Serotoninergic medullary raphespinal projection to the lumbar spinal cord in the rat: a retrograde immunohistochemical study.

Classically the raphespinal system has been regarded as a serotoninergic system; inhibition of spinal nociceptive transmission produced by stimulation of the medullary raphe nuclei is mediated partially by spinal serotoninergic receptors. However, recent evidence suggests that the raphe nuclei are not homogeneous populations of serotoninergic cells. The objective of the present study was to re-examine, in the rat, the serotoninergic raphespinal projection to the lumbar spinal cord, and to determine the relative contribution of serotoninergic raphespinal neurons to the total population of raphespinal neurons. Microinjections of wheat-germ agglutinin horseradish peroxidase conjugate coupled to colloidal gold into the lumbar spinal cord resulted in the retrograde labeling of 53% and 59% of the serotoninergic neurons in the raphe nuclei and in the para-raphe zone, respectively. Conversely, 47% and 28% of the retrogradely labeled neurons in the raphe and para-raphe zone, respectively, demonstrated serotonin-like immunoreactivity. Thus, contrary to previous reports, the present results suggest 1) that only about half of the serotoninergic neurons in the raphe nuclei and in the surrounding para-raphe zone project to the lumbar spinal cord, and 2) that a large proportion of the neurons in the raphe nuclei (53%) and in the surrounding para-raphe zone (72%) that project to the lumbar spinal cord are not serotoninergic.     

Origins and terminations of bulbospinal axons that contain serotonin and either enkephalin or substance-P in the North American opossum.

We have shown previously that some enkephalin, substance-P, and serotoninergic neurons in the medullary raphe and adjacent reticular formation project to the spinal cord in the opossum. In the present study we have combined the retrograde transport of True Blue and immunofluorescence histochemistry to determine whether methionine enkephalin or substance-P containing bulbospinal neurons are serotoninergic. Furthermore, we have used the same immunofluorescence protocol to determine whether spinal axons contain the same substances. Neurons that immunostained for both enkephalin and serotonin were observed in many brainstem nuclei. However, those that projected to the spinal cord were limited to the nuclei raphe magnus and obscurus, and the ventral part of nucleus reticularis gigantocellularis, pars ventralis. Neurons that immunostained for both substance P and serotonin were fewer in number, but some of the ones in the above nuclei and within the nucleus raphe pallidus, projected to the spinal cord. Spinal axons exhibiting both enkephalin- and serotonin-like immunoreactivity were observed in the superficial laminae of the dorsal horn, lamina X, and the intermediolateral cell column, whereas those showing both substance-P and serotonin-like immunoreactivity were seen primarily in lamina X, the intermediolateral cell column, and the ventral horn. Some of the axons in the ventral horn were in close apposition to presumed motoneurons. Comparison of the above results with those obtained from previous studies of bulbospinal projections has allowed us to infer the origins of axons that innervate different spinal targets.     

Substance P and neurokinin A, two coexisting tachykinins stimulating the release of [3H]5-HT from rat cerebral cortical slices.

Noxious heat applied to the footpad evokes the expression offos protein-like immunoreactivity in the rat spinal cord lumbar dorsal horn. Electrical stimulation in the medullary nucleus raphe magnus (NRM) while not evokingfos-like immunoreactivity itself, reduced significantly the number of neurons (to 50% of control) in the dorsal horn demonstratingfos protein-like immunoreactivity in response to noxious heating of the footpad. Thus descending projections from the medullary NRM appear to have direct inhibitory effects on dorsal horn neurons that receive nociceptive input from cutaneous thermal nociceptors.     

A thyrotropin-releasing hormone-containing system in the rat dorsal horn separate from serotonin

