Immunohistochemical distribution of enkephalin, substance P, and somatostatin in the brainstem of the leopard frog, Rana pipiens.

The brainstems of frogs contain many of the neurochemicals that are found in mammals. However, the clustering of nuclei near the ventricles makes it difficult to distinguish individual cell groups. We addressed this problem by combining immunohistochemistry with tract tracing and an analysis of cell morphology to localize neuropeptides within the brainstem of Rana pipiens. We injected a retrograde tracer, Fluoro-Gold, into the spinal cord, and, in the same frog, processed adjacent sections for immunohistochemical location of antibodies to the neuropeptides enkephalin (ENK), substance P (SP), and somatostatin (SOM). SOM+ cells were more widespread than cells containing immunoreactivity (ir) to the other substances. Most reticular nuclei in frog brainstem contained ir to at least one of these chemicals. Cells with SOM ir were found in nucleus (n.) reticularis pontis oralis, n. reticularis magnocellularis, n. reticularis paragigantocellularis, n. reticularis dorsalis, the optic tectum, n. interpeduncularis, and n. solitarius. ENK-containing cell bodies were found in n. reticularis pontis oralis, n. reticularis dorsalis, the nucleus of the solitary tract, and the tectum. The midbrain contained most of the SP+ cells. Six nonreticular nuclei (griseum centrale rhombencephali, n. isthmi, n. profundus mesencephali, n. interpeduncularis, torus semicircularis laminaris, and the tectum) contained ir to one or more of the substances but did not project to the spinal cord. The descending tract of V, and the rubrospinal, reticulospinal, and solitary tracts contained all three peptides as did the n. profundus mesencephali, n. isthmi, and specific tectal layers. Because the distribution of neurochemicals within the frog brainstem is similar to that of amniotes, our results emphasize the large amount of conservation of structure, biochemistry, and possibly function that has occurred in the brainstem, and especially in the phylogenetically old reticular formation.

Effect of aging on the substance P receptor, NK-1, in the spinal cord of rats with peripheral nerve injury.

Substance P (SP) levels in the spinal cords of very old rats are less than the levels in younger rats (Bergman et al., 1996). After injury to a peripheral nerve in young rats, immunoreactivity (ir) to the SP receptor, NK-1 (neurokinin-1), increases in the spinal cord ipsilateral to the injury and the increases are correlated with the development of thermal hyperalgesia (Goff et al., 1998). Thus we postulated that aged rats might display an increased sensitivity to thermal stimulation before peripheral nerve injury and that they might respond differently to injury than do younger rats. To test this hypothesis, we used the Bennett and Xie model (1988) of chronic constriction injury (CCI) to the sciatic nerve to induce a neuropathic pain condition. We investigated the effect of age on changes in NK-1 ir in superficial layers of the dorsal horn and on numbers of NK ir cells in deeper laminae at the L4-L5 levels of the spinal cord after CCI. NK-1 receptors were tagged immunohistochemically and their distribution quantified by use of computer-assisted image analysis. NK-1 ir changes were related to alterations in thermal and tactile sensitivity that developed after CCI in young, mature and aged (4-6, 14-16, and 24-26 months) Fischer F344 BNF1 hybrid rats. No differences in thermal or tactile sensitivity of young and aged rats were seen in the absence of nerve injury. After injury, aged rats developed thermal hyperalgesia and tactile allodynia more slowly than did the younger rats. NK-1 receptor ir and numbers of NK-1 ir cells in the dorsal horn increased with time post-injury in all three groups. NK-1 ir increases were correlated with the development of thermal hyperalgesia in those rats that displayed hyperalgesia. However, some rats developed an increased threshold to thermal stimuli (analgesia) and that also was correlated with increases in NK-1 ir. Thus NK-1 ir extent, while correlated with thermal sensitivity in the absence of injury, is not a specific marker for disturbances in one particular sensory modality; rather it increases with peripheral nerve injury per se.

Neuropathic pain in aged rats: behavioral responses and astrocytic activation.

