Christopher N. Honda

Cytotoxic targeting of isolectin IB4-binding sensory neurons

The isolectin I-B4 (IB4) binds specifically to a subset of small sensory neurons. We used a conjugate of IB4 and the toxin saporin to examine in vivo the contribution of IB4-binding sensory neurons to nociception. A single dose of the conjugate was injected unilaterally into the sciatic nerve of rats. The treatment resulted in a permanent selective loss of IB4-binding neurons as indicated by histological analysis of dorsal root ganglia, spinal cord, and skin from treated animals. Behavioral measurements showed that 7-10 days after the injection, conjugate-treated rats had elevated thermal and mechanical nociceptive thresholds. However, 21 days post-treatment the nociceptive thresholds returned to baseline levels. These results demonstrate the utility of the IB4-saporin conjugate as a tool for selective cytotoxic targeting and provide behavioral evidence for the role of IB4-binding neurons in nociception. The decreased sensitivity to noxious stimuli associated with the loss of IB4-binding neurons indicates that these sensory neurons are essential for the signaling of acute pain. Furthermore, the unexpected recovery of nociceptive thresholds suggests that the loss of IB4-binding neurons triggers changes in the processing of nociceptive information, which may represent a compensatory mechanism for the decreased sensitivity to acute pain.

Projections from the marginal zone and deep dorsal horn to the ventrobasal nuclei of the primate thalamus

&NA; It has been concluded recently that if a projection from the marginal zone to the ventral posterior lateral (VPL) nucleus exists, it is sparse. Given the importance of the marginal zone in nociception, this conclusion has raised doubts about the significance of the role of the ventrobasal complex in nociception. We have reexamined this projection using injections of the retrograde tracer, cholera toxin subunit B, into one side of the lateral thalamus in macaque monkeys. The injections were confined to the ventrobasal complex (with minimal spread to adjacent nuclei that do not receive spinal projections) in two animals. Many retrogradely labeled neurons were found in lamina I (as well as in lamina V) of the contralateral spinal and medullary dorsal horn. The results are consistent with the view that neurons in the marginal zone contribute prominently to the spinothalamic and trigeminothalamic projections to the VPL and ventral posterior medial (VPM) nuclei. This pathway is likely to be important for the sensory‐discriminative processing of nociceptive information with respect to the location and intensity of painful stimuli.

Differential distribution of calbindin-D28k and parvalbumin in somatic and visceral sensory neurons

The purpose of the present investigation was to determine whether calbindin-D28k and parvalbumin are distributed to different subpopulations of somatic and visceral sensory neurons. Immunofluorescent and retrograde techniques were combined to examine the distribution of calbindin- and parvalbumin-like immunoreactivity in the cell bodies of somatic and visceral primary afferent neurons in dorsal root ganglia L1-S1 of rats. Calbindin and parvalbumin were differentially distributed to essentially non-overlapping subpopulations of primary sensory neurons that could be distinguished by their segmental and size distributions, as well as by their innervation of somatic and visceral structures. Calbindin-like immunoreactivity was found in a population of smaller-sized cell bodies comprising approximately 14%of all dorsal root ganglion cells examined, with the proportions being greatest in L6 and S1. In contrast, parvalbumin was found in a population of larger-sized cells that made up about 11% of dorsal root ganglion cells and that were most concentrated in L4 and L5. Sensory neurons were further characterized by retrograde transport following the application of the neuroanatomical tracer FluoroGold to somatic (sural and gastrocnemius) and visceral (hypogastric and pelvic) nerves. Somatic tissues were innervated by a population of calbindin-containing as well as a separate population of parvalbumin-containing sensory neurons. In contrast, afferent neurons innervating visceral structures contained only a subpopulation of calbindin-containing neurons and very few parvalbumin-positive cells.

