Robert B. Leonard

Facilitation of the responses of primate spinothalamic cells to cold and to tactile stimuli by noxious heating of the skin

Abstract Spinothalamic tract cells in anesthetized monkeys were found to respond to noxious cold stimuli (18/19 cells tested), as well as to noxious heat and noxious mechanical stimuli. Responses to repetition of the noxious cold stimuli after a series of noxious heat stimuli were enhanced. However, subtraction of the enhanced background activity that resulted from damage of the skin revealed that the enhanced responses to noxious cold stimuli were due to superposition of the original responses upon an enhanced background activity, rather than to sensitization of the responses to noxious cold stimuli per se. Furthermore, the responses to innocuous mechanical stimuli applied either within the area that was damaged or outside this area were enhanced, provided the noxious heat was applied for a long enough time. Thus, damage to a region of skin can result in enhanced responsiveness of spinothalamic cells to stimuli applied in an undamaged region of the receptive field. The possible relationship between these observations and cutaneous hyperalgesia is discussed.

The relationship of the medullary catecholamine containing neurones to the vagal motor nuclei

We have re-examined in the rat the nuclear localization of the medullary catecholamine-containing cell groups (A1 and A2) and their relation to the vagal motor nuclei using a double labeling method. The vagal nuclei were defined by the retrograde transport of horseradish peroxidase applied to the cervical vagus, and noradrenergic and adrenergic neurons were stained with the peroxidase-antiperoxidase immunocytochemical method using an antibody to dopamine beta-hydrolase. The method allows visualization of both labels within single neurons. The neurons of the A2 group are primarily distributed in both the nucleus of the solitary tract and the dorsal motor nucleus of the vagus in a complex interrelationship that depends on the rostrocaudal level. Caudal to the obex, cells of the dorsal motor nucleus of the vagus are scattered among cells immunoreactive for dopamine beta-hydroxylase in the area considered to be the commissural subnucleus of the nucleus of the solitary tract. At levels near and slightly rostral to the obex, the dopamine beta-hydroxylase-positive cells are largely confined to nucleus of the solitary tract. However, the rostral third of the A2 group lies predominantly within dorsal motor nucleus, as defined by horseradish peroxidase labeled cells, with only a few cells in the nucleus of the solitary tract. A subset of the dopamine beta-hydroxylase positive cells within the rostral dorsal motor nucleus of the vagus are also vagal efferents. Our results suggest that a second population of dopamine beta-hydroxylase positive vagal efferents may exist ventrolaterally where neurons of the AI cell group intermingle with those of nucleus ambiguus.

Molecular probes of the vestibular nerve: I. Peripheral termination patterns of calretinin, calbindin and peripherin containing fibers

Vestibular afferents have different physiological properties that can be at least partially correlated with the morphology that the peripheral ending makes with type I and type II hair cells. If the location of the ending in the sensory epithelium is included, the correlations are further improved. It is also known that vestibular afferents can be immunohistochemically stained for a variety of different substances. We have concentrated on three of these markers, calretinin, calbindin and peripherin, because the sources of afferents to the vestibular nuclear complex that contain these substances are restricted, in two cases to the primary afferents. We demonstrate that calretinin is found only in the calyx-only afferents that are located at the apex of the cristae ampullaris and along the striola of the maculae. The area containing stained calyces is equal to or slightly smaller than the central zone of the cristae as defined by the Goldberg group [J. Neurophysiol. 60 (1988) 167]. Calbindin is also found in calyces at the apex of the cristae and along the striola of the otoliths. Examination of adjacent sections of all endorgans indicates that calbindin staining overlaps with calretinin, but is always several hair cells wider on each side. Peripherin also stains fibers in the neuroepithelium. The greatest density of staining is in the peripheral zone of the cristae, i.e. at the base and toward the planum semilunatum. We suggest that these substances are useful markers for specific sets of vestibular afferents.

