowed for development of allodynia and hyperalgesia. The three groups of animals received transplants of no cells, 10 6 RN46A-V1 (vector-only) or 10 6 RN46A-B14 cells at lumbar segments 2–3. Electrophysiological experiments were done two weeks later. Low-threshold mechanoreceptive, nociceptive-specific, and multireceptive cells ( n=394 total) were isolated at depths of 1–300 and 301–1000 μm in the lumbar enlargement. Responses to innocuous and noxious peripheral stimuli were characterized, and analyses of population responses were performed. Compared with normal animals, dorsal horn neurons of all types in hemisected animals showed increased responsiveness to peripheral stimuli. This was true for neurons on both sides of the spinal cord. After hemisection, the proportion of neurons classified as multireceptive cells increased, and interspike intervals of spontaneous discharges became less uniform after hemisection. Transplantation of RN46A-B14 cells restored evoked responses to near-control levels, normalized background activity, and returned the proportion of multireceptive cells to the control level. Restoration of normal activity was reversed with methysergide. These electrophysiological results corroborate anatomical and behavioral studies showing the effectiveness of serotonergic neural precursors in correcting phenomena associated with chronic central pain following spinal cord injury, and provide mechanistic insights regarding mode of action.

Antisense Ablation of Type I Metabotropic Glutamate Receptor mGluR1Inhibits Spinal Nociceptive Transmission

Electrophysiological and behavioral studies point to a role of group I metabotropic glutamate receptors (mGluR1 and mGluR5) in mediating spinal nociceptive responses in rats. However, antagonists with a high degree of specificity for each of these sites are not yet available. We, therefore, examined the effects of antisense deletion of spinal mGluR1 expression in assays of behavioral analgesia and of electrophysiological responses of dorsal horn neurons. Rats treated with an mGluR1 antisense oligonucleotide reagent, delivered continuously to the intrathecal space of the lumbar spinal cord, developed marked analgesia as measured by an increase in the latency to tail-flick (55 degreesC) over a period of 4-7 d. This correlated with a selective reduction in mGluR1, but not mGluR5, immunoreactivity in the superficial dorsal horn compared with untreated control rats, in parallel with a significant reduction in the proportion of neurons activated by the mGluR group I agonist 3, 5-dihydroxyphenylglycine (DHPG), whereas the proportion of cells excited by the mGluR5 agonist, trans-azetidine-2,4-dicarboxylic acid (t-ADA) remained unaffected. In contrast, rats treated with mGluR1 sense or mismatch probes showed none of these changes compared with untreated, control rats. Furthermore, multireceptive dorsal horn neurons in mGluR1 antisense-treated rats were strongly excited by innocuous stimuli to their peripheral receptive fields, but showed severe reductions in their sustained excitatory responses to the selective C-fiber activator mustard oil and in responses to DHPG.

Supraspinal inhibition of nociceptive dorsal horn neurones in the anaesthetized rat: tonic or dynamic?

1. Tonic inhibition of sensory spinal neurones is well known to descend from the rostroventral medulla. It is not clear if this inhibition is dynamically activated by peripheral noxious stimuli. 2. Transection of the ipsilateral dorsolateral funiculus (DLF) removed a descending inhibition of multireceptive spinal neurones and disproportionally prolonged the after-discharge component of their response to a noxious cutaneous stimulus. 3. Microinjection of GABA or tetracaine into the medullary nucleus gigantocellularis pars alpha (GiA) similarly prolonged the after-discharge in response to noxious stimuli. 4. Recordings of GiA cells, initially using minimal surgery, revealed that many had low levels of spontaneous activity and responded vigorously to noxious stimuli applied to any part of the body surface. One hour after the surgery necessary to expose the spinal cord, GiA cells had a high firing rate but responded weakly to noxious stimuli. 5. The response of GiA cells to noxious stimuli was abolished by transection of only the DLF contralateral to the stimulus. 6. It is concluded that the inhibition of multireceptive dorsal horn neurones from GiA is dynamically activated by noxious cutaneous stimuli via a projection in the contralateral DLF. Surgical exposure of the spinal cord tonically activates this inhibition and masks the dynamic component.

