Opiates and pain pathways: demonstration of enkephalin synapses on dorsal horn projection neurons.

The participation of the opiate peptide enkephalin in the neural circuitry of the dorsal horn was examined at the light and ultrastructural level through the use of the combined techniques of immunocytochemistry and retrograde transport of horseradish peroxidase. Enkephalin immunoreactive axonal endings made direct synaptic contact with the soma and proximal dendrites of dorsal horn thalamic projection neurons. This observation demonstrates that one major synaptic site of enkephalin modulation of the transfer of nociceptive information in the dorsla horn is on the projection neurons themselves.U

Altered nociceptive neuronal circuits after neonatal peripheral inflammation.

Nociceptive neuronal circuits are formed during embryonic and postnatal times when painful stimuli are normally absent or limited. Today, medical procedures for neonates with health risks can involve tissue injury and pain for which the long-term effects are unknown. To investigate the impact of neonatal tissue injury and pain on development of nociceptive neuronal circuitry, we used an animal model of persistent hind paw peripheral inflammation. We found that, as adults, these animals exhibited spinal neuronal circuits with increased input and segmental changes in nociceptive primary afferent axons and altered responses to sensory stimulation.

Activity-dependent neuronal plasticity following tissue injury and inflammation.

Increases in neuronal activity in response to tissue injury lead to changes in gene expression and prolonged changes in the nervous system. These functional changes appear to contribute to the hyperalgesia and spontaneous pain associated with tissue injury. This activity-dependent plasticity involves neuropeptides, such as dynorphin, substance P and calcitonin gene-related peptide, and excitatory amino acids, such as NMDA, which are chemical mediators involved in nociceptive processing. Unilateral inflammation in the hindpaw of the rat results in an increase in the expression of preprodynorphin and preproenkephalin mRNA in the spinal cord, which parallels the behavioral hyperalgesia associated with the inflammation. Cellular intermediate-early genes, such as c-fos, are also expressed in spinal cord neurons following inflammation and activation of nociceptors. Peripheral inflammation results in an enlargement of the receptive fields of many of these neurons. Dynorphin applied to the spinal cord also induces an enlargement of receptive fields. NMDA antagonists block the hyperexcitability produced by inflammation. A model has been proposed in which dynorphin, substance P and calcitonin gene-related peptide enhance excitability at NMDA receptor sites, leading first to dorsal horn hyperexcitability and then to excessive depolarization and excitotoxicity.

Spinal preprodynorphin mRNA expression in neonatal rats following peripheral inflammation.

Spinal nociceptive neural circuits undergo considerable changes during the postnatal period. This study showed that neonatal rats exhibited earlier upregulation and faster recovery of spinal preprodynorphin (PPD) mRNA than did the adults during complete Freund's adjuvant (CFA)-induced peripheral inflammation. These data suggest that the central nervous systems of neonates and adults respond differently to peripheral noxious inputs, a fact that should be considered when selecting pain treatment strategies for neonate populations.

Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene-related peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats.

This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age-dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP-like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception.

Calcitonin gene-related peptide immunoreactivity in the cat lumbosacral spinal cord and the effects of multiple dorsal rhizotomies.

This study determined the extent of the rostral projection of calcitonin gene-related peptide-like immunoreactive (CGRP-IR) primary afferents in the cat lumbosacral spinal cord. To do this we examined the distribution of CGRP-like immunoreactivity (CGRP-LI) contralateral and ipsilateral to multiple dorsal rhizotomies. In the contralateral dorsal spinal cord, CGRP-IR fibers were mostly observed in Lissauer's tract, the dorsal columns, and laminae I, II, and V. Fewer CGRP-IR fibers were observed in laminae III, IV, and VI and the area around the central canal. The location of the CGRP-LI suggests that the afferents arose from nociceptors. Unilateral dorsal rhizotomies of five consecutive segments in the lumbar enlargement caused a substantial although incomplete loss of CGRP-LI in the rhizotomized dorsal spinal cord ipsilateral to the lesions. The majority of the remaining CGRP-IR fibers were located in Lissauer's tract, the dorsal columns, and the lateral part of laminae I and V. Ventral rhizotomies or an ipsilateral hemisection in the most rostral rhizotomized segment, in addition to the dorsal rhizotomies, had no noticeable effect upon the density or location of the remaining CGRP-LI. These results suggest that the majority of the CGRP-LI within the rhizotomized region of spinal cord was contained within branches of small-diameter primary afferents that entered the spinal cord through intact dorsal roots located caudal to the rhizotomized segments of spinal cord. It is concluded that CGRP-IR small-diameter primary afferents are capable of projecting at least five segments beyond their segment of entry and supplying collaterals to the superficial and deeper layers of the dorsal horn involved in the processing of nociceptive information.

