Cytoarchitecture of the substantia nigra pars lateralis in the opossum (Didelphis virginiana): a correlated light and electron microscopic study.

BACKGROUND: The substantia nigra has been divided into three subdivisions. However, the cytoarchitecture of one of these subdivisions, the pars lateralis (SNl), has not been previously examined in detail at the light and electron microscopic levels in any species. In the adult opossum, the three nigral subdivisions can be easily distinguished as distinct, rostrocaudally oriented cell groups separated by neuron-free zones. Thus it was possible to determine the boundaries of the SNl unambiguously. This report covers the results of an examination of the morphology and organization of the SNl in the opossum. METHODS: Material from 13 opossums was used for this study. Eight of the animals had been previously stained for Nissl substance (n = 4) or impregnated by the Golgi technique (n = 4). The remaining five animals were prepared for electron microscopic studies using standard procedures. RESULTS: Two cell types were identified on the basis of morphological differences, small and medium-large neurons. Small neurons (10-18 microns long axis) have large nuclei with moderate amounts of heterochromatin and a thin rim of cytoplasm. They have long (up to 500 microns), spine-free dendrites. Medium-large neurons (18-54 microns long axis) have rounded nuclei with electron-lucent nucleoplasm. Few indentations of the nuclear envelope were observed. The surrounding cytoplasm has dense arrays of organelles. Nissl bodies are particularly prominent in the form of pyramids with their bases at juxtanuclear positions and their apices directed toward emerging dendrites. Dendrites of medium-large neurons are long (some > 1 mm in length), are primarily oriented in the frontal plane, and extend along the dorsal surface of or into the cerebral peduncle. Some cells have dendrites that are moderately spinous, whereas other neurons possess sparsely spinous dendrites. Relatively few synaptic profiles are observed to contact somata and proximal dendrites. CONCLUSION: This report provides added morphological support for the idea that the SNl is a distinct subdivision of the substantia nigra, a distinction previously made on the basis of the physiologically characterized relationship between the lateral substantia nigra and orienting behaviors and seizure-related function.

Immunocytochemical evidence for the involvement of glycine in sensory centers of the rat brain.

Glycine-like immunoreactivity was localized to a number of sites in the rat brain which are involved in processing sensory information. In the auditory and vestibular systems, glycine immunoreactivity was seen in dorsal and ventral cochlear nuclei, superior olive, trapezoid body, medial and lateral vestibular nuclei, and inferior colliculus. Staining in the visual system was seen in retina, dorsal lateral geniculate nucleus, and superior colliculus. The olfactory system exhibited staining in the olfactory bulb and accessory olfactory formation. Somatosensory centers with glycine immunoreactivity included the dorsal column nuclei, spinal trigeminal nucleus, principal sensory nucleus of V, reticular formation, and periaqueductal gray. Glycine-immunoreactive neurons were also seen in cerebellar cortex, deep cerebellar nuclei, hippocampus, cerebral cortex, and striatum. The distribution of staining indicates that glycine plays a major role in sensory centers with actions at both strychnine-sensitive and strychnine-insensitive receptors.

Organization of midbrain catecholamine-containing nuclei and their projections to the striatum in the North American opossum, Didelphis virginiana.

Presumptive catecholamine (CA) neurons in the opossum midbrain were identified by tyrosine hydroxylase immunohistochemistry. In the midline, small to moderate number of CA cells were present in the rostral third of the nucleus raphe dorsalis and throughout the nucleus linearis. Ventrolaterally, such cells were observed in the deep tegmental reticular formation, in all subnuclei of the ventral tegmental area, and in the three subdivisions of the substantia nigra. The CA cells in these areas conform to the dopamine cell groups, A8, A9, and A10 as described in the rat. In several areas there appeared to be no separation between the CA neurons belonging to cytoarchitecturally different nuclei. In order to determine which CA neurons gave rise to striatal projections, the neostriatum was injected with True Blue (TB), and sections through the midbrain were processed for tyrosine hydroxylase (TH) and visualized by immunofluorescence. Neurons containing both TB and TH were observed in each of the CA cell groups mentioned above. The distribution of these cells confirmed organizational features that may be unique to the opossum's substantia nigra. In addition, different patterns of labeling resulted from caudate versus putamen injections, suggesting a rudimentary medial to lateral topography in the organization of nigrostriatal projections. Although our results suggest that the organization of midbrain CA neurons in the opossum is similar to that in placental mammals, it is clear that differences exist.