We used the Bennett and Xie (1988) model of chronic neuropathic pain to study the effect of age on thermal and tactile sensitivity and on astrocytic activation in the dorsal horn of the spinal cord after nerve injury. Fischer 344 FBNF1 hybrid rats in three age groups, 4-6, 14-16, and 24-26 months, were studied. Rats were either unligated (day 0, control) or the left sciatic nerve was loosely ligated to cause a chronic constriction injury (CCI). CCI causes a neuropathic pain condition characterized by tactile allodynia and thermal hyperalgesia. Rats were behaviorally assessed for tactile and thermal sensitivity of their ligated and unligated hind paws up to 35 days postligation. Rats were sacrificed before or at various days postligation, and activated astrocytes were identified at the L4-L5 levels of their spinal cords by use of an antibody to glial fibrillary acid protein (GFAP). The number of GFAP-ir astrocytes in the dorsal horn of the spinal cord in the control, uninjured condition decreased with age (P < or = 0.001) but increased after CCI in all three age groups. After CCI, astrocytic activation in the cord was less robust in aged rats than in younger ones (P < or = 0.01). Not all the CCI rats displayed hyperalgesia to touch and to heat. Rats with an increased sensitivity to heat had increased levels of GFAP-ir in their cords; however, rats with decreased thermal sensitivity also displayed increased GFAP-ir. Thus the presence of activated astrocytes was not correlated with a single behavioral manifestation of neuropathic pain.

Gender and the behavioral manifestations of neuropathic pain.

A model of peripheral nerve injury was used to study gender differences in the development and progression of chronic constriction injury (CCI)-induced hyperalgesia and allodynia in male and female Fischer 344 FBNF1 hybrid rats. Rats were randomly assigned to one of the following treatment groups: (1) gonadally intact unligated males (male); (2) gonadally intact ligated males (male (CCI)); (3) castrated ligated males (male (CAS/CCI)); (4) gonadally intact unligated females (female); (5) gonadally intact ligated females (female (CCI)); and (6) ovariectomized ligated females (female (OVX/CCI)). A plantar analgesia meter and calibrated von Frey pressure filaments were used as the analgesiometric assays. In the absence of nerve injury, gonadally intact males responded significantly faster than females to a thermal nociceptive stimulus. The onset of the behavioral manifestations of unilateral ligation of the sciatic nerve did not differ as a function of sex or hormonal status (e.g., gonadally intact and gonadectomized male and female rats developed thermal hyperalgesia within 14 days post-CCI). Paw withdrawal latency (PWL) values of gonadally intact males returned to baseline control values after postligation day 14, whereas gonadally intact females, ovariectomized females and castrated males continued to elicit robust thermal hyperalgesic symptoms throughout the 35-day duration of the experiment. Allodynic responses to peripheral nerve injury were less variable across genders. These data suggest that the mechanisms underlying chronic nociceptive processing differ as a function of gender and gonadal hormone status.

Changes in spinal serotonin turnover mediate age-related differences in the behavioral manifestations of peripheral nerve injury.

The Bennett and Xie model of peripheral nerve injury was used to study the effects of aging on the onset and progression of sciatic nerve ligation (SNL)-induced thermal hyperalgesia and tactile-evoked allodynia in young, mature, and aged Fischer 344 FBNF1 male rats (4-6, 14-16, and 24-26 months old, respectively). A plantar analgesia meter and calibrated von Frey pressure filaments were employed as the analgesiometric assays. In the absence of nerve injury, aged rats were found to be more sensitive than younger animals to noxious thermal stimuli. Following the SNL surgery, thermal hyperalgesia was observed in all three age groups within 3 days. On post-SNL day 35, the paw-withdrawal latency values of the young and mature animals returned to presurgical baseline levels, while the aged rats continued to exhibit thermal hyperalgesia. Tactile-evoked allodynia was apparent within 3 days following peripheral nerve injury in the oldest cohort, but was delayed in the younger animals. On post-SNL days 0 (control), 3, 21, and 35, young, mature, and aged rats were sacrificed and high-performance liquid chromatography and electrochemical detection (HPLC/ECD) methods were used for neurochemical analyses of spinal serotonin (5-HT), norepinephrine (NE), and 5-hydroxyindoleacetic acid (5-HIAA). Spinal 5-HT and NE levels were not significantly altered by the aging process, nor were they affected by peripheral nerve injury. However, spinal 5-HT turnover from the aged animals was greater than that detected in spinal tissue from the younger counterparts. Differences in spinal 5-HT turnover may contribute to age-related variability in spinal nociceptive processing.

