Normal dendritic morphology of frog spinal motoneurons: a Golgi study.

Normal dendritic morphology of frog (Rana pipiens) lumbar motoneurons was studied using Golgi silver impregnation. Branching characteristics and quantitative measurements of dendrites were obtained using computer-aided serial reconstruction of a typical lumbar motoneuron over seven adjacent 80-micrometer transverse sections. Dendrites were classified based upon site of dendrite origin from the soma and distribution of the dendritic array within the spinal cord. Eight possible sites of dendritic origin from the soma were identified. Two dendrites, D1 and D2, are planar dendrites which arise from the dorsal aspect of the soma. They are moderately complex, reaching branch order 5-6, and are oriented predominantly in the transverse plane. Input to these dendrites is primarily segmental via dorsal root projections. Three dendrites, D3, D4, and D5, arise laterally from the soma and extend through the lateral funiculus toward the subpial region. Two dendrites, D6 and D7, arise ventrally. D6 extends ventrolaterally and is a simple dendrite reaching branch order 3-4. D7 aborizes extensively in the ventral funiculus and in the central gray, reaching a branch order of 8-9. This dendrite extends rostrally and caudally over a distance of at lest 560 micrometer. Another dendrite (D8) arises from the medial aspect of the soma and projects toward the central canal. Four sites (D1, D2, D6, and D7) almost invariably give rise to dendrites. Dendrites arise at D4 in 66% of the cells examined. Dendrites are found at D3, D5, and D8 much less frequently (6-21%). Total dendritic length (12,043 micrometer) and lengths of the individual dendrites, branch length versus branch order, and number of branches at increasing radii were examined, and Sholl analysis was performed.

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.

Brain stem origins of spinal projections in the lizard Tupinambis nigropunctatus.

In order to study brainstem origins of spinal projections, ten Tegu lizards (Tupinambis nigropunctatus) received complete or partial hemisections of the spinal cord at the first or second cervical segment. Their brains were processed for conventional Nissl staining. The sections were surveyed for the presence or absence of retrograde chromatolysis. Based on analysis and comparison of results from lesions in the various spinal cord funiculi, the following conclusions were reached: The interstitial nucleus projects ipsilaterally to the spinal cord via the medial longitudinal fasciculus, as does the middle reticular field of the metencephalon. The red nucleus and dorsal vagal motor nucleus both project contralaterally to the spinal cord via the dorsal part of the lateral funiculus. The superior reticular field in the rostral metencephalon and the ventrolateral vestibular nucleus project ipsilaterally to the spinal cord via the ventral funiculus. The dorsolateral metencephalic nucleus and the ventral part of the inferior reticular nucleus of the myelencephalon both project ipsilaterally to the spinal cord via the dorsal part of the lateral funiculus. Several brainstem nuclei in Tupinambis project bilaterally to the spinal cord. The ventrolateral metencephalic nucleus, for example, projects ipsilaterally to the cord via the medial longitudinal fasciculus and contralaterally via the dorsal part of the lateral funiculus. The dorsal part of the inferior reticular nucleus projects bilaterally to the spinal cord via the dorsal part of the lateral funiculus. The nucleus solitarius complex projects contralaterally via the dorsal part of the lateral funiculus but ipsilaterally via the middle of the lateral funiculus. The inferior raphe nucleus projects bilaterally to the spinal cord via the middle part of the lateral funiculus. These data suggest that supraspinal projections in reptiles, especially reticulospinal systems, are more highly differentiated than previously thought. On the other hand, recent findings in cat, opossum, and monkey reveal that the organization of supraspinal pathways in the Tegu lizard bears a striking resemblance to that observed in mammals.

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.

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.

The sources of supraspinal afferents to the spinal cord in a variety of limbed reptiles. I. Reticulospinal systems.