The early development of major projections to the dorsal striatum in the North American opossum.

We have employed immunocytochemical and axonal transport techniques to study the development of major projections to the dorsal striatum of the North American opossum. The opossum is born in a very immature state, 12-13 days after conception, and climbs into an external pouch where it remains attached to a nipple for several months. Its immaturity at birth and its protracted postnatal development make the opossum a good model for developmental studies. Although tyrosine hydroxylase-like immunoreactive (TH-LI), presumably dopaminergic, neurons were present in the ventral mesencephalon at birth (the presumptive substantia nigra and ventral tegmental area), there was no evidence for TH-LI axons in the striatal anlage. By postnatal day (PD)6, a few immunostained axons were found within the putamen. The subsequent growth of TH-LI axons into the striatum followed general caudal to rostral and ventrolateral to dorsomedial gradients and, at any age, they were most numerous in the areas exhibiting the greatest cytodifferentiation. By estimated (E)PD45, TH-LI axons were present in most, if not all, areas of the striatum. Serotoninergic (5-HT)-LI axons were found lateral to the presumptive striatum at birth but not within it. By PD7, however, a few 5-HT-LI axons could be identified in the putamen. The growth of 5-HT-LI axons into the striatum generally followed the same gradients described for TH-LI axons although at all ages their density was much less. Using the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), evidence was obtained for the existence of thalamostriatal projections by PD5 and for corticostriatal projections by PD10. Crossed corticostriatal projections were present by EPD23. Our results suggest that the development of major projections to the striatum occurs postnatally in the opossum, rather than prenatally as in placental animals. The timetable for striatal innervation is discussed in light of the developmental sequences established for other motor circuits.

Innervation of the pituitary gland by supraependymal neurons.

Retrograde transport of horseradish peroxidase (HRP) was used to identify supraependymal neurons projecting to the pituitary gland in the hamster. Supraependymal neurons overlying the median eminence were labeled by HRP injections into the neural and intermediate lobes of the pituitary gland; no neurons were labeled following HRP injections confined to the anterior lobe. Supraependymal neurons innervating the pituitary gland may provide a means whereby cerebrospinal fluid-borne substances modulate neuro-intermediate lobe function.

GABA-immunoreactivity in ganglion cells of the rat retina.

Ganglion cells in the rat retina were labeled with the fluorescent dye, Diamidino-yellow, by retrograde transport from the superior colliculus and subsequently reacted for GABA-like immunoreactivity with a rhodamine-conjugated antiserum. Examination of sectioned retinas by fluorescence microscopy showed double labeling in approximately 6% of the ganglion cells. The presence of GABA in these neurons suggests that they may be involved in providing direct inhibitory input to the rat tectum.

The development of selected rubral connections in the North American opossum.

We have employed axonal transport and degeneration techniques to study the development of selected rubral connections in the North American opossum. Opossums were chosen for study because they are born in an immature state, 12 days after conception, and have a lengthy postnatal development. The results of our studies suggest that: (1) the red nucleus innervates the spinal cord early in development, but not as early as some areas of the brainstem; (2) rubrospinal development occurs postnatally in the opossum; (3) rubrospinal axons do not grow synchronously into the spinal cord, but are added over time; (4) rubrospinal development follows rough rostral to caudal and lateral to medial gradients; (5) the red nucleus is innervated by the cerebellum well before it receives projections from the cerebral cortex; and (6) cortical axons do not grow into the red nucleus until after rubrospinal axons have reached most of their adult targets.

Development of brainstem and cerebellar projections to the diencephalon with notes on thalamocortical projections: studies in the North American opossum.