In this study, we report the identification of a thyrotropin-releasing hormone (TRH)-containing system in the dorsal horn of the rat spinal cord. This system is distinct from the TRH and serotonin (5-hydroxytryptamine, 5-HT) cotransmitter supraspinal system that has projections to the intermediolateral (IML) and ventral columns. Spinal cord sections from untreated rats, and those treated with colchicine or 5,7-dihydroxytryptamine (5,7-DHT) were processed using peroxidase-antiperoxidase (PAP) immunocytochemistry with nickel intensification. Results of the 5,7-DHT treatment were verified by quantifying TRH and 5-HT by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC), respectively. Prominent immunocytochemical staining for TRH in the dorsal horn was seen in varicose fibers mainly in lamina II and superficial lamina III of the dorsal horn of the spinal cord of control rats. A few fibers were seen ascending into lamina I. A moderate number of fibers that were immunoreactive for 5-HT were primarily in laminae I and II. The distribution of TRH- and 5-HT-containing neurites in the IML and the ventral horn agreed with previously published reports. Rats treated with colchicine showed many small round TRH immunoreactive cells that were limited to laminae II/III of the dorsal horn. TRH immunoreactivity in the dorsal horn and IML was resistant to the effects of the selective serotonin neurotoxin, 5,7-DHT, while the ventral horn was depleted of TRH staining. Serotonin was almost completely eliminated in all spinal cord laminae. Quantitative biochemical studies showed significant, but non-parallel reductions of TRH and 5-HT in cervical, thoracic and lumbar spinal cord. These studies demonstrate the existence of TRH-containing cell bodies and terminals in the dorsal horn of the rat spinal cord. These findings provide evidence that a TRH-containing system exists in the dorsal horn of the rat and that it is distinct from the descending medullary raphe system that contains 5-HT; suggest that a population of TRH-containing fibers that project to the IML may not contain 5-HT; and confirm previously published results that 5-HT and TRH coexist in terminals in the ventral horn of the spinal cord.     

Thyrotropin-releasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata with special emphasis on the bulbospinal tract.

The distribution of thyrotropin-releasing hormone (TRH)-like immunoreactivity (LI) has been studied in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata by the use of indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. Furthermore, double-labeling experiments were performed in order to study colocalization of 5-hydroxytryptamine (5-HT)- and substance P-LI. A dense innervation of TRH-immunoreactive (IR) varicose fibers was found in the ventral horn motor nuclei, in the region surrounding the central canal, in the intermediolateral cell column, and in the dorsal horn laminae II and III. In addition, cell bodies harboring TRH-LI were found in the dorsal horn laminae II-IV. In the ventral horn, many of the large cell bodies and their proximal dendrites were totally encapsulated by TRH-IR fibers. From double-labeled sections a high degree of coexistence could be established between TRH-/5-HT-LI, TRH-/substance P-LI, and 5-HT-/substance P-LI in fibers in the motor nuclei; as a consequence, a large proportion of these fibers should harbor TRH-/5-HT-/substance P-LI. A coexistence between TRH-/5-HT-LI could also be demonstrated in the intermediolateral cell column. However, no unequivocal coexistence could be found between TRH-/substance P-LI and 5-HT-/substance P-LI in this region. In the dorsal horn, no clear coexistence could be encountered for any of the above indicated combinations. Electron microscopic analysis of material from the lumbar lateral motor nucleus demonstrated TRH-IR terminals making synapses with large cell bodies and dendrites. In addition, contacts lacking synaptic specializations could also be verified. In the medulla oblongata, with the use of the PAP technique, a large number of cell bodies containing TRH-LI were encountered in the midline raphe nuclei and in nucleus reticularis lateralis. A similar distribution pattern could be found for 5-HT-LI, but no cell bodies containing substance P-LI could be seen in these regions. Chemical analysis of specimens from cervical, thoracic, and lumbar spinal cord revealed higher concentrations of TRH- and 5-HT-LI in the ventral quadrants, whereas substance P-LI dominated in the dorsal quadrants. Thus, the concentrations of TRH-, 5-HT-, and substance P-LI was in accordance with the observed regional variation in density of IR-fibers and varicosities found in the spinal cord. We have shown that TRH-LI has a distribution in the monkey spinal cord and medulla oblongata similar to that previously demonstrated in other species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain

Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrtrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide. © Wiley-Liss, Inc.     

Evidence for a projection from the B9 serotonergic cell group to the median raphe nucleus

Following injections of several fluorescent retrograde tracers into the median raphe nucleus large numbers of retrogradely labeled cells were observed in the ventral mesencephalic tegmentum. Subsequent immunocytochemical processing for serotonin-like immunoreactivity suggested that a large component of this projection originates from serotonergic B9 cells. Although tracer injections into the dorsal raphe did not result in similar labeling of B9 cells, evidence was obtained suggesting the existence of a small serotonergic projection from the median to the dorsal raphe.     

Calcitonin gene-related peptide in monkey spinal cord and medulla oblongata.