Evidence for roles of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) receptors in modulating the responses of rat dorsal horn neurons to sensory inputs

The extracellularly recorded electrophysiological activity of single multireceptive dorsal horn neurons was markedly increased by ionophoretic administration of vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase activating polypeptide (PACAP)-38. Some cells responded selectively to PACAP-38 (suggesting mediation by a PACAP receptor), whereas others responded to both VIP and PACAP-38 (suggesting a VIP 1 and/or VIP 2 receptor). Most non-nociceptive cells were unaffected by PACAP-38 and all were unaffected by VIP. The selectivity of VIP/PACAP receptor antagonists was established on cloned rat VIP 1, VIP 2 and PACAP receptors in vitro before their utilization to indicate the likely involvement of VIP 1, and possibly PACAP receptors, in VIP- and PACAP-38-mediated responses of dorsal horn neurons. The VIP/PACAP receptor antagonists inhibited responses of multireceptive cells to sustained innocuous (brush) and noxious (mustard oil) stimuli, with a selectivity suggesting the involvement of VIP 1 and PACAP receptors, although the participation by VIP 2 receptors cannot be excluded. These data implicate both VIP and PACAP in regulating the basal responsiveness of multireceptive dorsal horn neurons to sensory stimuli.

The pontine parabrachial region mediates some of the descending inhibitory effects of stimulating the anterior pretectal nucleus

Electrical stimulation of the anterior pretectal nucleus (APtN) elicits antinociception by inhibiting the responses of spinal multireceptive neurones to noxious stimuli. This descending inhibition is mediated, in part, by activating cells in the ventrolateral medulla. Neuronal tract tracing has previously shown that the APtN also projects directly to the pontine parabrachial region (PPR). The PPR, investigated by Katayama et al. ( Brain Res., 296 (1984) 263–283), corresponds to the cholinergic cell group Ch5 of Mesulam et al. ( Neuroscience, 10 (1983) 1185–1201). In this study, the pathway from APtN to PPR was investigated using urethane anaesthetised rats. Electrical stimulation (single square wave 0.2 ms pulses, 1–10 V, 5 Hz) of the APtN potently excites 40% of the cells recorded in the PPR. In the reverse experiment, stimulation of the PPR at the same parameters excited 36% of the cells recorded in the APtN. The contribution of this pathway to the spinal inhibitory effects of APtN stimulation was then examined. Unanaesthetised animals received electrical stimulation to the APtN (35 μ A r.m.s., 15 s) and the increase in tail-flick latencies was measured. Bilateral electrolytic lesions of the PPR caused a 67% reduction of the antinociceptive effect of APtN stimulation. In urethane anaesthetised rats, microinjection of tetracaine into the PPR blocked the inhibition of multireceptive dorsal horn neurones caused by APtN stimulation (20 s train of 50 μA square wave 0.1 ms pulses, 100 Hz). In conclusion, these experiments strongly sugget that the PPR may be an important part of a descending antinociceptive pathway originating in the APtN.

Alterations in neuronal excitability and the potency of spinal mu, delta and kappa opioids after carrageenan-induced inflammation

These electrophysiological results show that the development of inflammation following peripheral injection of carrageenan into the paw is accompanied by alterations in the magnitude of the C-fibre evoked response of multireceptive dorsal horn neurones. The evoked response of the dorsal horn cells was found to either increase or decrease in the 3 h following the carrageenan injection, and the direction of this change was related to the degree of wind-up exhibited by the cell. Regardless of whether a cell was facilitated or inhibited by carrageenan, mu, delta and kappa opioids applied topically onto the spinal cord (equivalent to an intrathecal injection) exhibited increased antinociceptive potency. This increased effectiveness was especially marked for the mu opioid, morphine, which showed a 30-fold increase in potency. Interestingly the facilitations seen with the lowest doses of the mu and kappa opioids in this model in normal animals were absent after carrageenan. In addition, a very low dose of spinal naloxone caused a small but significant reduction in the C-fibre evoked responses. These results demonstrate that following peripheral inflammation, functional changes develop in both spinal transmission and modulatory systems. Alterations in the antinociceptive potency of opioid agonists occurs, with the mu agonist, morphine, showing the greatest change.

