Development of the optic nerve of the opossum (Didelphis virginiana)

The development of the optic nerve of a marsupial, the North American opossum, was examined in 24 animals from postnatal days 5 to 78 (P5-P78): gestation is 13 days. The estimated number of axons increased from 24,000 at P5, to 267,000 at P27, approximately 2.7 times the mean number in the adult. Following P27, axon numbers decreased rapidly to 140,000 at P40, then decreased more slowly, attaining adult values between P50 and P59. Thus, the opossum is similar to placental mammals examined in evidencing an overproduction and later attenuation to adult values in the number of axons in the optic nerve during development. Monocular enucleation of 3 animals at P17, 10 days before peak axon counts, resulted in a mean population increase of 24,000 (range 19,000-30,000) above the normal adult mean. Additionally, a 4th animal monocularly enucleated on P7, 3 days prior to the arrival of migrating fibers to central target sites, had a similar value of 26,500 supernumerary axons. Our findings in the opposum, when coupled with previous reports in other mammals, suggest that binocular interactions during development account only for optic nerve axon loss approximately equal in magnitude to the ipsilateral projection from one eye.     

The distribution of ganglion cells in the retina of the north american opossum (Didelphis virginiana)

The distribution of ganglion cells in the retina of the opossum was determined from whole-mounted retinae stained with cresyl violet. Isodensity lines were approximately circular with a peak density of 2,000 to 2,700 cells/mm² in superior temporal retina (area centralis). The total number of retinal ganglion cells was estimated to be 72,000 to 135,000 (mean 101,026) in retinae ranging from 125 to 187 mm² in total area. Three groups of ganglion cells were distinguished on the basis of soma size and retinal topography. Large cells (24 to 32 μm diameter) were fairly evenly distributed across the retina. Medium cells (12 to 23 μm diameter) were more numerous in the superior temporal quadrant than in other regions of the retina. Small cells (7 to 11 μm diameter) were prominent in all retinal regions, but particularly in nasal and inferior retina. An analysis of topographical differences in soma size distribution suggests that the medium size cells can be further subdivided into small-medium and large-medium groups.     

Developmental plasticity of ascending spinal axons: Studies using the north american opossum,Didelphis virginiana

The objectives of the present study were to determine if axons of all ascending tracts grow through the lesion after transection of the thoracic spinal cord during development in the North American opossum, and if so, whether they reach regions of the brain they normally innervate. Opossum pups were subjected to transection of the mid-thoracic cord at PD5, PD8, PD12, PD20, or PD26 and injections of Fast Blue (FB) into the lower thoracic or upper lumbar cord 30–40 days or 6 months later. In the PD5 transected cases, labeled axons were present in all of the supraspinal areas labeled by comparable injections in unlesioned, age-matched controls. In the experimental cases, however, labeled axons appeared to be fewer in number and in some areas more restricted in location than in the controls. When lesions were made at PD8, labeled axons were present in the brain of animals allowed to survive 30–40 days prior to FB injections but they were not observed in those allowed to survive 6 months. When lesions were made at PD12 or later, labeled axons were never found rostral to the lesion. It appears, therefore, that axons of all ascending spinal pathways grow though the lesion after transection of the thoracic cord in developing opossums and that they innervate appropriate areas of the brain. Interestingly, the critical period for such growth is shorter than that for most descending axons, suggesting that factors which influence loss of developmental plasticity are not the same for all axons.     

Conduction of velocity groups in the optic nerve of the North American opossum (Didelphis virginiana): retinal origins and central projections

The axonal conduction velocity groups in the optic nerve of the North American opossum were analyzed electrophysiologically and related to soma size groups of ganglion cells in terms of their retinal origin and laterality of projection. On the basis of analysis of field potentials and single unit responses recorded at the optic disc, three velocity groups were identified (d1, d2, and d3) and estimated to have average conduction velocities of 12, 8, and 5 meters/second. From recordings of the field potential around the perimeter of the optic disc, it was found that the d1 group was equally represented at all points around the disc, whereas the d2 group was largest in amplitude in superior temporal regions. Electrical stimulation of the optic tracts indicated that axons in the d1 group project either ipsilaterally or contralaterally, whereas the d2 group projects predominantly ipsilaterally, and the d3 group projects predominantly contralaterally. In order to relate these physiological data directly to soma size groups, horseradish peroxidase (HRP) was injected into one optic tract, and subsequently the retinae were processed for peroxidase reaction product in the ganglion cells. Labeled cells were seen in contralateral nasal, contralateral temporal, and ipsilateral temporal retina. Cells in all size classes were labeled in contralateral nasal retina. In contralateral temporal retina, labeled cells were either 10-17 micrometers diameter (small and medium) or 23-27 micrometer diameter (large), whereas in ipsilateral temporal retina, most labeled cells (94%) were 15-30 micrometer diameter (medium and large). The correspondence between these conduction velocity groups and the soma size groups described in the preceding paper (Rapaport et al., "81) is discussed.     