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers and cell bodies was studied in the spinal cord and the medulla oblongata of the grey monkey (Macaca fascicularis) using peroxidase-antiperoxidase (PAP) immunohistochemistry. At all levels of the spinal cord many CGRP-IR motoneurons and fibers were seen in the motor nuclei. In the medulla, CGRP-IR cell bodies were encountered in nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus, nucleus reticularis lateralis as well as in the area dorsal to the inferior olive. Bulbar motoneurons were much more intensely stained than spinal cord motoneurons, indicating higher levels of CGRP-like immunoreactivity (LI) at the medullary level. The concentration of CGRP-LI measured by radioimmunoassay showed higher levels in the dorsal quadrants as compared to the ventral quadrants, but the dorsal/ventral ratio was lower than has previously been reported from the rat. The present results demonstrate that using the PAP technique CGRP-LI can be visualized in a larger number of spinal cord motoneurons of the monkey than earlier revealed by immunofluorescence. Moreover, the finding supports the view that the CGRP-IR nerve endings in the spinal motor nuclei originate from cell bodies in the medullary raphe nuclei.     

Some anatomical observations on the projections from the hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the cat

The hypothalamus is closely involved in a wide variety of behavioral, autonomic, visceral, and endocrine functions. To find out which descending pathways are involved in these functions, we investigated them by horseradish peroxidase (HRP) and autoradiographic tracing techniques. HRP injections at various levels of the spinal cord resulted in a nearly uniform distribution of HRP-labeled neurons in most areas of the hypothalamus except for the anterior part. After HRP injections in the raphe magnus (NRM) and adjoining tegmentum the distribution of labeled neurons was again uniform, but many were found in the anterior hypothalamus as well. Injections of 3H-leucine in the hypothalamus demonstrated that: The anterior hypothalamic area sent many fibers through the medial forebrain bundle (MFB) to terminate in the ventral tegmental area of Tsai (VTA), the rostral raphe nuclei, the nucleus Edinger-Westphal, the dorsal part of the substantia nigra, the periaqueductal gray (PAG), and the interpeduncular nuclei. Further caudally a lateral fiber stream (mainly derived from the lateral parts of the anterior hypothalamic area) distributed fibers to the parabrachial nuclei, nucleus subcoeruleus, locus coeruleus, the micturition-coordinating region, the caudal brainstem lateral tegmentum, and the solitary and dorsal vagal nucleus. Furthermore, a medial fiber stream (mainly derived from the medial parts of the anterior hypothalamic area) distributed fibers to the superior central and dorsal raphe nucleus and to the NRM, nucleus raphe pallidus (NRP), and adjoining tegmentum. The medial and posterior hypothalamic area including the paraventricular hypothalamic nucleus (PVN) sent fibers to approximately the same mesencephalic structures as the anterior hypothalamic area. Further caudally two different fiber bundles were observed. A medial stream distributed labeled fibers to the NRM, rostral NRP, the upper thoracic intermediolateral cell group, and spinal lamina X. A second and well-defined fiber stream, probably derived from the PVN, distributed many fibers to specific parts of the lateral tegmental field, to the solitary and dorsal vagal nuclei, and, in the spinal cord, to lamina I and X, to the thoracolumbar and sacral intermediolateral cell column, and to the nucleus of Onuf. The lateral hypothalamic area sent many labeled fibers to the lateral part of the brainstem and many terminated in the caudal brainstem lateral tegmentum, including the parabrachial nuclei, locus coeruleus, nucleus subcoeruleus, and the solitary and dorsal vagal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

The difference of osteocalcin-immunoreactive neurons in the rat dorsal root and trigeminal ganglia: co-expression with nociceptive transducers and central projection

The co-expression of osteocalcin (OC) with the capsaicin receptor (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the dorsal root (DRG) and trigeminal ganglia (TG). Virtually all OC-immunoreactive (ir) DRG neurons were devoid of VR1- and VRL-1-immunoreactivity (ir). In the TG, 14.1% of OC-ir neurons were also immunoreactive for VR1. Only 1.7% of OC-ir TG neurons co-expressed VRL-1-ir. The distribution of OC-ir was also examined in the spinal cord and trigeminal sensory nuclei. In the spinal cord, the superficial laminae of the dorsal horn were devoid of OC-ir. The neuropil was weakly stained in other regions of the spinal horns. The medullary dorsal horn (MDH) contained numerous OC-ir varicose fibers in laminae I and II. These fibers were occasionally observed originating from the spinal trigeminal tract. The neuropil was weakly stained in deeper laminae of the MDH, and the rostral parts of the trigeminal sensory nuclei. The present study suggests that OC-ir TG nociceptors send their unmyelinated axons to the superficial laminae of the MDH.     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.     