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotoninergic axonal contacts on identified cat spinal dorsal horn neurons and their correlation with nucleus raphe magnus stimulation.

This study examined the distribution of serotoninergic (5-HT) immunoreactive axonal contacts on spinal laminae I and II neurons by combining the intracellular horseradish peroxidase (HRP) method with immunocytochemistry. In addition, the 5-HT distribution was correlated with effects produced by electrical stimulation within the nucleus raphe magnus (NRM). Responses of lamina I neurons and lamina II stalked cells to noxious stimulation were markedly suppressed during NRM stimulation. In contrast, responses of nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells remained unchanged during nucleus raphe magnus stimulation. These inhibitory influences were positively correlated with the distribution of 5-HT immunoreactive contacts on these neurons. Nociceptive lamina I neurons and lamina II stalked cells received a significantly greater number of contacts (average of 74 and 63, respectively) than either nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells (average of 25 and eight contacts, respectively). Irrespective of cell type, most 5-HT contacts occurred on dendritic shafts rather than spines. These data reveal a differential distribution of 5-HT contacts on neurons in spinal laminae I and II, and indicate that at least a portion of the NRM modulation of dorsal horn neuronal activity is serotoninergic and concentrated on the dendritic shafts of nociceptive lamina I neurons and lamina II stalked cells.

Inflammation and hyperalgesia in rats neonatally treated with capsaicin: effects on two classes of nociceptive neurons in the superficial dorsal horn.

To address the mechanisms of hyperalgesia and dorsal horn plasticity following peripheral tissue inflammation, the effects of adjuvant-induced inflammation of the rat hindpaw on behavioral nociception and nociceptive neuronal activity in the superficial dorsal horn were examined in neonatally capsaicin-treated rats 6-8 weeks of age. Capsaicin treatment resulted in an 82% loss of unmyelinated fibers in L5 dorsal roots, a dramatic reduction of substance P-like immunoreactivity in the spinal cord, and a significant decrease in the percentage of dorsal horn nociceptive neurons that responded to C-fiber stimulation and noxious heating of the skin. The thermal nociceptive threshold was significantly increased in capsaicin-treated rats, but behavioral hyperalgesia to thermal stimuli still developed in response to inflammation. Following inflammation, there was a significant decrease in mechanical threshold and an increase in response duration to mechanical stimuli in both vehicle- and capsaicin-treated rats, suggesting that a state of mechanical hyperalgesia was also induced. The capsaicin treatment appears to have differential effects on nociceptive specific (NS) and wide-dynamic-range (WDR) neurons in inflamed rats. Expansion of the receptive fields of nociceptive neurons, a measure of the effect of inflammation-induced CNS plasticity, was less extensive for NS than for WDR neurons in capsaicin-treated rats. Compared to vehicle-treated rats, a smaller population of NS neurons, but a similar percentage of WDR neurons, had background activity in inflamed capsaicin-treated rats. C-fiber strength electrical stimulation of the sciatic nerve produced expansion of the receptive fields in a greater portion of NS neurons (53%, P < 0.05) in capsaicin- than in vehicle-treated rats (32%). There was no difference in stimulation-induced expansion of the receptive fields for WDR neurons between vehicle- or capsaicin-treated rats. An N-methyl-D-aspartate receptor antagonist, MK-801, attenuated the behavioral hyperalgesia and reduced the receptive field size of dorsal horn neurons in inflamed capsaicin- and vehicle-treated rats. The data suggest that while capsaicin-sensitive primary afferents may be involved in neuronal plasticity induced by peripheral tissue inflammation, changes in the capsaicin-insensitive WDR and NS populations are sufficient to produce thermal and mechanical hyperalgesia after the loss of capsaicin-sensitive primary afferents.

Sex differences and phases of the estrous cycle alter the response of spinal cord dynorphin neurons to peripheral inflammation and hyperalgesia.