The postnatal development of the optic nerve in hamsters: an electron microscopic study

The postnatal development of the optic nerve in the golden hamster has been examined using the electron microscope. The number of the optic fibres present in the optic nerve showed an initial increase during the first day after birth but it declined afterwards and tapered off after Day 16. At its peak on postnatal Day 1, there was an average of 314,629 axons in the optic nerve but when the animal reached adulthood only 109,587 fibres remained amounting to about 65% loss of the total fibre population. The period of axon loss coincided with the appearance of large patches of degenerated profiles in the optic nerve. The occurrence of the optic fibre loss has been implied to correlate with the time when the retinal projections were undergoing a dynamic reorganization at the target sites during the process of establishing the adult patterns of retinofugal connections.     

Rubrobulbar projections of the opossum (Didelphis virginiana)

The course and distribution of rubral pathways to the pons and medulla were determined for the opossum by employing the Nauta-Gygax and Fink-Heimer techniques on the brain stems of animals with lesions either within the red nucleus or involving the fibers emanating from it. Control material was provided by previous studies on corticomesencephalic and tectal efferent pathways and by the brains of specimens subjected to deep midbrain lesion which did not involve the red nucleus. A predominantly crossed rubrobulbar pathway coursed through the brain stem as described by Voris and Hoerr ('32) and distributed to the nucleus “K” of Meessen and Olszewski ('49), to neurons interspersed between the fiber bundles of the motor root of the trigeminal nerve, to the parabrachial nucleus of the brachium conjunctivum, the parvocellular reticular formation, the ventral and medial portions of the spinal trigeminal nucleus (nucleus oralis and interpolaris), the lateral and intermediate portions of the motor nucleus of the facial nerve, the lateral reticular nucleus, the ventral external arcuate nucleus and the subnucleus reticularis dorsalis medullae oblongatae. The possible significance of these connections in the opossum is discussed.     

Raphespinal projections in the north american opossum: Evidence for connectional heterogeneity

Retrograde transport studies revealed that the nuclei pallidus, obscurus, and magnus raphae as well as the adjacent reticular formation innervate the spinal cord in the opossum. HRP-lesion experiments showed that a relatively large number of neurons within the nucleus obscurus raphae and closely adjacent areas of the nucleus reticularis gigantocellularis project through the ventrolateral white matter and that many cells within the nucleus magnus raphae, the nucleus reticularis gigantocellularis pars ventralis, and the nucleus reticularis pontis pars ventralis contribute axons to the dorsal half of the lateral funiculi. Neurons within the rostral pole of the nucleus magnus raphae and the adjacent nucleus reticularis pontis pars ventralis may project exclusively through the latter route. Each of the above-mentioned raphe and reticular nuclei contain nonindolaminergic as well as indolaminergic neurons (Crutcher and Humbertson, 1978). When True-Blue was injected into the spinal cord and the brain processed for monoamine histofluorescence evidence for True-Blue was found in neurons of both types. Injections of ³H-leucine centered within the nuclei pallidus and obscurus raphae and/or the closely adjacent nucleus reticularis gigantocellularis labeled axons within autonomic nuclei and laminae IV-X. Labeled axons were particularly numerous within the intermediolateral cell column and within laminae IX and X. Injections of the caudoventral part of the nucleus magnus raphae or the adjacent nucleus reticualris gigantocellularis pars ventralis labeled axons in the same areas as well as within laminae I-III. When the injection was placed within the rostal part of the nucleus magnus raphae or the adjacent nucleus reticularis pontis pars ventralis axons were labeled within laminae I-III and external zones of laminae IV-VII, but not within lamina IX. The immunohistofluorescence method revealed evidence for indolaminergic axons in each of the spinal areas labeled by injections of ³H-leucine into the raphe and adjacent reticular formation. They were particularly abundant within the intermediolateral cell column and within laminae IX and X. These data indicate that raphe spinal systems are chemically and connectionally heterogeneous.     

The organization of monoamine neurons within the brainstem of the north american opossum (didelphis virginiana)

The distribution of monoamine-containing neurons within the brain of the opossum is described using the Falck-Hillarp histofluorescence technique. Catecholamine-containing neurons are organized into four groups. The medulla contains one group which is located dorsolateral to the lateral reticular nucleus and ventrolateral to the dorsal vagal nucleus. The second collection is found within the pons and includes both the locus coeruleus and a region continuous with it referred to as the nucleus coeruleus, pars α. The third aggregate includes the substantia nigra, the ventral tegmental area, and the mesencephalic reticular formation and a fourth group is located within the periventricular and dorsal paraventricular nuclei of the hypothalamus. The indoleamine-containing cell bodies are distributed within the nuclei raphe obscurus, pallidus, magnus, dorsalis, and the nuclei linearis and superior centralis except at certain pontine levels where they appear laterally within the reticular formation. A number of small intensely fluorescent (SIF) cells are present within the connective tissue surrounding the brain and its blood vessels as well. Although certain differences are present, the didtribution of monoamine neurons in the American opossum conforms generally to that described for the placental mammals studied to date.     