Serotonergic projections to the caudal brain stem: a double label study using horseradish peroxidase and serotonin immunocytochemistry

Cells of origin of serotonergic and non-serotonergic projections to the caudal brain stem in the primate were examined using a double label technique. Following HRP injections into medullary raphe nuclei and the adjacent reticular formation double labeled cells were found in the dorsal raphe nucleus, the central superior nucleus and the ventrolateral tegmentum. Retrogradely labeled cells that did not stain for serotonin-like immunoreactivity were found primarily in the periaqueductal gray (PAG) and the mesencephalic and pontine reticular formation. The results are discussed in relation to the descending pathway(s) mediating the effects of PAG stimulation.     

Immunohistochemical localisation of the voltage gated potassium ion channel subunit Kv3.3 in the rat medulla oblongata and thoracic spinal cord

Voltage gated K+ channels (Kv) are a diverse group of channels important in determining neuronal excitability. The Kv superfamily is divided into 12 subfamilies (Kv1-12) and members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique expression pattern. Since the localisation of Kv subunits is important in defining the roles they play in neuronal function, we have used immunohistochemistry to determine the distribution of the Kv3.3 subunit in the medulla oblongata and spinal cord of rats. Kv3.3 subunit immunoreactivity (Kv3.3-IR) was widespread but present only in specific cell populations where it could be detected in somata, dendrites and synaptic terminals. Labelled neurones were observed in the spinal cord in laminae IV and V, in the region of the central canal and in the ventral horn. In the medulla oblongata, labelled cell bodies were numerous in the spinal trigeminal, cuneate and gracilis nuclei whilst rarer in the lateral reticular nucleus, hypoglossal nucleus and raphe nucleus. Regions containing autonomic efferent neurones were predominantly devoid of labelling with only occasional labelled neurones being observed. Dual immunohistochemistry revealed that some Kv3.3-IR neurones in the ventral medullary reticular nucleus, spinal trigeminal nucleus, dorsal horn, ventral horn and central canal region were also immunoreactive for the Kv3.1b subunit. The presence of Kv3.3 subunits in terminals was confirmed by co-localisation of Kv3.3-IR with the synaptic vesicle protein SV2, the vesicular glutamate transporter VGluT2 and the glycine transporter GlyT2. Co-localisation of Kv3.3-IR was not observed with VGluT1, tyrosine hydroxylase, serotonin or choline acetyl transferase. Electron microscopy confirmed the presence of Kv3.3-IR in terminals and somatic membranes in ventral horn neurones, but not motoneurones. This study provides evidence supporting a role for Kv3.3 subunits in regulating neuronal excitability and in the modulation of excitatory and inhibitory synaptic transmission in the medulla oblongata and spinal cord.     

Immunohistochemical detection of calcium/calmodulin-dependent protein kinase II in the spinal cord of the rat and monkey with special reference to the corticospinal tract