Dextromethorphan and levorphanol on dorsal horn nociceptive neurones in the rat

Intrathecal administration of dextromethorphan and levorphanol and intravenous injection of dextromethorphan were tested on the electrophysiological response of deep multireceptive dorsal horn neurones to peripheral stimuli. Both blockade of C-fibre input to the cells and wind-up, the increase in C-flbre firing with repeated stimulus, were recorded. p ]Intrathecal injection of levorphanol (0.25–100 μg) had a typical opioid effect, blocking the C-fibre input. Its affect on wind-up was dose-dependent, paralleled precisely the blocking effect on the C-fibre input and both effects were reversed by naloxone. p ]Unlike levorphanol and other opiates, intrathecal administration of dextromethorphan (50–500 μg) blocked the C-fibre input and Aβ response in parallel and was not reversed by naloxone. Wind-up was reduced by a maximum of 56% at the largest dose tested. Intravenous injection of dextromethorphan (5 mg/kg) also produced a reduction in wind-up but not in the C-fibre response.

Spinal somatostatin superfusion in vivo affects activity of cat nociceptive dorsal horn neurons: Comparison with spinal morphine

A controversy exists concerning the role of the neuropeptide somatostatin for the transmission or inhibition of nociceptive information in the spinal cord. To better correlate electrophysiological effects of somatostatin at single cell level with results obtained with intrathecal injections of somatostatin in behaving animals and human pain patients we applied somatostatin to the spinal cord by controlled superfusion of the recording segment in vivo. The hypothesis of an opiod link and possible neurotoxic effects of somatostatin were also addressed. In cats deeply anaesthetized with pentobarbitone, halothane and nitrous oxide, extracellular recordings were made from 27 neurons located in laminae I–VI. All neurons responded to both innocuous mechanical and noxious radiant heat stimuli applied to the glabrous skin of the ipsilateral hindpaw. The dorsal surface of the spinal cord was superfused at the recording segment by means of a Perspex chamber (7 × 7 mm). Somatostatin superfusions at 1.2 μM had no effect on responses to noxious heat. Responses were, however, depressed by somatostatin at 61 μM to59.7 ± 5.1% of control and by somatostatin at 1.53 mM to39.9 ± 9.5% of control. This inhibition was not antagonized by the μ-opiate antagonist naloxone applied to the spinal cord at concentrations of 2.7 mM, either together with somatostatin, or after the inhibition by somatostatin had fully developed. Neuronal responses were linear functions of the skin temperatures for stimulation intensities between 42°C and 52°C. The slopes of these stimulus response functions were reduced during somatostatin superfusion at 61 μM to46.8 ± 9.3% of control, without changing the temperature thresholds for responding (42.5 ± 0.6°C). Somatostatin superfusion at 61 μM had no effect on the number of action potentials evoked by innocuous skin brushing, or by electrical stimulation of primary afferent A-fibres in cutaneous nerves. The amplitude of intraspinally recorded field potentials evoked by these electrical nerve stimuli was also unaffected by somatostatin. The inhibition of nociceptive spinal dorsal horn neurons by spinally administered morphine was assessed in eight experiments. Morphine reduced noxious heat-evoked responses to42.1 ± 9.6% of control at 0.3 mM and to51.8 ±6.9% of control at 3.0 mM. The slopes of the stimulus-response functions were reduced by morphine at 0.3 mM to53.1 ± 11.3% of control, without changing the temperature thresholds (42.7°C). Naloxone superfusion (2.7 mM) reliably antagonized the inhibition by morphine. Brush-evoked responses were not, or much less, affected by spinal morphine. It is concluded that both, somatostatin and morphine selectively depress nociceptive responses of multireceptive dorsal horn neurons by a direct, spinal site of action, providing a neuronal basis for their antinociceptive effects in behaving animals and in pain patients. The effects of somatostatin and morphine are qualitatively and quantitatively similar, although the inhibition by morphine, but not by somatostatin, is mediated via spinal μ-opiate receptors.