The neuromodulatory interactions of sex steroids with the opioid system may result in sex differences in pain and analgesia. Dynorphin is an endogenous kappa-opioid peptide that is upregulated in an animal model of peripheral inflammation and hyperalgesia and is possibly regulated by circulating levels of sex steroids. The present study compared behavioral responses of male, cycling female, and gonadectomized Sprague-Dawley rats in a model of persistent pain. Cycling female rats were behaviorally tested over a 14-day period, and their estrous cycles were monitored by daily vaginal smears. Thermal hyperalgesia was measured by paw withdrawal latencies taken prior to and 24-72 h after rats received a unilateral hindpaw injection of complete Freund's adjuvant (CFA). Prior to CFA administration, there was no significant difference in paw withdrawal latencies between male rats, cycling female rats, and ovariectomized female rats. Following CFA administration, female rats in proestrus exhibited significantly increased hyperalgesia compared with male rats, ovariectomized female rats, and female rats in other estrous stages (P

Neuropathic pain from an experimental neuritis of the rat sciatic nerve.

Painful peripheral neuropathies involve both axonal damage and an inflammation of the nerve. The role of the latter by itself was investigated by producing an experimental neuritis in the rat. The sciatic nerves were exposed at mid-thigh level and wrapped loosely in hemostatic oxidized cellulose (Oxycel) that on one side was saturated with an inflammatory stimulus, carrageenan (CARRA) or complete Freund's adjuvant (CFA), and on the other side saturated with saline. In other rats, a myositis was created by implanting Oxycel saturated with CFA into a pocket made in the biceps femoris at a position adjacent to where the nerve was treated. Pain-evoked responses from the plantar hind paws were tested before treatment and daily thereafter. Statistically significant heat- and mechano-hyperalgesia, and mechano- and cold-allodynia were present on the side of the inflamed nerve (CARRA or CFA) for 1-5 days after which responses returned to normal. There were no abnormal pain responses on the side of the saline-treated nerve, and none in the rats with the experimental myositis. The abnormal pain responses were inhibited by N-methyl-D-aspartate receptor blockade with MK-801, but were relatively resistant to the dose of morphine tested (10 mg/kg). Light microscopic examination of CARRA-treated nerves, harvested at the time of peak symptom severity, revealed that the treated region was mildly edematous and that there was an obvious endoneurial infiltration of immune cells (granulocytes and lymphocytes). There was either a complete absence of degeneration, or the degeneration of no more than a few tens of axons. Immunocytochemical staining for CD4 and CD8 T-lymphocyte markers revealed that both cell types were present in the epineurial and endoneurial compartments. The endoneurial T-cells appeared to derive from the endoneurial vasculature, rather than from migration across the nerve sheath. We conclude that a focal inflammation of the sciatic nerve produces neuropathic pain sensations in a distant region (the ipsilateral hind paw) and that this is not due to axonal damage. The neuropathic pain is specific to inflammation of the nerve because it was absent in animals with the experimental myositis and in those receiving sham-treatment. These results suggest that an acute episode of neuritis-evoked neuropathic pain may contribute to the genesis of chronically painful peripheral neuropathies, and that a chronic (or chronically recurrent) focal neuritis might produce neuropathic pain in the absence of significant (or clinically detectable) structural damage to the nerve. The model that we describe is likely to be useful in the study of the neuroimmune factors that contribute to painful peripheral neuropathies.

The effects of a non-competitive NMDA receptor antagonist, MK-801, on behavioral hyperalgesia and dorsal horn neuronal activity in rats with unilateral inflammation.

The involvement of NMDA receptors in rats with peripheral inflammation and hyperalgesia was evaluated by administration of the non-competitive NMDA receptor antagonist, MK-801. Inflammation and hyperalgesia was induced by intradermal injection of complete Freund's adjuvant (CFA) or carrageenan into the left hind paw. The latency of paw withdrawal from a thermal stimulus was used as a measure of hyperalgesia in awake rats. MK-801 (1.6 mg/kg, i.p., or 31.5 micrograms, intrathecal) significantly attenuated thermal hyperalgesia and reduced its duration in comparison to saline-injected rats (P less than 0.05). The receptive field size of nociceptive-specific and wide-dynamic-range neurons in the superficial and deep spinal dorsal horn recorded 24 h after injection of CFA was significantly reduced to 73 +/- 6% (P less than 0.05, n = 8) and 74 +/- 4% (P less than 0.05, n = 8) of control values, respectively, by a cumulative dose of 3 mg/kg of MK-801 (i.v.). MK-801 (2 mg/kg) prevented the expansion of the receptive fields of dorsal horn neurons recorded 5 +/- 0.4 h (n = 5) after intradermal injection of CFA as compared to saline-injected rats (P less than 0.05). MK-801 had no significant effect on receptive field size of dorsal horn neurons in rats without CFA-induced inflammation but blocked a transient expansion of the receptive fields induced by 1 Hz, C-fiber intensity electrical stimulation of the sciatic nerve. The background activity and noxious heat-evoked response of dorsal horn neurons in rats with CFA-induced inflammation were primarily inhibited and noxious pinch-evoked activity was both facilitated and inhibited by the administration of MK-801. These results support the hypothesis that NMDA receptors are involved in the dorsal horn neuronal plasticity and behavioral hyperalgesia that follows peripheral tissue inflammation.