Microglial proliferation in the spinal cord of aged rats with a sciatic nerve injury.

Nerve injury may lead to chronic neuropathic pain syndromes. We determined whether the extent of central nervous system microglial activation that accompanies nerve injury is age dependent and correlated with behavioral manifestations of pain. We used the Bennett and Xie sciatic nerve chronic constriction injury model (Bennett, G.J., Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1998) 87-107) to induce neuropathic pain in three age cohorts of Fischer 344 FBNF1 hybrid rats (4-6, 14-16, and 24-26 months). Rats were assessed for thermal sensitivity (hyperalgesia) of their hind paws pre-injury (day 0) and up to 35 days post injury. On various days post injury, the L4-L5 levels of their spinal cords were reacted for localization of an antibody to OX-42, a marker for microlgia. OX-42 immunoreactivity (ir) was quantified by use of a Bioquant density analysis system. OX-42 ir was heavy in areas of sciatic nerve primary afferent terminations and in the motor columns of its neurons. Aging increases OX-42 ir in the absence of injury. After injury, OX-42 ir increased further, but the increases over control levels decreased with age. Ligation-induced analgesia and hyperalgesia were both correlated with the increases in OX-42 ir, regardless of age.

Strain differences in neuropathic hyperalgesia.

The purpose of this study was to investigate strain-related differences in the onset and maintenance of thermal hyperalgesia following the induction of peripheral nerve injury in two inbred strains of rats (Fischer 344 and Lewis) and two outbred strains of rats (Sprague-Dawley and Wistar). Neuropathic pain was induced via unilateral ligation of the left sciatic nerve with chromic gut sutures. A plantar analgesia meter was used to measure paw-withdrawal latency from the ligated vs. unligated hind paws of inbred vs. outbred strains of rats to investigate strain-related differences in nerve injury-induced thermal hyperalgesia. The results demonstrated no significant effects of animal strain on presurgical paw-withdrawal latency values. Following the sciatic nerve ligation (SNL) surgery, a significant hyperalgesic response was elicited from the Sprague-Dawley and Wistar rats (outbred strains) for at least 28 days. Conversely, data analyses from the inbred strains failed to demonstrate significant hyperalgesic responses to peripheral nerve injury, with the exception of postsurgical day 10. These data emphasize the importance of considering the strain of the rat being investigated before extrapolating the results from animals experiments to treatment strategies for humans with chronic neuropathic pain.

Aging and neuropathic pain.

Although chronic neuropathic pain disorders are more prevalent in the senescent population, little is known about how the aging process alters the thermal hyperalgesic sensitivity to peripheral nerve injury. In this study, neuropathic pain was induced in young, mature and aged FBNF1 hybrid rats via unilateral ligation of the left sciatic nerve. The extent to which the aging process affects the thermal hyperalgesic responsiveness of these animals was investigated. The results demonstrate that the aging process differentially alters nociceptive processing.

Immunohistochemistry and spinal projections of the reticular formation in the northern leopard frog, Rana pipiens.

Over 30 nuclei have been identified in the reticular formation of rats, but only a small number of distinct reticular nuclei have been recognized in frogs. We used immunohistochemistry, retrograde tracing, and cell morphology to identify nuclei within the brainstem of Rana pipiens. FluoroGold was injected into the spinal cord, and, in the same frogs, antibodies to enkephalin, substance P, somatostatin, and serotonin were localized in adjacent sections. We identified many previously unrecognized reticular nuclei. The rhombencephalic reticular formation contained reticularis (r.) dorsalis; r. ventralis, pars alpha and pars beta; r. magnocellularis; r. parvocellularis; r. gigantocellularis; r. paragigantocellularis lateralis and dorsalis; r. pontis caudalis, pars alpha and pars beta; nucleus visceralis secundarius; r. pontis oralis, pars medialis and pars lateralis; raphe obscurus; raphe pallidus; raphe magnus; and raphe pontis. The mesencephalic reticular formation contained locus coeruleus-subcoeruleus, r. cuneiformis, r. subcuneiformis, raphe dorsalis-raphe centralis superior, and raphe linearis. Thus, the reticular formation of frog, which is an anamniote, is organized complexly and is similar to the reticular formation in amniotes. Because many of these nuclei may be homologous to reticular nuclei in mammals, we used mammalian terminology for frog reticular nuclei.

