Leukotriene B4 produces hyperalgesia that is dependent on polymorphonuclear leukocytes.

Leukotriene B4, at the same intracutaneous doses as bradykinin, reduced the nociceptive threshold in the rat paw. The mechanism of leukotriene B4-induced hyperalgesia was distinguished from that of the hyperalgesia elicited by prostaglandin E2 and bradykinin by its dependence on polymorphonuclear leukocytes and independence of the cyclooxygenation of arachidonic acid.

GABAergic regulation of noradrenergic spinal projection neurons of the A5 cell group in the rat: an electron microscopic analysis.

Recent studies have demonstrated an important contribution of the A5 noradrenergic cell group of the rostral medulla in the regulation of nociceptive messages at the level of the spinal cord. These noradrenergic controls parallel those arising from the serotonin-containing neurons of the nucleus raphe magnus. In the present study, we used postembedding immunogold staining to identify GABA-immunoreactive terminals that synapse upon identified spinally projecting noradrenergic neurons of the A5 cell group in the rat. A5 projection neurons were identified by Fluoro-Gold transport from the spinal cord; sections containing retrogradely labelled cells were then immunoreacted for tyrosine hydroxylase (TH) to identify the catecholamine-containing, presumed noradrenergic, neurons. Double-labelled A5 cells were intracellularly filled with Lucifer Yellow (LY) and then the LY was photo-oxidized to an electron-dense product. Seven intracellularly filled TH-immunoreactive projection neurons were studied with postembedding immunocytochemistry. Each A5 neuron received a significant GABA-immunoreactive terminal input. Out of a pooled total of 151 terminal profiles found in apposition to intracellularly labelled somatic and dendritic profiles, 31 (20.5%) were GABA-immunoreactive. The proportion of GABA-immunoreactive terminals that contacted somatic profiles (12/72; 17%) was similar to the proportion that contacted TH-labelled dendritic profiles (19/79; 24%). There was a discernible synaptic specialization in about 50% of the labelled terminals that contacted the TH projection neuron. Both symmetric and asymmetric synaptic specializations were found. Labelled terminals contained round or pleiomorphic vesicles, but not flat vesicles; many also contained dense-core vesicles. Our results indicate that noradrenergic neurons of the A5 cell group, which contribute to both antinociceptive and cardiovascular controls through their projection to the spinal cord, are regulated by local GABAergic, presumably inhibitory, mechanisms. Whether the initiation of A5 neuron activity results from a lifting of tonic GABAergic inhibitory control, as has been proposed for the neurons of the nucleus raphe magnus, remains to be determined.

Leukotriene and prostaglandin sensitization of cutaneous high-threshold C- and A-delta mechanonociceptors in the hairy skin of rat hindlimbs.

Single C- and A-delta fibers were isolated from dissected filaments of the saphenous nerve in pentobarbital anesthetized rats and the corresponding cutaneous receptive fields mapped with calibrated von Frey hairs. Nociceptors were characterized by their responses to noxious mechanical, thermal and chemical stimuli, including intradermal injections of leukotriene B4, prostaglandin E2, bradykinin and capsaicin. Leukotriene B4 decreased the mean mechanical threshold by a maximum of 80% within 10 min and for more than 3 h after intradermal injection of 75 ng of leukotriene B4. The degrees of sensitization of a fiber by leukotriene B4 and prostaglandin E2 were highly correlated. A potentiation effect also was observed, in that injection of prostaglandin E2 or leukotriene B4 1 h after the other eicosanoid further lowered the mechanical threshold of a sensitized fiber, whereas fibers that were not sensitized by leukotriene B4 were unaffected by prostaglandin E2. The sensitizing action of leukotriene B4 and prostaglandin E2 was directed to multiple classes of cutaneous nociceptors including 73% of C-polymodal, 60% of C-mechano-heat, 42% of C-mechano-cold nociceptors and 70% of A-delta high-threshold mechanonociceptors. The pain-evoking substances bradykinin and capsaicin activated 81% and 88%, respectively, of the sensitized C-polymodal nociceptors, 17% and 84% of the sensitized-C-mechano-heat nociceptors, 12% and 37% of the sensitized C-mechano-cold nociceptors, and 17% and none of the sensitized A-delta high-threshold mechanociceptors. The responses of C-fibers to bradykinin and capsaicin were highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization of tyrosine hydroxylase- and serotonin-immunoreactive brainstem neurons with axon collaterals to the periaqueductal gray and the spinal cord in the rat.

Retrograde tracing and immunocytochemistry were used to examine the axon collateralization of brainstem serotonin (5-HT) and norepinephrine (NE) cells to the periaqueductal gray (PAG) and spinal cord. Tyrosine hydroxylase (TH)-immunofluorescent neurons which collateralize to the PAG and the cervical spinal cord were found in all brainstem catecholamine cell groups previously shown to contain neurons which project to the spinal cord, including the A5 and A7 cell groups, locus coeruleus, subcoeruleus and the C1 cell group. Many TH-immunofluorescent cells which project to the PAG but not to the spinal cord were also found. The region of the nucleus raphe magnus (NRM) also contained many neurons retrogradely labeled from the PAG. These overlapped with the distribution of spinally projecting 5-HT-immunofluorescent cells in the NRM, however, less than 1% of the PAG projecting cells in this region were 5-HT-immunofluorescent. In contrast, many 5-HT-immunofluorescent cells in the more rostral nucleus raphe pontis and nucleus raphe dorsalis were retrogradely labeled from the PAG but not from the spinal cord. Finally, a population of neurons in the NRM and adjacent reticular formation and in the region of several pontomedullary catecholamine cell groups collateralized to the PAG and spinal cord, but were neither 5-HT nor TH-immunofluorescent. Taken together, these findings raise the possibility that the noradrenergic contribution to the spinal antinociceptive effects produced by PAG electrical stimulation results, in part, from antidromic activation of brainstem noradrenergic neurons that have axon collaterals projecting to the PAG and spinal cord. In contrast, the 5-HT contribution to the spinal antinociceptive effects produced by PAG electrical stimulation is more likely to derive, as previously proposed, from orthodromic activation of raphe-spinal serotonergic axons.

Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 membrane.

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation produced and opioid induced analgesia. In the present study the opioid binding characteristics of the PAG were examined with an in vitro radioligand binding technique. [3H]Ethylketocyclazocine (EKC), 2 nM, was used as a tracer ligand to nonselectively label mu, delta, and kappa binding sites in PAG enriched P2 membrane. The mu selective ligand [D-Ala2,N-methylPhe4,Glyol5]enkephalin (DAGO) competed with [3H]EKC for more than one population of binding sites with both high and low affinity. In contrast the delta selective ligand [D-Pen2,D-Pen5]enkephalin (DPDPE) and the kappa selective ligand trans-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, methane sulfonate, hydrate (U50,488H) each competed with [3H]EKC for a single population of binding sites with low affinity. DPDPE and U50,488H also competed with 2 nM [3H]DAGO for a single population of binding sites with similar low affinity. DAGO and not DPDPE competed with 2 nM [3H][D-Ala2,D-Leu5]enkephalin (DADLE) with high affinity. 2 nM [3H]DPDPE did not substantially label PAG enriched P2 membrane, and 1 nM DAGO competed with all specific [3H]DPDPE binding which was observed. These binding data are consistent with the presence of a single population of mu selective high affinity binding sites in PAG enriched P2 membrane to which delta ligands and kappa ligands have low affinity.

Erythrocyte nuclei resemble dying neurons in embryonic dorsal root ganglia.

Cell death or apoptosis is regarded as an important feature of mammalian neural development, but the evidence for this generalization depends on the assumption that cell death can be clearly recognized. The usual profile of a dying neuron is a deeply stained pyknotic homogeneous sphere. In this paper we present evidence that such profiles in embryonic rat T6 and L4 dorsal root ganglia are not dying neurons but rather nuclei of immature red blood cells. This observation, combined with recent work showing that the methods previously used for counting normal or dying neurons are biased, indicates that the classic work establishing the importance of apoptosis needs to be repeated.

The origin of brainstem noradrenergic and serotonergic projections to the spinal cord dorsal horn in the rat.

Although it has been proposed that the locus coeruleus is the predominant, if not exclusive, brainstem origin of the noradrenergic innervation of the spinal dorsal horn, pharmacological studies argue otherwise. In this study we made localized injections of the retrograde tracer wheatgerm agglutinin conjugated to apo-horseradish peroxidase gold (WGA:apoHRP-Au), in conjunction with immunocytochemical labeling for tyrosine hydroxylase (TH) or serotonin (5-HT), to identify the brainstem source of the noradrenaline (NA) and 5-HT innervation of the dorsal horn of the rat. Our studies were concentrated in the C5 spinal segment. The pattern of labeling was only studied in animals in which the tracer injection was restricted to the dorsal horn. In these rats, TH-immunoreactive neurons in widespread regions of the brainstem, including the locus coeruleus, subcoeruleus, A5, and A7 cell groups, were found to project to the dorsal horn. In terms of absolute numbers of double-labeled cells, no one noradrenergic cell group predominated. As expected, dorsal-horn-projecting 5-HT-immunoreactive neurons were found within the 5-HT populations of the rostroventromedial medulla and caudal pons, including the nucleus raphe magnus, nucleus paragigantocellularis (PGi), and ventral portions of the nucleus gigantocellularis (Gi). The majority of retrogradely labeled 5-HT-immunoreactive cells were, however, located off the midline, in the ipsilateral PGi and ventral Gi. Finally, a large number of retrogradely labeled, non-5-HT cells were found intermingled among the 5-HT cells of this region. Our results provide evidence that the noradrenergic regulation of nociceptive transmission at the spinal cord level arises from direct spinal projections of several brainstem noradrenergic cell groups.

Ventral spinal cord inhibition of neurite outgrowth from embryonic rat dorsal root ganglia.

Organotypic culture of embryonic rat lumbar spinal cord and dorsal root ganglia has been used to demonstrate an inhibitory effect of ventral spinal cord on neurite growth from dorsal root ganglion explants. When dorsal root ganglion explants from 14-15 day old embryos were cultured alone or in close proximity to a dorsal cord explant, the pattern of dorsal root ganglion neurite outgrowth was typically radial. However, when E14-15 dorsal root ganglion explants were cocultured for 22-24 hours in proximity to a ventral spinal cord explant from the same embryo, few, if any, dorsal root ganglion neurites grew in the direction of the ventral cord explant. This inhibitory effect appeared to be developmentally regulated; it was diminished or absent in cocultures prepared from 18 day old embryos. In contrast, in cocultures of dorsal cord and ventral cord explants from E14-15 embryos, dorsal cord neurites grew abundantly toward the ventral cord explant suggesting that the inhibition is not likely to be due to a nonspecific neurotoxic effect and that the activity responsible selectively inhibits dorsal root ganglion neurite outgrowth. We conclude that a diffusible, primary afferent inhibitory factor(s) produced by embryonic ventral horn may be responsible for the inhibition. Our results are discussed with respect to the possible involvement of inhibition in the normal development of primary afferent innervation of the spinal cord.