An autoradiographic study of midbrain-diencephalic projections to the inferior olivary nucleus in the opossum (Didelphis virginiana)

Techniques of intra-axonal transport were utlizied to elucidate the organization of diencephalic and midbrain projections to the inferior olivary nucleus of the Virginia opossum. Retrograde transport of horseradish peroxidase injected into the olive suggests that terminals within it arise from the subparafascicular nucleus of the caudal thalamus, the nucleus of Darkschewitsch, the fields of Forel, the interstitial nucleus of Cajal, the periaqueductal grey, the caudal pretectal nucleus, the tegmentum dorsomedial to the red nucleus, the red nucleus (minimal), the nucleus linearis, as well as the dorsolateral midbrain tegmentum and tectum (Henkel et al., '75). Tritiated leucine injections were made into each of the above-mentioned cell groups so that the olivary terminals of their axons could be demonstrated autoradiographically. In general, the projection systems show three basic patterns of organization. Ventromedial areas of the midbrain, including the ventral periaqueductal grey, the interstitial nucleus of Cajal, part of the red nucleus and the tegmentum dorso-medial to it, provide a substantial and topographically organized projection to the principal nucleus of the olive, as well as minor inputs to the accessory nuclei. Secondly, neurons within the subparafascicular nucleus, the nucleus of Darkschewitsch and the fields of Forel project most heavily to parts of the medial accessory nucleus, although they also provide input to the other major subdivisions of the olive. Third, axons from the dorsolateral tegmentum and tectum completely avoid the principal nucleus, while supplying small regions of the accessory nuclei.     

Efferent tectal pathways of the opossum (Didelphis virginiana)

Efferent tectal pathways have been determined for the opossum, Didelphis virginiana, by employing the Nauta-Gygax technique ('54) on animals with tectal lesions of varying sizes. The superior colliculus projected tectothalamic fascicles to the suprageniculate nucleus, the central nucleus of the medial geniculate body, the lateral posterior thalamus, the pretectal nucleus, the ventral lateral geniculate nucleus, the fields of Forel and zona incerta, the parafascicular complex, the paracentral thalamic nucleus and in some cases to restricted areas of the anterior thalamus. Degenerating fibers from superior collicular lesions showed profuse distribution to the deeper layers of the superior colliculus on both sides and to the midbrain tegmentum, but only minimally to the red nucleus and substantia nigra. Fibers of tectal origin did not distribute to the motor nuclei of the oculomotor or trochlear nerves. At pontine levels, efferent fascicles from the superior colliculus were present as an ipsilateral tectopontine and tectobulbar tract and as a crossed predorsal bundle. The tectopontine tract ended mostly within the lateral and ventral basal pontine nuclei, whereas the ipsilateral tectobulbar tract distributed to certain specific areas of the reticular formation throughout the pons and medulla, minimally to the most medial portion of the motor nucleus of the facial nerve and to the nucleus of the inferior olive. The predorsal tract contributed fascicles to certain nuclei of the pontine raphe, extensively to the medial reticular formation of the pons, to the central and ventral motor tegmental nuclei of the reticular formation within the pons and medulla, to the paraabducens region, minimally to cells within restricted portions of the motor nucleus of the facial nerve, to certail specific regions of the caudal medulla and to the cervical cord as far caudally as the fourth segment. The tectospinal fascicles were few but some ended related to the spinal accessory nucleus and the ventral medial nucleus of the ventral horn. Lesions of the inferior colliculus resulted in degenerating fibers which distributed rostrally to the rostral nucleus of the lateral lemniscus and parabrachial region, to the suprageniculate nucleus, the parabigeminal nucleus and to the central nucleus of the medial geniculate body. The inferior colliculus also contributed fibers to the ipsilateral tectopontine and tectobulbar tracts. The latter bundle was traced as far caudally as the medulla and may arise from cells of the superior colliculus which are situated dorsal to the nucleus of the inferior colliculus.     