Brainstem neurons with descending projections to the spinal cord of two elasmobranch fishes: thornback guitarfish, Platyrhinoidis triseriata, and horn shark, Heterodontus francisci.

We studied two cartilaginous fishes and described their brainstem supraspinal projections because most nuclei in the reticular formation can be identified that way. A retrogradely transported tracer, horseradish peroxidase or Fluoro-Gold, was injected into the spinal cord of Platyrhinoidis triseriata (thornback guitarfish) or Heterodontus fransisci (horn shark). We described labeled reticular cells by their position, morpohology, somatic orientation, dendritic processes, and laterality of spinal projections. Nineteen reticular nuclei have spinal projections: reticularis (r.) dorsalis, r. ventralis pars alpha and beta, r. gigantocellularis, r. magnocellularis, r. parvocellularis, r. paragigantocellularis lateralis and dorsalis, r. pontis caudalis pars alpha and beta, r. pontis oralis pars medialis and lateralis, r. subcuneiformis, r. peduncularis pars compacta, r. subcoeruleus pars alpha, raphe obscurus, raphe pallidus, raphe magnus, and locus coeruleus. Twenty nonreticular nuclei have spinal projections: descending trigeminal, retroambiguus, solitarius, posterior octaval, descending octaval, magnocellular octaval, ruber, Edinger-Westphal, nucleus of the medial longitudinal fasciculus, interstitial nucleus of Cajal, latral mesencephalic complex, periventricularis pretectalis pars dorsalis, central pretectal, ventromedial thalamic, posterior central thalamic, posterior dorsal thalamic, the posterior tuberculum, and nuclei B, F, and J. The large number of distinct reticular nuclei with spinal projections corroborates the hypothesis that the reticular formation of elasmobranches is complexly organized into many of the same nuclei that are found in frogs, reptiles, birds, and mammals.

Raphe nuclei in three cartilaginous fishes, Hydrolagus colliei, Heterodontus francisci, and Squalus acanthias.

The vertebrate reticular formation, containing over 30 nuclei in mammals, is a core brainstem area with a long evolutionary history. However, not all reticular nuclei are equally old. Nuclei that are widespread among the vertebrate classes are probably ones that evolved early. We describe raphe nuclei in the reticular formation of three cartilaginous fishes that diverged from a common ancestor over 350 million years ago. These fishes are Hydrolagus colliei, a holocephalan, Squalus acanthias, a small-brained shark, and Heterodontus francisci, a large-brained shark. Nuclear identification was based on immunohistochemical localization of serotonin and leu-enkephalin, on brainstem location, and on cytoarchitectonics. Raphe nuclei are clustered in inferior and superior cell groups, but within these groups individual nuclei can be identified: raphe pallidus, raphe obscurus, and raphe magnus in the inferior group and raphe pontis, raphe dorsalis, raphe centralis superior, and raphe linearis in the superior group. Hydrolagus lacked a dorsal raphe nucleus, but the nucleus was present in the sharks. The majority of immunoreactive cells are found in the superior group, especially in raphe centralis superior, but immunoreactive cells are present from spinal cord to caudal mesencephalon. The distribution and cytoarchitectonics of serotoninergic and enkephalinergic cells are similar to each other, but raphe nuclei contain fewer enkephalinergic than serotoninergic cells. The cytoarchitectonics of immunoreactive raphe cells in cartilaginous fishes are remarkably similar to those described for raphe nuclei in mammals; however, the lack of a raphe dorsalis in Hydrolagus indicates that either it evolved later than the other raphe nuclei or it was lost in holocephalan fishes.

Immunohistochemical localization of serotonin, leu-enkephalin, tyrosine hydroxylase, and substance P within the visceral sensory area of cartilaginous fish.