Horseradish peroxidase was injected into various levels of the spinal cord of turtles (Pseudemys and Chrysemys), lizards (Tupinambis, Iquana, Gekko, Sauromelus, and Gerrhonotus), and a crocodilian (Caiman). The results suggest that brainstem reticulospinal projections in limbed reptiles rival mammalian reticulospinal systems in complexity. The reptilian myelencephalic reticular formation can be divided into four distinct reticulospinal nuclei. Reticularis inferior pars dorsalis (RID) contains multipolar neurons which project bilaterally to the spinal cord. Reticularis inferior pars ventralis (RIV), which is only found in lizards and crocodilians, contains fusiform neurons with horizontally running dendrites and it projects ipsilaterally to the spinal cord. Reticularis ventrolateralis (RVL), which is found only in field lizards, contains triangular neurons whose dendrites parallel the ventrolateral edge of the brainstem and it projects ipsilaterally to the spinal cord. The myelencephalic raphe (RaI) varies considerably. RaI of turtles contains large reticulospinal neurons which form a continuous population with more laterally situated RID cells. RaI of lizards contains a few small reticulospinal neurons. RaI of the crocodilian Caiman contains giant reticulospinal neurons with laterally directed dendrites. The caudal metencephalic reticular formation of reptiles can be divided into two distinct reticulospinal nuclei. Reticularis medius (RM) contains large neurons with long, ventrally directed dendrites; it projects ipsilaterally to the spinal cord. Reticularis medius pars lateralis (RML) contains small neurons with laterally directed dendrites; it projects contralaterally to the spinal cord. The rostral mesencephalic and caudal mesencephalic reticular formation of reptiles can be divided into three distinct reticulospinal nuclei. Reticularis superior pars medialis (RSM) consists mostly of small, spindle-shaped neurons which project bilaterally to the spinal cord. In the lizard Tupinambis, however, large multipolar, ipsilaterally projecting neurons are occasionally seen in RSM. Reticularis superior pars lateralis (RSL) contains large, ipsilaterally projecting neurons with long, ventrolaterally directed dendrites. SRL in lizards can be divided into a dorsomedial portion, which projects ipsilaterally to the spinal cord, and a ventrolateral portion which projects contralaterally. The locus ceruleus-subceruleus field (LC-SC) contains small spindle-shaped neurons which project bilaterally to the spinal cord. Labelled reticulospinal neurons were also observed in the rostral metencephalic raphe (RaS) of the turtle brainstem. These cells are small, spindle-shaped neurons which resemble the small cells of the adjacent RSM field.

Afferent and efferent components of the hypoglossal nerve in the grass frog, Rana pipiens.

In amphibians, the spinomedullary region of the central nervous system is compressed rostrocaudally because of the absence of a neck. In Ranid frogs, the hypoglossal nerve emerges as the ventral ramus of the second spinal nerve. The first spinal nerve, though present in tadpoles, is absent as a separate nerve in adults. To investigate the central nervous system components of the hypoglossal nerve in Rana pipiens, we soaked identified, transected branches of this nerve in horseradish peroxidase, a retrograde and anterograde tracer. We found that the hypoglossal nerve in these frogs originates from two efferent nuclei located in the caudal medulla, a medial and a lateral one. Afferent fibers, primarily from the tongue, are also found in the hypoglossal nerve and travel in the dorsolateral funiculus of the spinal cord, descending to thoracic levels of the cord. Efferents to intrinsic tongue muscles and the genioglossus muscle originate in the medial medullary nucleus. Efferents to the sternohyoid muscle, which travel through the hypoglossal nerve, originate in the lateral medullary nucleus. Since in mammals the sternohyoid muscle is innervated by the first spinal nerve, we have obtained experimental evidence that the hypoglossal nerve in Rana pipiens contains components of this spinal nerve.

Organization within the cranial IX-X complex in ranid frogs: a horseradish peroxidase transport study.