Antinociceptive effects of dorsal column stimulation in the rat: involvement of the anterior pretectal nucleus.

1. The effects of stimulating A fibres in the dorsal columns on the responses of dorsal horn neurones to intense cutaneous stimuli were studied in the rat anaesthetized with urethane. 2. Multireceptive cells deep in the lumbar dorsal horn were excited for 5-10 ms by dorsal column stimulation and subsequently responses to noxiously hot water placed on the cutaneous receptive field were reduced for the following 4-5 min. Seven of the cells studied projected to the brain via the contralateral anterolateral funiculus. 3. If the discharge of the multireceptive neurones was raised by ionophoretic application of DL-homocysteic acid, a brief period of inhibition lasting for 100-150 ms was seen following a single stimulus to the dorsal columns. Studies were conducted to determine if this brief inhibition could account for the long-lasting inhibition of responses to high-threshold stimuli. 4. Dorsal columns were transected at cervical levels. Stimulation caudal to the transection evoked only the brief excitation and subsequent inhibition for 100-150 ms. No long-lasting inhibition of high-threshold cutaneous afferent input was seen. 5. Stimulation of the dorsal columns rostral to transection did not evoke the brief excitation or inhibition of multireceptive dorsal horn neurones. However, the 4-5 min inhibition of responses to high-threshold cutaneous stimuli was present. 6. The long-lasting inhibition of responses to high-threshold stimuli by dorsal column stimulation was blocked by microinjection of gamma-aminobutyric acid into the anterior pretectal nucleus (APTN) but not by microinjections into adjacent areas of the brain. 7. Ipsilateral lesions of the dorsolateral funiculus at the cervical level also blocked the long-lasting inhibitory effects of dorsal column stimulation. 8. It is concluded that the brief excitation and inhibition of multireceptive dorsal horn neurones is due to antidromic action potentials passing caudally in the dorsal columns to activate spinal segmental mechanisms. The longer-lasting inhibition of responses to high-threshold cutaneous stimuli is due to action potentials ascending in the dorsal columns to activate cells in the APTN which in turn activate a descending inhibition mediated by the dorsolateral funiculus.

Heterogenous effects of serotonin in the dorsal horn of rat: the involvement of 5-HT 1 receptor subtypes

The effect of ionophoretically applied serotonin (5-HT) was tested on cutaneous sensory responses of multireceptive dorsal horn neurones in the anaesthetized rat. Three types of 5-HT action were discerned: selective inhibition of nociceptive responses (10/18 cells), non-selective inhibition of responses to both noxious and innocous stimuli as well as to excitatory amino acids (4/18 cells) and non-selective excitation of evoked responses (1/18 cells). A few cells (3/18) were unaffected by 5-HT. The use of agonists, shown to discriminate between subtypes of 5-HT 1 receptor revealed that a 5-HT 1A receptor agonist mimicked the non-selective effects of 5-HT, whereas a 5-HT 1B receptor agonist mimicked the selective antinociptive effects of 5-HT. A 5-HT 2 receptor agonist, in contrast, was without effect. Both the selective and the non-selective effects were reversed by a 5-HT 1 receptor antagonist, but not a 5-HT 2 antagonist.

Fluoxetine and pargyline on the nucleus raphe magnus inhibition of cat multireceptive dorsal horn neurones

1. 1. To determine whether serotonin (5-HT) is involved in the nucleus raphe magnus (NRM) inhibition of cat multireceptive dorsal horn neurones, the NRM inhibition was tested with drugs (fluoxetine and pargyline) that should enhance 5-HT synaptic activity. 2. 2. Fluoxetine and pargyline systemically administered, individually or concurrently, decreased rather than increased the inhibition. 3. 3. The results do not support the hypothesis that NRM stimulation-produced inhibition is mediated by 5-HT release.