We examined the distribution of immunoreactivity to serotonin (5-HT), leu-enkephalin (LENK), tyrosine-hydroxylase (TH), and substance P (SP) within the primary visceral sensory region of cartilaginous fish. Two genera of sharks, Squalus and Heterodontus, a skate, Raja, a ray, Myliobatis, and a holocephalian, Hydrolagus, were used. Cranial nerves, VII, IX, and X enter the visceral sensory complex from the lateral aspect and divide it into lobes. Based on sagittally cut sections, there are four lobes in Hydrolagus and five in Squalus, corresponding to the number of gill arches. The neurochemicals are differentially distributed within each lobe. LENK+ and 5-HT+ fibers are located in all regions within the visceral sensory complex. SP+ fibers are extremely dense in a dorsolateral subdivision and do not extend as far ventrally as 5-HT+ and LENK+ fibers. The lobes lack 5-HT+ cells, but contain a few LENK+ and SP+ cells. Many TH+ cells are distributed in dorsomedial portions of the complex, but there are few TH+ fibers. Thus, the visceral sensory area of cartilaginous fish contains several divisions that can be distinguished by their neurochemical content.

Distribution of tyrosine hydroxylase, serotonin, and leu-enkephalin immunoreactive cells in the brainstem of a shark, Squalus acanthias.

The central nervous system location of neurochemicals that are widely distributed among extant animals may give us clues to changes that occurred in the brains of these animals during evolution. We have been studying the brains of cartilaginous fishes, a heterogeneous group whose central nervous system varies considerably. Squalus acanthias, the spiny dogfish shark, was chosen to represent the squalomorphs, a group of living sharks known to possess many primitive characters. The distribution of tyrosine hydroxylase (TH+), serotonin (5-HT+), and leu-enkephalin (LENK+) positive cells within the brainstem of Squalus was determined by use of antibodies to these substances. All the major raphe groups described for mammals were found in Squalus. The 5-HT+ cells in raphe nuclei were more uniformly distributed in Squalus than in Heterodontus, the horn shark. Other nuclei that were 5-HT+ and LENK+, and that have been identified in mammals, included reticularis paragigantocellularis lateralis, a B9 cell group, and reticularis magnocellularis. The postcommissural nucleus and pretectal area contained 5-HT+ and LENK+ cells. These cells have been described in a holocephalian, in teleosts, and in reptiles but not in other elasmobranchs or in mammals. Cells that were TH+ were located in prominent A1/A2, A6 (locus coeruleus), A9 (substantia nigra), and A10 (ventral tegmental area) cell groups, and in a very small A5 group. We conclude that the variation in chondrichthian brainstems exceeds that in mammals, and we suggest that this variation is related to life-style and the long evolutionary history of these fishes.

The red nucleus and mesencephalic tegmentum in a ranid amphibian: a cytoarchitectonic and HRP connectional study.

Movement control in vertebrates is a complex function that is known to involve several parallel systems. In amphibians, which lack the isocortical structures shown in mammals to initiate and control voluntary movements, supraspinal motor control systems have received surprisingly little attention. Because amphibians lack a corticospinal equivalent, coordination and control of all movement strategies must take place in non-cortical, supraspinal integrating centers. The rubro-cerebello-rubrospinal circuit is likely to represent a major motor control system in such vertebrates. In this anatomical investigation four mesencephalic tegmentospinal projection nuclei are described in ranid amphibians (Rana catesbiana and Rana pipiens): reticular formation, accessory optic complex, interstitial nucleus of Cajal, and the red nucleus. The red nucleus, which shows no distinct somatotopic organization, can be distinguished because it is the only one of the four that is predominantly contralateral in its projections. Horseradish peroxidase injections into the tegmentum and the cerebellum demonstrated that the red nucleus also maintains reciprocal connections with the cerebellum via the deep cerebellar nucleus. These connections could not be localized to any distinct region in the deep cerebellar nuclear mass, suggesting that this represents a single cerebellar recipient nucleus. Thus, anuran amphibians are shown to possess the major pathways that comprise the rubro-cerebello-rubrospinal circuitry in mammals.

Immunohistochemical localization of serotoninergic, enkephalinergic, and catecholaminergic cells in the brainstem and diencephalon of a cartilaginous fish, Hydrolagus colliei.