Cranial nerves IX and X in frogs have been described as originating from a nuclear group referred to as the IX-X complex. We studied the central nervous system components of this complex in Rana pipiens and R. catesbiana by labeling peripheral branches of cranial nerves IX and X and identifying the central nervous system contributions of these branches. Various peripheral nerves (IX and the cardiac, gastric, pulmonary, and laryngeal branches of X) were identified and soaked in horseradish peroxidase (HRP). One to 2 weeks later, the frogs were killed and processed for HRP by the tetramethylbenzidine method. Glossopharyngeal efferents originated from a small ventrolateral cell group found at the level of IX root exit. Vagal efferents formed a single column of cells in a ventrolateral position from the level of the brainstem exist of the vagus nerve (approximately 2,000 micrometers above the obex) to 200 micrometers below the obex (values given are for an 80-g frog). This cell group was separate from and just caudal to efferent cells of the glossopharyngeal nerve. Within the vagal portion of the column, cells projecting through the gastric branch were found throughout the rostral-caudal extent of the nucleus. "Cardiac" cells tended to be more rostral than "pulmonary" cells, and both groups of cells were located in the middle of the nucleus. "Laryngeal" cells were located more caudally in the nucleus. This peripheral representation within the vagal nucleus corresponds more closely to the organization found in the mammalian nucleus ambiguus, rather than to the apparent lack of organization found in the mammalian dorsal motor nucleus. Afferents of IX and X entered slightly rostral to the ventral roots of their respective nerves and descended in two tracts. The majority entered the tractus solitarius and descended in a medial position to cervical spinal cord. A portion of the afferents from the vagus nerve crossed the midline in the lower myelencephalon just dorsal to the central canal and ascended a short distance on the contralateral side. Within the solitary tract, vagal afferents were located in a ventrolateral position as they descended to below the obex. Glossopharyngeal afferents filled the remainder of the tract. A smaller portion of afferents from both IX and X did not enter the solitary tract but descended in the spinal tract of V and the dorsolateral funiculus of the spinal cord (Lissauer's tract) to thoracic levels. Afferents of IX also formed a rostral bundle which extended in the solitary tract to the caudal metencephalon.

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.

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.

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.

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 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.

A Golgi impregnation technique for thin brain slices maintained in vitro.

A technique for using routine rapid Golgi impregnation procedures on very thin freshly fixed slices (less than 0.5 mm) of brain tissue is described. The technique was particularly successful with hippocampal slices that were maintained and stimulated in vitro prior to fixation. Thin tissue slices were surrounded by thicker sections of tissue to form a 5 mm thick bundle. The tissue bundle was then processed by a rapid Golgi procedure, 5 days each in chromate osmium and silver nitrate solutions. At the end of this time the thin tissue slices were unwrapped from their thicker protecting tissue sections, embedded in celloidin, cut at 60-100 micrometer thickness on a sliding microtome and mounted in permount under cover glass. Qualitative light microscopic analysis of the rapid Golgi impregnated slices revealed fully impregnated cell bodies, dendrites, dentritic spines, axons and axonal varicosities with minimal background artifact. In contrast, unprotected thin tissue slices showed only a dense black artifact without cells or processes.

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.

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.

The organization of the reptilian formation: a comparative study using Nissl and Golgi techniques.

The brainstem reticular formation has been studied in 16 genera representing 11 families of reptiles. Measurements of Nissl-stained reticular neurons revealed that they are distributed along a continuum, ranging in length from 10 micrometer to 95 micrometer. Reticular neurons in crocodilians and snakes tend to be larger than those found in lizards and turtles. Golgi studies revealed that reticular neurons possess long, rectilinear, sparsely branching dendrites. Small reticular neurons (less than 31 micrometers length) possess fusiform or triangular somata which bear two or three primary dendrites. These dendrites have a somewhat simpler ramification pattern when compared with those of large reticular neurons ( greater than 30 micrometers length). Large reticular neurons generally possess perikarya which are triangular or polygonal in shape. The somata of large reticular neurons bear an average of four primary dendrites. The dendrites of reptilian reticular neurons ramify predominantly in the transverse plane and are devoid of spines or excrescences. The dendritic ramification patterns observed in the various repitilian reticular nuclei were correlated with known input and output connections of these nuclei. Nissl and golgi techniques were used to divide the reticular formation into seven nuclei. A nucleus reticularis inferior (RI) is found in the myelencephalon, a reticularis medius (RM) in the caudal two-thirds of the metencephalon, and a reticularis superior (RS) in the rostral metencephalon and caudal mesencephalon. Reticularis inferior can be subdivided into a dorsal portion (RID) and a ventral portion (RIV). All reptilian groups possess RID and RM but RIV is lacking in turtles. Reticularis superior can be subdivided into a large-celled lateral portion (RSL) and a small-celled medial portion (RSM). All reptilian groups possess RSM and RSL, but RSL is quite variable in appearance, being best developed in snakes and crocodilians. The myelencephalic raphe nucleus is also quite variable in its morphology among the different reptilian families. A seventh reticular nucleus, reticularis ventrolateralis (RVL), is found only in snakes and in teiid lizards. It was noted that the reticular formation is simpler (fewer numbers of nuclei) in the representative of older reptilian lineages and more complex (greater numbers of nuclei) in the more modern lineages. Certain reticular nuclei are present or more extensive in those families which have prominent axial musculature.

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.

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.

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.