Immunocytochemical demonstration of serotonergic cells, terminals and axons in the spinal cord of the stingray, dasyatis sabina

Serotonin-like immunoreactivity, as demonstrated by the PAP method, was contained within cells as well as fibers and terminals, in the spinal cord of the stingray. Serotonergic spinal neurons were seen on 43% of the sections examined and were restricted to the ventral white matter. Immunoreactive terminals and varicosities were densely distributed over the spinal gray matter at all segmental levels, and stained fibers were seen in all portions of the white matter with the exception of the medial anterior and dorsal funiculi.

Molecular probes of the vestibular nerve

Abstract An unambiguous delineation of the exact numbers and/or proportions of calyx-only, dimorph, and bouton-only vestibular afferents is needed to continue studies concerning vestibular integration in the nervous system. Here, we take advantage of immunocytochemical properties of three groups of vestibular afferents. We utilize calretinin to delineate the calyx-only population, and peripherin to stain the bouton-only afferents. An additional subgroup of afferents that stain with calbindin, but not calretinin is also introduced. The size of the cells that stain with these markers was determined. Cells that are calbindin-positive overlap the sizes of Nissl-stained somata. Cells that stain with peripherin or calretinin are non-overlapping with calretinin cells being the largest and peripherin-positive cells the smallest. Twenty percent of the ganglion cells were peripherin positive, another 20% stained with calretinin antibodies, 30% stained with calbindin, and all cells in Scarpa’s ganglion stained with parvalbumin. Most of the calretinin-positive cells also stained with calbindin. One-third of the calbindin-positive population stained only with calbindin. These studies indicate that the calyx- and bouton-only populations of vestibular afferents in gerbil comprise at least 40% of the nerve. In addition, at least 10% of the nerve also stains with calbindin and neither calretinin nor peripherin. Based on indirect evidence, we hypothesize that these are a subpopulation of dimorph afferents. This study has provided an anatomical instrument (in addition to intracellular physiological methods) to study separate populations of vestibular afferents.

Identification of the midbrain locomotor region and its relation to descending locomotor pathways in the Atlantic stingray, Dasyatis sabina

The midbrain locomotor region (MLR) in the Atlantic stingray, Dasyatis sabina, was identified and characterized. Stimulation (50-100 microA, 60 Hz) of the midbrain in decerebrated, paralyzed animals was used to elicit locomotion monitored as alternating activity in nerves innervating an antagonist pair of elevator and depressor muscles. Effective sites for evoking locomotion in the midbrain included parts of several nuclei: the caudal portion of the interstitial nucleus of the medial longitudinal fasciculus and the caudomedial parts of the cuneiform and subcuneiform nuclei. This region did not include the red nucleus, any parts of the optic tectum or the medial or lateral mesencephalic nuclei. Electrical stimulation in the MLR evokes locomotion in either the ipsi- or contralateral pectoral fin, whereas stimulation in the medullary reticular formation evokes locomotion only in the contralateral fin. Lesion experiments were performed to identify the location of descending pathways from the midbrain to the medullary reticular formation. To abolish locomotion evoked by electrical stimulation in the MLR, the medial reticular formation in the rostral medulla had to be lesioned bilaterally, or the ipsilateral medial medullary reticular formation and fibers projecting from the MLR to the contralateral midbrain had to be disrupted. Injections of HRP into the magnocellular/gigantocellular reticular formation confirmed that this area received bilateral projections from the MLR. The MLR of the Atlantic stingray appears to be similar to the lateral component of the mammalian MLR and to the MLR in other non-mammalian vertebrates.