We localized serotonin (5-HT), leu-enkephalin (LENK), and tyrosine hydroxylase (TH) immunoreactive cells in the brain of a holocephalian, Hydrolagus colliei, by use of antibodies made in rabbit and the peroxidase-antiperoxidase technique. Only three locations contained TH+ cells, the caudal myelencephalon, the locus coeruleus, and the diencephalon. Of these locations, the diencephalon contained the most cells and the locus coeruleus the least cells. The caudal TH+ myelencephalic cells formed a single large group that spanned both the dorsal and ventral portions of the brain (A1A2). The diencephalic TH+ cells were located in the posterior tuberculum, in the ventromedial and ventrolateral thalamic nuclei, and in the inferior lobe of the hypothalamus. Hydrolagus differed from mammals and the elasmobranchs, their sister group, in that no substantia nigra (A9), ventral tegmental area (A10), or A5 cell group was found. Distribution of LENK+ and 5-HT+ cells were similar to each other; the raphe nuclei contained most of the 5-HT+ and LENK+ cells. These 5-HT+ and LENK+ cells were found at all rostrocaudal levels of the myelencephalon. The nucleus reticularis magnocellularis, reticularis paragigantocellularis lateralis, the ventral met- and mesencephalon (B7 and B9 cell groups), the hypothalamus, and the pretectal area contained additional 5-HT+ and LENK+ cells. The solitary complex contained LENK+ cells but not but 5-HT+ cells. A dorsal raphe nucleus, which is the largest 5-HT+ cell group in mammals, was absent in Hydrolagus. A dorsal raphe nucleus is present in one galeomorph shark radiation but is absent in three radiations of batoids (rays, skates, and guitarfish). Thus even within cartilaginous fish, there are differences in the distribution of neurochemicals and possibly nuclei within their brains.

Localization of serotonin, tyrosine hydroxylase, and leu-enkephalin immunoreactive cells in the brainstem of the horn shark, Heterodontus francisci.

In previous studies on reptiles and elasmobranchs, we determined that some reticular groups are either absent or may be displaced compared to their locations in mammals. For example, nucleus raphe dorsalis, the largest serotoninergic cell group in mammals, is not present in rays, skates, or guitarfish. In the present study, we chose heterodontid sharks, a sister group to these batoids, for an out-group comparison of this and other characters. We identified cells in the brainstem of Heterodontus francisci by use of antibodies against tyrosine hydroxylase, serotonin, or leu-enkephalin and compared the distribution of these nuclei to descriptions in mammals and other elasmobranchs. The majority of tyrosine hydroxylase-positive cells were found in the midbrain tegmentum (A8-A10) and the hypothalamus. In addition, putative A1, A2, A5, A7 (noradrenergic) groups were found in the metencephalon and myelencephalon. Serotonin-positive cells were found in raphe nuclei and scattered lateral to the raphe. We identified probable homologues to raphe pallidus, raphe obscurus, raphe magnus, and raphe centralis superior (B8) cell groups, which have been described in mammals. A cluster of cells dorsomedial to the medial longitudinal fasciculus was identified as raphe dorsalis. The distributions of leu-enkephalin and serotonin immunoreactive cells were similar to each other, but the tyrosine-hydroxylase immunoreactive cells rarely intermingle with the former two immunoreactive cell types. Other reticular groups that contained both serotonin- and leu-enkephalin-positive cells included reticularis (r.) ventralis, r. magnocellularis, r. paragigantocellularis lateralis, r. pontis caudalis, and r. pontis oralis medialis and lateralis. Thus, this shark contains many of the major brainstem raphe and catecholaminergic cell groups described for rats, but the relative distribution of the immunopositive cell groups differs in mammals and cartilaginous fish.

Serotoninergic and enkephalinergic cell groups in the reticular formation of the bat ray and two skates.

The distribution of cells which were immunohistochemically positive for leu-enkephalin (LENK+) or serotonin (5-HT+), two substances widely distributed in the reticular formation, was determined in two species of skates (Raja binoculata and Raja rhina) and a bat ray (Myliobatis californica). The Rajoids are closely related to the Rhinobatoids which contains Platyrhinoidis, an elasmobranch that does not have a nucleus raphe dorsalis. Myliobatis was chosen for an outgroup comparison. Most of the nuclei that were 5-HT+ were also LENK+. The greatest number of labeled cells was in the hypothalamus bordering the third ventricle and in the neurointermediate lobe of the pituitary. The mesencephalon was rich in cells in the ventral tegmental area bordering the red nucleus. In both genera, there were numerous 5-HT+ and LENK+ fusiform cells paralleling the ventral surface of the metencephalon and myelencephalon. These cells were located in several reticular nuclei but were especially prominent in nucleus reticularis (n.r.) pontis oralis, n.r. magnocellularis, and n.r. paragigantocellularis lateralis. The latter nucleus contained fewer LENK+ than 5-HT+ cells. In both genera, 5-HT+ and LENK+ cells were located in raphe pallidus, raphe obscurus, raphe magnus, raphe centralis superior, and raphe linearis. Minor differences in distribution of the remaining 5-HT+ and LENK+ cell groups were found, but these representative elasmobranchs lack a dorsal raphe nucleus which, in mammals, is the largest serotoninergic group.