IMMUNOHISTOCHEMICAL LOCALIZATION OF DELTA OPIOID RECEPTORS IN PERIPHERAL TISSUES

The distribution of delta opioid receptor (DOR) immunoreactivity (ir) was examined in various peripheral tissues of Sprague‐Dawley rats and macaque monkeys, including glabrous and hairy skin, corneas, eyelids, and the lip. DOR‐ir was observed in all tissues examined. In addition to the presence of DOR‐immunoreactive fibers in subcutaneous nerve bundles and the papillary dermis, we report the existence of positively labeled fibers and terminals in close association with peripheral structures not traditionally assigned a primarily nociceptive function, such as hair follicles, glandular apparatus, and blood vessels. In every case, staining was restricted to small‐diameter axons that appeared to terminate as free nerve endings. To further classify DOR‐immunoreactive fibers, we examined the extent of colocalization between DOR and three commonly used neuronal subtype markers; tyrosine hydroxylase (TH), calcitonin gene‐related peptide (CGRP), and RT‐97, a monoclonal antibody which preferentially labels neurons with myelinated axons. Additional double‐labeling experiments using the nonspecific neuronal marker Protein Gene Product 9.5 were performed in glabrous skin to determine the percentage of total fiber count that displayed DOR‐ir. No colocalization was observed between DOR and RT‐97, indicating that DOR‐ir is localized to unmyelinated axons. In addition, DOR colocalized with CGRP, but did not colocalize with TH. Taken together, these data support the hypothesis that delta opioid receptors in peripheral tissues are associated with sensory fibers, but not with the terminals of postganglionic sympathetic neurons. J. Comp. Neurol. 408:567–579, 1999.

Pruriceptive spinothalamic tract neurons: physiological properties and projection targets in the primate

Itch of peripheral origin requires information transfer from the spinal cord to the brain for perception. Here, primate spinothalamic tract (STT) neurons from lumbar spinal cord were functionally characterized by in vivo electrophysiology to determine the role of these cells in the transmission of pruriceptive information. One hundred eleven STT neurons were identified by antidromic stimulation and then recorded while histamine and cowhage (a nonhistaminergic pruritogen) were sequentially applied to the cutaneous receptive field of each cell. Twenty percent of STT neurons responded to histamine, 13% responded to cowhage, and 2% responded to both. All pruriceptive STT neurons were mechanically sensitive and additionally responded to heat, intradermal capsaicin, or both. STT neurons located in the superficial dorsal horn responded with greater discharge and longer duration to pruritogens than STT neurons located in the deep dorsal horn. Pruriceptive STT neurons discharged in a bursting pattern in response to the activating pruritogen and to capsaicin. Microantidromic mapping was used to determine the zone of termination for pruriceptive STT axons within the thalamus. Axons from histamine-responsive and cowhage-responsive STT neurons terminated in several thalamic nuclei including the ventral posterior lateral, ventral posterior inferior, and posterior nuclei. Axons from cowhage-responsive neurons were additionally found to terminate in the suprageniculate and medial geniculate nuclei. Histamine-responsive STT neurons were sensitized to gentle stroking of the receptive field after the response to histamine, suggesting a spinal mechanism for alloknesis. The results show that pruriceptive information is encoded by polymodal STT neurons in histaminergic or nonhistaminergic pathways and transmitted to the ventrobasal complex and posterior thalamus in primates.

Effect of morphine sulphate eye drops on hyperalgesia in the rat cornea

&NA; In addition to their traditional role in centrally mediated analgesia, opiate compounds produce significant effects when administered peripherally. Using a recently characterized model of acute chemical injury to the rat cornea (Pain (in press)), we assessed the effects of morphine sulphate eye drops on corneal inflammation and hyperalgesia. Topical application of a 5 &mgr;M morphine sulphate eye drop preparation attenuated capsaicin‐induced blinking in a concentration‐dependent manner. However, morphine had no effect on capsaicin‐induced blinking when applied to healthy, non‐inflamed rat cornea. In addition, 5 &mgr;M morphine given every 2 h following cauterization retarded the development of both stromal edema and the infiltration of immune cells. Both the analgesic and anti‐inflammatory effects of morphine were prevented by the opioid receptor antagonists naloxone, CTAP, and naltrindole. We conclude that morphine acts on &mgr; and &dgr; opioid receptors located in the rat cornea to attenuate inflammation and hyperalgesia.