Use of Calcium-Binding Proteins to Map Inputs in Vestibular Nuclei of the Gerbil

We wished to determine whether calbindin and/or calretinin are appropriate markers for vestibular afferents, a population of neurons in the vestibular nuclear complex, or cerebellar Purkinje inputs. To accomplish this goal, immunocytochemical staining was observed in gerbils after lesions of the vestibular nerve central to the ganglion, the cerebellum, or both. Eleven to fourteen days after recovery, the brain was processed for immunocytochemical identification of calretinin and calbindin. After lesion of the vestibular nerve, no calretinin staining was seen in any of the vestibular nuclei except for a population of intrinsic neurons, which showed no obvious change in number or staining pattern. Calbindin staining was reduced in all nuclei except the dorsal part of the lateral vestibular nuclei. The density of staining of each marker, measured in the magnocellular medial vestibular nucleus, was significantly reduced. After the cerebellar lesion, no differences in calretinin staining were noted. However, calbindin staining was greatly reduced in all nuclei. The density of staining, measured in the caudal medial vestibular nucleus, was significantly lower. After a combined lesion of the cerebellum and vestibular nerve, the distribution and density of calretinin staining resembled that after vestibular nerve section alone, whereas calbindin staining was no longer seen. This study demonstrates that calretinin and calbindin are effective markers for the identification of vestibular afferents. J. Comp. Neurol. 386:317–327, 1997.

Immunocytochemical demonstration of serotonergic neurons and processes in the retina and optic nerve of the stingray,Dasyatis sabina

Neurons and processes in the stingray retina can be stained using PAP immunohistochemistry and an antibody to serotonin, without pharmacological pretreatment. Most of the cell bodies are in the inner nuclear layer while the processes ramify in the inner plexiform layer suggesting the presence of a population of serotonin containing amacrine cells in this species. Scattered immunopositive axons were observed in the optic nerve from the optic chiasm to the optic nerve head.

Chromatophore motoneurons in the brain of the squid, Lolliguncula brevis: a HRP study

The location of the motoneuron somata controlling activity of the chromatophore muscles was studied in the squid Lolliguncula brevis. Retrograde transport of horseradish peroxidase from injection sites in the skin or in the mantle muscle established that the chromatophore motoneurons are situated in the subesophageal mass of the brain while at least some of the mantle muscle motoneurons are in the stellate ganglia. Motoneurons to chromatophores in the mantle have their somata in the posterior subesophageal mass, mainly in the chromatophore or fin lobes. Motoneurons to chromatophores in the head are located in the anterior pedal lobes and those to the chromatophores in the arms project mainly from the anterior chromatophore lobes. However, some neurons in the posterior chromatophore lobes project to the head or arm regions. A few cells in both the anterior and posterior chromatophore lobes project contralaterally. Somata in other lobes of the subesophageal mass are also labelled by injections in the skin or in the mantle muscle. Evidence presented here suggests that some of the neurons labelled outside the chromatophore lobes are chromatophore motoneurons.

Organization of spinal motor nuclei in the stingray, Dasyatis sabina

The Atlantic stingray, Dasyatis sabina, has enlarged pectoral fins consisting of a series of antagonist dorsal (elevator) and ventral (depressor) muscles. Each muscle is divided into superficial and deep components. The retrograde transport of horseradish peroxidase (HRP) was used to determine the organization of motoneuron pools innervating fin and epaxial muscles. HRP applied to a single peripheral nerve labeled motoneurons within a single spinal segment. Following intramuscular injection of HRP, 3 distinct cell groups were identified in the transverse plane. Motoneurons innervating elevator muscles were lateral in the ventral horn, while motoneurons innervating depressor muscles were dorsomedial. The epaxial muscles were found to be innervated by a distinct cell column along the ventral border of the ventral horn. Separate injections of the superficial and deep bundles of the elevator muscle resulted in considerable overlap in the distribution of labeled motoneurons. Soma areas for both elevator and depressor motoneurons were unimodally distributed. The mean cell diameters were 33.6 and 31.8 micron respectively. Motoneurons innervating the superficial and deep bundles of elevator muscle also had similar size distributions. The location of motoneurons innervating elevator and depressor fin muscles in the stingray supports the hypothesis that motoneurons innervating muscle derived from the dorsal premuscle mass are located laterally in the ventral horn while motoneurons innervating muscle derived from the ventral premuscle mass are located medially.