Distribution of tyrosine hydroxylase- and serotonin-immunoreactive cells in the central nervous system of the thornback guitarfish, Platyrhinoidis triseriata.

Brainstem reticular nuclei of amniotes (mammals, birds and reptiles) may share a common phylogenetic origin as demonstrated by their many shared features (hodology, cytoarchitectonics, presence of neurochemicals). By studying characteristics of these nuclei in outgroups of amniotes, we hope to obtain clues about the phylogeny of the reticular formation. In this paper we report the distribution of immunoreactivity to tyrosine hydroxylase (TH) and serotonin (5-HT) in the brain of an elasmobranch, the thornback guitarfish, Platyrhinoidis triseriata. Our working hypothesis is that if morphologically and immunohistochemically similar cell groups are present, they are homologous to cell groups in amniotes. Thus we have used mammalian terminology. The dorsal and lateral pallium of the telencephalon and many diencephalic nuclei contained TH+ cells. In the mesencephalon, TH+ cell groups were located in raphe linearis, the ventral tegmentum and substantia nigra. The rhombencephalon contained TH+ cells in a putative locus coeruleus (A6), and a subcoeruleus group. Probable A5, A2/C2 and A1/C1 groups were also located. A few 5-HT+ cells were located in the telencephalon and many were found in the diencephalon. In the mesencephalon, 5-HT+ cells were located in the nucleus reticularis pedunculopontinus pars dissipatus (B9). Metencephalic cells were found in reticularis pontis oralis lateralis and medialis, the reticulotegmental nucleus, nucleus centralis superior (B8), reticularis magnocellularis and reticularis pontis caudalis. In the myelencephalon, 5-HT+ cells were contained in raphe pallidus, reticularis paragigantocellularis lateralis and reticularis ventralis pars alpha. The cell shapes, locations, and neurochemical content of Platyrhinoidis reticular groups were very similar to those of amniotes. This elasmobranch has most of the 5-HT+ and TH+ cell groups found in mammals with the major exception that no 5-HT+ cells were in a nucleus which might correspond to raphe dorsalis.

Immunohistochemical localization of substance P, somatostatin, enkephalin, and serotonin in the spinal cord of the northern leopard frog, Rana pipiens.

Using the indirect antibody peroxidase-antiperoxidase method of Sternberger, we localized substance P (SP), somatostatin (SOM), enkephalin (ENK), and serotonin (5HT, 5-hydroxytryptamine) in the spinal cord of Rana pipiens. This is the first study to demonstrate all four substances in adjacent sections of frog spinal cord. The distribution patterns of ENK, SP, SOM, and 5HT in our study differ from that described for laminae I and II in amniotes. A high density of ENK, SP, and SOM fibers is present in a band ventral to the dorsal terminal field of cutaneous primary afferent fibers and slightly overlapping the ventral terminal field of muscle primary afferent fibers. However, a high density of 5HT fibers is present in the dorsal terminal field.

Evolution of the reticular formation.

The reticular formation of mammals contains numerous nuclei which can be recognized by their projection patterns, cytoarchitectonics, and neuropeptide/neurotransmitter content. We have identified reticular nuclei in representatives from numerous reptilian groups and ascertained presence or absence of these reticular nuclei in an attempt to use neuronal occurrence as a tool to determine phylogenetic relationships. Recently these studies have been extended to two elasmobranchs, a galeomorph shark and a ray. In this report, we concentrate on three medullary spinal projecting reticular nuclei, reticularis gigantocellularis, reticularis magnocellularis, and reticularis paragigantocellularis. We found that all three nuclei were present in rats, lizards, and elasmobranchs, but one nucleus was absent in crocodilians, and two nuclei were absent in turtles. Thus brain organization may give us clues to phylogenetic relationships. Moreover, these three reticular nuclei exhibited remarkably similar cellular morphology in mammals, reptiles, and elasmobranchs.