The North American opossum is born in a very immature state, 12 days after conception, and climbs into an external pouch where it remains attached to a nipple for an extended period of time. We have taken advantage of the opossum's embryology to study the development of brainstem and cerebellar projections to the diencephalon as well as the timing of diencephalic projections to somatosensory motor areas of neocortex. The techniques employed included immunocytochemistry for serotonin, the retrograde and orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, and the selective impregnation of degenerating axons. Our results suggest that serotoninergic axons, presumably from the dorsal raphe and superior central nuclei, are present in the diencephalon at birth. Axons from the bulbar reticular formation, the vestibular complex, the trigeminal sensory nuclei, and the dorsal column nuclei reach at least mesencephalic (and probably diencephalic) levels by postnatal day (PND) 3, whereas those from the cerebellar nuclei may not grow into comparable levels until PND 5. The dorsal column and cerebellar nuclei innervate the ventral nuclei of the thalamus by estimated postnatal day (EPND) 17 and all of the diencephalic nuclei supplied in the adult animal by EPND 26. Diencephalic axons enter ventrolateral (face) areas of presumptive somatosensory motor cortex by PND 12, but do not reach dorsomedial (limb) regions until EPND 21. At both ages, diencephalic axons are limited to the cortical subplate and marginal zone; they do not innervate an identifiable internal granular layer until considerably later. Our results suggest that axons from the brainstem and cerebellum grow into the diencephalon early in development, but that they do not influence the cerebral cortex until relatively late. When the results of the present study are compared with those reported previously on the development of ascending spinal (Martin et al., '83) and corticofugal (Martin et al., '80; Cabana and Martin, '85b,c) projections, it appears that specific components of major somatosensory and motor circuits develop according to different timetables.

Development of the basilar pons in the North American opossum: dendrogenesis and maturation of afferent and efferent connections.

The present study provides data on temporal factors that may play a role in the development of precerebellar-cerebellar circuits in the North American opossum. In this study the basilar pons and cerebellum are analyzed from birth, 12-13 days after conception, to approximately postnatal day (PD) 80 at which time the brainstem and cerebellum have a mature histological appearance. In Nissl preparations, the basilar pons was first seen at PD 7 as a small cluster of tightly packed cells. Analysis of Golgi impregnations revealed that dendritic growth occurred between PD 25-80. During this period, dendrites gradually increased in length and in the complexity of their branching pattern. Horseradish peroxidase (HRP) was placed into the cerebellar and cerebral cortices in order to examine the development of efferent and afferent projections of the basilar pons, respectively. Evidence for the growth of pontine axons into the cerebellum was first detected on PD 17. Neurons located dorsally within the basilar pons appear to be the first neurons retrogradely labeled with horseradish peroxidase. By PD 27 retrogradely labeled neurons are found throughout the basilar pons. Afferent fibers from the cerebral cortex are not seen within the neuropil of the nucleus until after PD 25 and by PD 29, they have greatly expanded their terminal fields. Degeneration techniques reveal that afferent fibers from the cerebellum arrive by PD 19 and increase in number until PD 30 when their adult distribution is achieved. These data suggest that the time of afferent arrival from the cerebral cortex and deep cerebellar nuclei is closely correlated in time with the initiation of dendritic maturation and the outgrowth of pontocerebellar axons. Afferent axons from the cerebral cortex and deep cerebellar nuclei reach the basilar pons and afferents from the basilar pons grow into the cerebellum when the dendrites of the respective target neurons are very immature. Thus, the time of axon arrival in these circuits may be an important factor in determining their synaptic location on individual neurons. The data derived from the present study is compared to those obtained in previous studies on the inferior olive. The results of this comparison provide evidence for a similar sequence of events, but a differential timetable for the development of specific connections within precerebellar-cerebellar circuits.

Origin and topography of thalamocaudate projections in the opossum.

The origins of the thalamocadaute projections in the North American opossum include the intralaminar nuclei (parafascicular complex, the paracentral, centralis and central lateral nuclei) and the intermedial dorsal, medial dorsal and interanterior dorsal nuclei. Of these, portions of the intralaminar nuclei exhibit rudimentary elements of a topographically organized projection. However, it is obvious that the patterns observed here are much less specific than those reported in other mammalian species. We suggest that the question of topographic specificity may relate to the overlapping of sensory and motor representations in the neocortex.

Observations on the early development of ascending spinal pathways. Studies using the North American opossum.

The development of ascending spinal pathways has been studied in the North American opossum using degeneration methods and the retrograde transport of horseradish peroxidase. Axons from caudal thoracic and/or lumbosacral levels of the spinal cord reach the lateral reticular nucleus, the inferior olivary complex, the reticular formation of the medulla and pons as well as the cerebellum very early in development. Innervation of the nucleus gracilis occurs somewhat later. Spinal axons grow into most of the caudal brain stem areas they occupy in the adult animal, including the nucleus gracilis, before there is convincing evidence that they reach the thalamus. Although spinal axons enter the cerebellum early in development their adult distribution with its characteristic discontinuities appears relatively late.

Organization of projections from the gracile, medial cuneate and lateral nuclei in the North American opossum. Horseradish peroxidase study of the cells projecting to the cerebellum, thalamus and spinal cord.