Calcium/ calmodulin-dependent protein kinase II is a prominent enzyme in the mammalian brain that phosphorylates a variety of substrate proteins. In the present study, monoclonal antibodies that specifically recognize either the α or the β isoforms of this enzyme were used to determine the distribution of these isoforms within the rat and monkey spinal cord. In the rat, the corticospinal tract consists of two components: the dorsal corticospinal tract, which occupies the ventralmost aspect of the dorsal funiculus; and the ventral corticospinal tract, which occupies an area adjacent to the ventral median fissure. Both dorsal and ventral corticospinal tract fibers were strongly immunopositive for the α-antibody. Unilateral ablation of the sensorimotor cortex of the rat eliminated the α-immunoreactive staining in the contralateral dorsal corticospinal tract. The neuropil in the superficial laminae of the dorsal horn (Rexed's laminae I and II) was densely stained with the α-antibody, whereas the neuropil in laminae IV-X was immunonegative. Dense α-immunopositive neurons were also distributed in the head of the dorsal horn (laminae I-IV). In contrast to the strong α-immunoreactivity seen in the dorsal corticospinal tract fibers, only very weak β-immunoreactivity was observed in this tract. Moderate β-immunoreactive products were distributed homogenously throughout the neuropil of the gray matter, although the neuropil of the superficial laminae of the dorsal horn (laminae I and II) was stained more strongly than the other regions of the gray matter (laminae III-X). Neuronal components in all laminae were immunopositive for the β-antibody. Thus, motoneurons in the ventral horn, which were immunonegative for the α-antibody, were immunopositive for the β-antibody. This selective distribution pattern of immunoreactivity of α- and β-antibodies in the rat was also present in the monkey spinal cord, although the α-immunopositive corticospinal tract fibers in the monkey descended in the lateral funiculus as the lateral corticospinal tract instead of passing through the dorsal funiculus, as is the case in the rat. The differential distribution of immunoreactivity in the spinal cord suggests that these two isoforms of calcium/ calmodulin-dependent protein kinase II may have different functional roles in the spinal cord. © Wiley-Liss, Inc.     

Serotonin- and substance P-containing projections to the nucleus tractus solitarii of the rat

The objective of the present study was to determine the location of the neurons that give rise to serotonin- and substance P-containing terminals in the nucleus tractus solitarii. This was done by injecting rhodamine-filled latex microspheres into the nucleus tractus solitarii of rats to retrogradely label neuronal cell bodies and by processing sections from the brains of these animals to determine whether the labelled neurons contained serotonin or substance P immunoreactivity. Serotonin-immunoreactive neurons that projected to the nucleus tractus solitarii were found in the nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, and in the ventral medulla, lateral to the pyramidal tract. Substance P-immunoreactive neurons that projected to the nucleus tractus solitarii were found in similar areas but were proportionately less numerous in the nucleus raphe magnus and proportionately more numerous in the nucleus raphe pallidus. It is concluded that neurons in the medullary raphe nuclei, some of which presumably utilize serotonin or substance P as a neurotransmitter, could regulate autonomic function via direct projections to the nucleus tractus solitarii.     

Collateral projection of substance P receptor expressing neurons in the medullary dorsal horn to bilateral parabrachial nuclei of the rat

Employing a combination of fluorescent retrograde double labeling and immunofluorescence histochemistry for substance P receptor (SPR), we examined the collateral projection from single SPR-like immunoreactive neurons in the medullary dorsal horn (caudal subnucleus of the spinal trigeminal nucleus) to bilateral parabrachial nuclei in the rat. After injection of fast blue (FB) or diamidino yellow (DY) into the right or left parabrachial nucleus, respectively, single-labeled FB or DY neurons and double-labeled FB/DY neurons were observed mainly bilaterally in laminae I and II of the medullary dorsal horn. Some of the single-labeled FB or DY and double-labeled FB/DY neurons showed SPR-like immunoreactivity, especially in lamina I. In lamina I of left medullary dorsal horn which is ipsilateral to the DY injection into the PBN, the percentages of double-labeled FB/DY neurons to the total number of FB- or DY-labeled neurons were 34.0% or 20.2%, triple-labeled FB/DY/SPR neurons to the total number of FB/DY double-labeled or SPR-like immunoreactive neurons were 22.0% or 2.4%, respectively. In lamina I of the right medullary dorsal horn which is ipsilateral to the FB injection into the PBN, the percentages of double-labeled FB/DY neurons to the total number of FB- or DY-labeled neurons were 12.9% or 59.3%, triple-labeled FB/DY/SPR neurons to the total number of double-labeled FB/DY or SPR-like immunoreactive neurons were 24. 6% or 3.9%, respectively. The results suggest that some of the single SPR expressing neurons in the medullary dorsal horn might be innervated by substance P containing primary afferent fibers and transmit sensory information diffusely to bilateral parabrachial nuclei by way of their axonal collaterals.     