Nitric oxide synthase-containing neurons in sensory ganglia of the rat are susceptible to capsaicin-induced cytotoxicity.

Nitric oxide synthase in lumbar dorsal root ganglia of neonatal rat was studied by reduced nicotinamide adenine dinucleotide phosphate diaphorase and in situ hybridization histochemistry. Induction of nitric oxide synthase in neonatal capsaicin-treated rats after sciatic axotomy was compared with the axotomy-induced nitric oxide synthase increase observed in vehicle-treated littermates. In neonatal capsaicin-treated animals, the number of neurons constitutively labeled by reduced nicotinamide adenine dinucleotide phosphate diaphorase was greatly reduced as compared to vehicle-treated littermates. Nitric oxide synthase messenger RNA was not readily identified constitutively in dorsal root ganglion neurons. Seven days after sciatic transection the induction of reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthase messenger RNA found in the vehicle-treated group was not observed in the capsaicin group. The presence of nitric oxide synthase in dorsal root ganglion neurons thus does not appear to protect against Ca(2+)-mediated capsaicin-induced cytotoxicity. However, since some nitric oxide synthase dorsal root ganglion neurons persist after the capsaicin neurotoxicity, nitric oxide synthase expression must occur in a neurochemically diverse subpopulation of small (< 1000 microns2) neurons. The capsaicin sensitivity of most nitric oxide synthase dorsal root ganglion neurons indicates that they have unmyelinated axons and are likely to be involved in nociception.

Preproenkephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and is co-localized with Fos and Fos-related proteins.

Increased levels of preproenkephalin mRNA in spinal cord neurons induced by peripheral tissue inflammation were examined using in situ hybridization histochemistry. In addition, in situ hybridization histochemistry was combined with immunocytochemistry to determine whether increases in preproenkephalin mRNA were co-localized in spinal cord neurons with increases in immunoreactivity for Fos and Fos-related proteins coded by the immediate-early proto-oncogene, c-fos, and related genes. Dorsal horn laminae I-II, V-VI and VII showed a greater than 200% increase in preproenkephalin mRNA-labeled neurons on the inflamed side as compared to the contralateral control. Inflammation also induced Fos-like immunoreactivity in cell nuclei, mainly in the superficial laminae I-II and the neck of the dorsal horn (laminae V-VI). Few labeled nuclei were detected on the contralateral side. Inflammation resulted in double-labeling of neurons ipsilateral to the inflamed limb whereas they were almost completely absent on the contralateral side. Double-labeled neurons were most frequently found in laminae V-VI. Double-labeled laminae I-II neurons were concentrated in the medial two-thirds of the dorsal horn, the site that receives innervation from the inflamed limb. There were also many double-labeled neurons in laminae VII. Over 90%, 82% and 69% of all neurons expressing preproenkephalin mRNA co-localized Fos immunoreactivity in laminae V-VI, I-II, and VII, respectively. However, the number of neurons expressing increased Fos immunoreactivity was substantially greater than the subpopulation of double-labeled neurons. Our findings indicated that peripheral inflammation induces an increase in preproenkephalin mRNA levels in spinal cord neurons and that most neurons exhibiting preproenkephalin mRNA labeling also co-localized Fos and Fos-related immunoreactivity. These data are consistent with evidence supporting the role of Fos and Fos-related proteins in the regulation of transcription of the preproenkephalin gene in spinal neurons.

Descending modulation of opioid-containing nociceptive neurons in rats with peripheral inflammation and hyperalgesia.