Using the horseradish peroxidase technique on the North American opossum, we were able to locate the neurons within the dorsal column and lateral cuneate nuclei which innervate the cerebellum and thalamus as well as those within the dorsal column nuclei which project spinalward. The medial and lateral cuneate nuclei supply axons to the anterior lobe, the paramedian lobule and the pyramis of the cerebellum and the lateral nucleus provides an additional projection to the uvula. The cerebellar projections from these nuclei arise from neurons located rostral to the obex. The thalamic projections from the gracile and medial cuneate nuclei originate from neurons throughout their rostral to caudal extent, although most of them are located just rostral to the obex. Neurons within the lateral cuneate nucleus which innervate the thalamus are found at intermediate rostrocaudal levels where most of them approximate the medial cuneate nucleus. The medial cuneate also projects to at least lumbar levels of the spinal cord in the opossum and neurons giving rise to such connections are found at the level of the obex and caudal to it. Neurons within the dorsal part of the dorsal column nuclei were labelled only after thalamic injections. Our results in the opossum are compared with those obtained in several placental mammals.

The pineal region in the opossum, Didelphis virginiana. I. Ultrastructural observations.

In the pineal region of the opossum, Didelphis virginiana, two types of cells predominate: 1) pinealocytes, and 2) fibrous astrocytes. Pinealocytes are characterized by the presence of prominent Golgi bod'ies, numerous clear and dense-cored vesicles, sensory cilia (9+0), vesicle-crowned rods, and condensation of a maternial that was always associated with the rough endoplasmic reticulum. In addition, two other cell types are occasionally seen. These include 1) neuron-like cells, and 2) darker staining cells of unknown identity. The endoplasmic reticulum of the darker staining cells is typically expanded and filled with an amorphous substance. Although the pineal region is small in size, the present findings suggest that pinealocytes in this species are metabolically active cells displaying a secretory function. Moreover, the presence of sensory cilia (9+0) and vesicle-crowned rods indicates that pinealocytes of the opossum are phylogenetically related to the photoreceptor cells found in the pineal organ of lower vertebrates.

Small neurons in the parafascicular complex in the opossum: a golgi study.

Golgi preparations of the caudal intralaminar area in the opossum contain two varieties of small neurons. The first variety, the short axon cells possess intrinsic beaded axons and a few axon-like processes. The second type, although possessing certain features of the short axon cells, has a more extensive denritic arborization and gives rise to an axon initial segment.

The neurons in the centromedian-parafascicular complex of the monkey (Macaca mulatta): a Golgi study.

The neurons of the nucleus centrum medianum and the neurons of the nucleus parafascicularis were studied in Golgi preparations of the adult monkey (Macaca mulatta). The cell bodies of the prinicipal neurons in the nucleus centrum medianum have a few somatic spines and vary in shape: some are cubical with protruding angles; some are egg-shaped; some are elongated and sausage-shaped. Four to six slightly branched dendrites of unequal thickness radiate from the cell body. Some dendrites extend for nearly 500 microns; all have dendritic spines. In the nucleus parafascicularis there are two varieties of principal neurons: (1) neurons with somatic spines and (2) neurons without somatic spines. The neurons with somatic spines are most numerous. They have polygonal-shaped cell bodies, prominent somatic spines and processes, larger than spines but considerably smaller than dendrites. These processes bear spines and are designated here "microdendrites." Spines and occasionally a "microdendrite" are found on the axon-hillocks. Five to six dendrites of unequal thickness emerge from the cell bodies. Some extend for more than 500 microns; all have prominent dendritic spines. The neurons without somatic spines are relatively few. Usually three exceptionally long, slightly branched dendrites, one apical and two basal, emerge from their elongated, slim cell bodies. Some dendrites extend for more than 800 microns; all have few scattered spines. The Golgi type II neurons found in both of these intralaminar nuclei have small cell bodies and a few, relatively long, undulating dendrites, which bear bulbous dendritic appendages and beaded axon-like processes. Distally on these dendrites, where the appendages and processes are more numerous, the dendritic appendages and axon-like processes form complex entanglements. Distally on these dendrites, where the appendages and processes are more numerous, the dendritic appendages and axon-like processes form complex entanglements. Beaded axons are found on some but not all of the cell bodies. Morphologically these neurons resemble the local interneurons that have been described in various thalamic nuclei.