Distribution of substance P-like Immunoreactivity in the chameleon brain

The distribution of substance P-like immunoreactivity in the chameleon brain and spinal cord was studied with immunohistochemical methods using polyclonal antibodies against substance P. In the telencephalon, immunoreactive cell bodies and fibers were located primarily in the striatum and in the globus pallidus. In addition, few substance P-like fibers were observed in the cortical areas, in the septum, and in the amygdala. In the diencephalon, a high density of immunostained neurons and fibers were seen in the periventricular and ventrolateral hypothalamus. Another group of cell bodies was located in the optic tectum and particularly in the stratum griseum central. A large number of immunoreactive fibers were also detected in the thalamic nuclei and in the median eminence. In the mesencephalon, few immunoreactive neurons were observed in the ventral tegmental area, in the substantia nigra, and in the nucleus reticularis isthmi. These latter nuclei, the periventricular area, the posterior commissure, the nucleus lentiformis mesencephali, the oculomotor nucleus, and the raphe nuclei contained a dense plexus of substance P immunoreactive fibers. No immunoreactive cell bodies were observed in raphe nuclei. In the spinal cord, no substance P-like immunoreactive neurons were observed, but a large number of substance P immunostained fibers were seen in the dorsal and lateral part of the dorsal horn and surrounding the dorsal parts of the central canal. The results of the present study are discussed with respect to those obtained in other species of reptiles, the main differences concerning the lateral septum, the habenula, the area of the paraventricular organ, and the raphe nuclei.     

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.     

Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord.

Previous experiments have shown that noxious stimulation increases expression of the c-fos proto-oncogene in subpopulations of spinal cord neurons. c-fos expression was assessed by immunostaining for Fos, the nuclear phosphoprotein product of the c-fos gene. In this study, we examined the effect of systemic morphine on Fos-like immunoreactivity (FLI) evoked in the formalin test, a widely used model of persistent pain. Awake rats received a subcutaneous 150 microliters injection of 5% formalin into the plantar aspect of the right hindpaw. The pattern of nuclear FLI was consistent with the known nociceptive primary afferent input from the hindpaw. Dense labeling was recorded in the superficial dorsal horn (laminae I and IIo) and in the neck of the dorsal horn (laminae V and VI), areas that contain large populations of nociceptive neurons. Sparse labeling was noted in lamina IIi and in the nucleus proprius (laminae III and IV), generally considered to be nonnociceptive areas of the cord. Fos immunoreactivity was also evoked in the ventromedial gray, including laminae VII, VIII, and X. There was no labeling in lamina IX of the ventral horn. Since FLI was time dependent and distributed over several spinal segments, we focused our analysis where maximal staining was found (L3-L5) and at the earliest time point of the peak Fos immunoreactivity (2 hr). Twenty minutes prior to the formalin injection, the rats received morphine (1.0, 2.5, 5.0, or 10 mg/kg, s.c.) or saline vehicle. Two hours later, the rats were killed, their spinal cords removed, and 50 microns transverse sections of the lumbar enlargement were immunostained with a rabbit polyclonal antiserum directed against Fos. Prior treatment with morphine sulfate profoundly suppressed formalin-evoked FLI in a dose-dependent and naloxone-reversible manner. The dose-response relationship of morphine-induced suppression of FLI varied in different laminae. To quantify the effect of morphine on FLI, labeled neurons in sections taken from the L4/5 level of each rat were plotted with a camera lucida and counted. Staining in the neck of the dorsal horn (laminae V and VI) and in more ventral laminae VII, VIII, and X, was profoundly suppressed by doses of morphine which also suppress formalin-evoked behavior. Although the labeling was also significantly reduced in laminae I and II, at the highest doses of morphine there was substantial residual labeling in the superficial dorsal horn. These data indicate that analgesia from systemic opiates involves differential regulation of nociceptive processing in subpopulations of spinal nociceptive neurons.     

Ascending dopaminergic projections from the dorsal raphe nucleus in the rat

The projections of putative dopamine containing cells within the dorsal raphe nucleus (DR) were studied using a combination of tyrosine hydroxylase (TH) immunocytochemistry and fluorescent retrograde tracing. Substantial numbers of TH-immunoreactive cells in the DR were found to project to the nucleus accumbens. Progressively smaller numbers of cells were found to project to the lateral septum and medial prefrontal cortex. Very few TH-immunoreactive cells projected to the dorsal striatum, and none to the substantia nigra. TH-immunoreactive cells did not display serotonin-like immunoreactivity. These findings indicate that the projection pattern of TH-immunoreactive cells within the dorsal raphe more closely resembles that of dopaminergic cells within the ventral tegmental area (VTA) than that of serotonergic cells within the DR.