Spinal NK1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation

Hyperalgesia is a characteristic of inflammation and is mediated, in part, by an increase in the excitability of spinal neurons. Although substance P does not appear to mediate fast synaptic events that underlie nociception in the spinal cord, it may contribute to the hyperalgesia and increased excitability of spinal neurons during inflammation induced by complete Freunds adjuvant. We examined the role of endogenous substance P in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation by determining the effect of a selective NK1 receptor antagonist (RP67580) on the nociceptive flexor reflex in adult rats. Experiments were conducted 2 or 3 days after injection of adjuvant. Animals exhibited moderate thermal hyperalgesia at this time. The flexor reflex was evoked by electrical stimulation of the sural nerve and was recorded in the ipsilateral hamstring muscles. The flexor reflex ipsilateral to the inflamed hindpaw was enhanced approximately two-fold compared to the flexor reflex evoked in untreated animals as determined by the number of potentials and the duration of the reflex. The enhanced reflex in adjuvant-treated animals was most likely due to an increase in the excitability of spinal interneurons because short-latency activity in the hamstring muscles did not differ between untreated animals and adjuvant-treated animals following electrical stimulation of the L5 dorsal root or the nerve innervating the muscle with a stimulus that was 1.3-1.5 times the threshold for excitation of A-fibers. Intrathecal administration of RP67580 (2.3 and 6.8 nmol) attenuated the flexor reflex evoked in adjuvant-treated animals, but had no effect in untreated animals. Intravenous or intraplantar injection of RP67580 (6.8 nmol) did not affect the flexor reflex in adjuvant-treated animals indicating a spinal action of the drug following intrathecal administration. RP68651, the enantiomer of RP67580, was without effect at doses up to 6.8 nmol, indicating that the effects of comparable doses of RP67580 were due to an action of the drug at NK1 receptors. However, intrathecal administration of 23 nmol of both drugs attenuated the reflex in adjuvant-treated and control animals indicating that effects of RP67580 at this dose were not mediated entirely by its action at NK1 receptors. Overall, these data suggest that endogenous substance P has a role in the increased excitability of spinal interneurons observed during persistent inflammation and support the hypothesis that substance P released in the spinal cord contributes to the hyperalgesia that accompanies adjuvant-induced persistent, peripheral inflammation.

Effects of high-frequency alternating current on axonal conduction through the vagus nerve

High-frequency alternating current (HFAC) is known to disrupt axonal conduction in peripheral nerves, and HFAC has much potential as a therapeutic approach for a number of pathological conditions. Many previous studies have utilized motor output as a bioassay of effects of HFAC on conduction through medium- to large-diameter motor axons. However, little is known about the effectiveness of HFAC on smaller, more slowly conducting nerve fibres. The present study tested whether HFAC influences axonal conduction through sub-diaphragmatic levels of the rat vagus nerve, which consists almost entirely of small calibre axons. Using an isolated nerve preparation, we tested the effects of HFAC on electrically evoked compound action potentials (CAPs). We found that delivery of charge-balanced HFAC at 5000 Hz for 1 min was effective in producing reversible blockade of axonal conduction. Both Aδ and C components of the vagus CAP were attenuated, and the degree of blockade as well as time to recovery was proportional to the amount of HFAC current delivered. The Aδ waves were more sensitive than C waves to HFAC blockade, but they required more time to recover.

Proteomic Analysis Uncovers Novel Actions of the Neurosecretory Protein VGF in Nociceptive Processing

Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury.

Co-administration of δ- and μ-opioid receptor agonists promotes peripheral opioid receptor function.

&NA; Enhancement of peripheral opioid analgesia following tissue injury or inflammation in animal models is well‐documented, but clinical results of peripheral opioid therapy remain inconsistent. Previous studies in the central nervous system have shown that co‐administration of &mgr;‐ and &dgr;‐opioid receptor agonists can enhance analgesic outcomes; however, less is known about the functional consequences of opioid receptor interactions in the periphery. The present study examines the effects of intraplantar injection of the &mgr;‐ and &dgr;‐opioid receptor agonists, morphine and deltorphin, alone and in combination on behavioral tests of nociception in naïve rats and on potassium‐evoked release of CGRP from sciatic nerves of naïve rats. Neither drug alone affected nociceptive behaviors or CGRP release. Two separate measures of mechanical nociceptive sensitivity remained unchanged after co‐administration of the two drugs. In contrast, when deltorphin was co‐injected with morphine, dose‐dependent and peripherally restricted increases in paw withdrawal latencies to radiant heat were observed. Similarly, concentration‐dependent inhibition of CGRP release was observed when deltorphin and morphine were administered in sequence prior to potassium stimulation. However, no inhibition was observed when morphine was administered prior to deltorphin. All combined opioid effects were blocked by co‐application of antagonists. Deltorphin exposure also enhanced the in vivo and in vitro effects of another &mgr;‐opioid receptor agonist, DAMGO. Together, these results suggest that under normal conditions, &dgr;‐opioid receptor agonists enhance the effect of &mgr;‐opioid receptor agonists in the periphery, and local co‐administration of &dgr;‐ and &mgr;‐opioid receptor agonists may improve results of peripheral opioid therapy for the treatment of pain.