The failure of morphine to attenuate spinal cord nociceptive transmission through supraspinal actions in the cat

1. 1. Morphine sulphate was perfused between the third ventricle and cisterna magna in α-chloralose anaesthetized cats while recording from multireceptive dorsal horn neurones activated by peripheral noxious radiant heat. 2. 2. Morphine concentrations of 10 −5 and 10 −4 M were without effect, while 10 −3 M resulted in an increase in the spinal cord neuronal responses. 3. 3. The results do not support the notion that morphine activates a supraspinal mediated descending inhibition which antagonizes spinal cord nociceptive transmission.

Methysergide and spinal inhibition from electrical stimulation in the periaqueductal grey

In barbiturate anaesthetized cats, electrical stimulation in the periaqueductal grey (PAG) selectively inhibited the excitation of multireceptive dorsal horn neurons by noxious heating of the skin or by electrical stimulation of unmyelinated primary afferents. Intravenous methysergide (0.2–1.0 mg/kg) had opposing effects on uninhibited responses, increasing excitation by noxious heat but reducing responses to C fibre stimulation. Evidence was obtained that the increases to nuxious heat resulted from increased firing of peripheral nociceptors secondary to decreased cutaneous blood flow. Intravenous methysergide reduced inhibition from PAG stimulation. When administered electrophoretically from micropipettes however, methysergide did not reduce such inhibition. The mechanism whereby systemic methysergide reduces PAG spinal inhibition is unknown and cannot necessarily be related to a blockade of spinally released 5-hydroxytryptamine.

Lack of interaction between methionine enkephalin and d-phenylalanine on nociceptive and non-nociceptive responses of dorsal horn neurones of the cat

In the spinal cord of the barbiturate anaesthetized cat, D-phenylalanine (DPA) depressed spontaneous and amino acid-induced firing of multireceptive dorsal horn neurones. The excitation of these neurons by noxious and non-noxious cutaneous stimuli was non-selectively depressed by DPA. DPA did not potentiate the depressant action of methionine enkephalin on these cells. Although naloxone reduced the depressant action of DPA, this was associated with increases in spontaneous firing. Spinal reflexes in this preparation are tonically inhibited by opioid peptides. Intravenous DPA (20–70 mg/kg) did not potentiate this inhibition but produced small increases in spinal reflexes. Collectively these data do not support the hypothesis that analgesia from DPA results from potentiation of endogenously released methionine enkephalin.

Spinal vs supraspinal actions of morphine on cat spinal cord multireceptive neurons

To examine whether morphine elicits a supraspinal mediated spinal inhibition of nociceptive transmission, several investigators have compared the effects of morphine on nociceptive transmission in animals with the spinal cord intact vs transected or cold-blocked. The results have been conflicting, possibly due to different methods of analysis. For example, some investigators have found i.v. administered morphine produces a greater percentage decrease in nociceptive transmission when the spinal cord is intact compared to the transected state. Therefore, they concluded that morphine elicits a supraspinal-mediated inhibition. Conversely, others have reported that the increase in noxious stimulus-evoked responses of dorsal horn neurons upon cold blocking the spinal cord was reduced by i.v. morphine. They therefore concluded that morphine decreases descending inhibition. We tested the effects of i.v. morphine on spinal cord multireceptive neurons in the presence and absence of descending inhibition. Using the above methods of analysis, our results were found to be consistent with their findings which indicate that the method of analysis used is critical to the interpretation reached. To determine how these calculations would be altered by a depressant effect on the spinal cord neurons only, we performed similar experiments iontophoresing γ-aminobutyric acid (GABA) onto these dorsal horn neurons. The similarity between the morphine and GABA data suggests that the effects of systemically administered morphine on multireceptive dorsal horn neurons can be adequately explained by a spinal cord site of action.