Inflammation and hyperalgesia induce a dramatic up-regulation of opioid messenger RNA and peptide levels in nociceptive neurons of the spinal dorsal horn. Descending axons modulate nociceptive transmission at the spinal level during inflammatory pain, and may play a role in the development of persistent pain. The role of descending bulbospinal pathways in opioid-containing nociceptive neurons was examined. Removal of descending inputs to the spinal cord was performed by complete spinal transection at the midthoracic level. Seven days after spinal transection, rats received a unilateral hindpaw injection of complete Freund's adjuvant, a noxious stimulus that produces inflammation and hyperalgesia. Tissues from the L4 and L5 segments of the spinal cord were removed and analysed by northern blotting and immunocytochemistry. Spinal transection resulted in a further increase in both dynorphin and enkephalin messenger RNA content following complete Freund's adjuvant injection. There was a similar distribution and number of dynorphin-immunoreactive cells in transected rats compared to rats which received sham surgery. These data suggest that increased dynorphin messenger RNA ipsilateral to inflammation, in rats without descending axons, was due to increased expression within the same cells and not to recruitment of additional dynorphin-expressing cells. This reflects a greater dynamic response of nociceptive neurons to noxious stimuli in the absence of descending modulation. Therefore, the net effect of descending afferents on spinal nociceptive circuits may be to reduce the response of opioid-containing neurons to noxious stimulation from the periphery.

Immunocytochemical evidence for a serotoninergic innervation of dorsal column postsynaptic neurons in cat and monkey: light- and electron-microscopic observations.

Dorsal column postsynaptic neurons in the lumbosacral enlargements of cats and a monkey were retrogradely labeled by placing horseradish peroxidase on their severed axons in the thoracic dorsal columns. After visualizing the retrogradely-labeled neurons, the tissue was immunocytochemically stained with an antiserum directed against serotonin. Immunoreactive axonal varicosities contacted the perikarya and proximal dendrites of every retrogradely-labeled neuron examined in cat (mean 61 contacts/cell) and nearly every neuron in the monkey (mean 18 contacts/cell). Electron microscopy showed that the immunoreactive axonal varicosities contained pleomorphic (round to oval) agranular vesicles and formed symmetrical synapses on retrogradely-labeled neurons. It is concluded that dorsal column postsynaptic neurons are innervated directly by the brain stem's descending, serotoninergic system(s).

Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons.

Fos and Fos-related proteins are increased in spinal dorsal horn neurons following noxious stimulation. The laminar location of neurons that exhibit this increase is coincident with those that exhibit an increase in dynorphin in a rat model of peripheral inflammation and hyperalgesia. In order to determine whether the increase in Fos or related proteins and dynorphin occurs in the same dorsal horn neurons, two kinds of double-labeling methods were used: in situ hybridization histochemistry to label dynorphin mRNA autoradiographically, and immunocytochemistry to label Fos and Fos-related proteins, or a double immunocytochemical method that labeled Fos and Fos-related proteins and dynorphin peptide with distinct chromagens. With both methods more than 80% of the neurons in laminae I, II, V and VI exhibiting an increase in either dynorphin mRNA or peptide following peripheral inflammation also colocalized increased nuclear Fos-like immunoreactivity. However, the number of neurons displaying increased Fos-like immunoreactivity was substantially greater than the number of neurons colocalizing increased dynorphin. These data suggest that the activation of nuclear Fos and Fos-related proteins may be related to the induction of dynorphin gene expression in a subpopulation of spinal cord neurons following peripheral inflammation and hyperalgesia.

Descending modulation of Fos expression after persistent peripheral inflammation.

The effects of spinal transection on Fos protein expression following complete Freund's adjuvant-induced hindpaw inflammation and hyperalgesia were studied. Fos-like immunoreactivity (LI) was used as a measure of neuronal activity in dorsal horn nociceptive pathways. Induction of Fos-LI in spinally transected rats with 3 days of hindpaw inflammation was significantly increased in ipsilateral lumbar (L 4,5) spinal cord, when compared with control animals with similar inflammation but an intact spinal cord. The contralateral side of spinally transected rats also showed significant induction of Fos-LI compared to the non-inflamed side of control rats. Since Fos induction provides an indication of the level of neuronal activity in response to a given stimulus, these results suggest that the net effect of brain stem descending pathways is to dampen central hyper-excitability in the spinal cord induced by tissue inflammation and hyperalgesia.

Increased Fos induction in adult rats that experienced neonatal peripheral inflammation.

The response to noxious stimulation was compared in adult rats that had peripheral inflammation as neonates and untreated rats. On postnatal day 1, rat pups experienced complete Freund's adjuvant (CFA)-induced inflammation of the left hind paw. At 8 weeks of age, these rats and neonatal untreated rats received a bilateral injection of CFA into their hind paws. Fos-like immunoreactivity (Fos-LI) was used as a measure of neuronal activity in dorsal horn nociceptive pathways. A significant increase in Fos-LI was found on the left side of the lumbar spinal cord of neonatal treated rats as compared to neonatal untreated rats. These results suggest that the experience of neonatal peripheral inflammation may result in an increase in the response of spinal cord neurons to peripheral inflammation as adults.

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord.

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.