An electron microscopic study of rubrospinal projections to the lumbar spinal cord of the opossum

The rubrospinal system is a major suprasegmental input to the important interneuronal pool at the base of the lumbar dorsal horn in the North American opossum. After appropriate lesions, rubral axons and their synaptic terminals were found in electron micrographs of lamina IV, V and VI as well as within the dorsal extreme of lamina VII. Degenerating terminals contact small diameter dendrites in the lateral terminal zone and large dendritic profiles in the medial terminal zone. Correlating these data with the dendritic arborizations of interneurons in Golgi preparations and with existing physiologic studies, it appears that interneurons in the intermediate and medial aspects of lamina V, VI and VII receive rubral input on both their proximal and distal dendrites.     

Observations on the development of brainstem-spinal systems in the north american opossum

The North American opossum is born 12 to 13 days after conception and is available for 90 days or more in an external pouch where it can be observed and experimentally manipulated. It is of particular interest that the hindlimbs of the newborn opossum are very immature and remain immobile for a week or more after birth. Degeneration techniques reveal that immature brainstem axons are present within the marginal zone of the lumbosacral cord before hindlimb movements begin (our stage I) and material processed for formaldehyde induced fluorescence shows that some of them transport monoamines. Several lines of evidence suggest that part of the fluorescent axons arise within the nucleus locus coeruleus. At this early stage the electron microscope reveals that all brainstem-spinal axons are small (0.1-0.4 μm in diameter) and unmyelinated. By the time random hindlimb movements can be observed (stage II), brainstem axons, including those transporting monoamines, can be demonstrated to have grown into limited areas of the intermediate zone of the lumbosacral cord and to arise from most of the areas contributing to them in the adult animal (horseradish peroxidase technique). Such axons are still immature and it is not yet clear that they have formed synaptic terminals. Brainstem axons continue to grow into the intermediate zone of the lumbosacral cord for some time and come to occupy all of their adult territories before thoracic transection produces obvious change in hindlimb motility (beginning of stage III). It is still another 20 days or so before thoracic transection produces spinal shock comparable to that in the adult animal. The relatively mature use of the hindlimbs and the full expression of spinal shock correlate with changes in the technique and survival time needed to demonstrate degenerating brainstem axons in experimental material.     

Afferent connections to neocortex in the opossum (Didelphis virginiana)

Afferent axons to the opossum's neocortex were carefully traced to their areas of termination using the Nauta-Gygax silver technique for degenerating axons. The two major systems of incoming fibers arise primarily from contralateral neocortex (commissural fibes), and ipsilateral thalamus (thalamocortical fibers). Following complete transection of the anterior commissure-corpus callosum amalgam, degenerating fibers could be traced to almost all areas of neocortex. The same pattern resulted from total removal of neocortex from one cerebral hemisphere. Commissural fibers arising from neocortex cross the midline in teh posterior two-thirds of the anterior commissure before terminating in contralateral neocortcial areas. The vast majority of fibers terminated in the deep cortical layers IV, V, VI. There was degeneration in layer III and occasionally in layer II, but essentially never in layer, I. A small central portion of striate cortex receives very few commissural fibers. Following thalamectomy, degenerating fibers could be traced to all areas of neocortex. One system of thalamocortical fibers terminates coextensively with commissural fibers in the deep layers IV, V, VI. A second system of thalamic projection arises from thalamic cells, traverses the cortical layers in parallel fascicles at 90⁰ to the pial surface, bifurcates and ends in the thick outer plexiform lamina (layer I) of neocortex. These fibers run for some distance parallel to the pial surface before terminating among apial dendrites of neurons from all cell layers. This system of layr I thalamocortical connectiions appears in all neocortical areas, even in primary receptive cortex in teh opossum. There is little evidence of the highly specialized axonal arborizatiions that characterize layer IV thalamic terminations in the primary receptive cortex of eutherian mammals.     

Development of an astrocytic response to lesions of the spinal cord in the north american opossum: An immunohistochemical study using anti-glial fibrillary acidic protein

We have shown previously that rubral axons grow around a lesion of their pathway in developing opossums and that a critical period exists for that plasticity. The critical period begins when rubral axons first reach the level of the lesion and ends sometime between postnatal days (PD) 26 and 30. The aim of the present study was to examine the development of an astrocytic response to lesioning the spinal cord to determine if there is a temporal correlation between the development of such a response and the end of the critical period. The astrocytic response was examined immunohistochemically, 2 and 4 weeks after hemisecting the thoracic spinal cord, using an antibody to glial fibrillary acidic protein (GFAP). A response was first seen at PD21 in the 2-week series. The response was relatively mild, however, and limited to the white matter. When the lesion was made at PD26. the response was still restricted to the white matter, but hypertrophied astrocytes were found at the gray/white matter junction and cystic cavities were present. When the lesion was made at PD41, the response had spread to the gray matter and it occupied a larger area rostral and caudal to the lesion than at earlier ages. The animals allowed to survive 4 weeks after lesioning were subjected to a second operation 4-5 days before sacrifice so that Fast Blue could be injected bilaterally two to three segments caudal to the lesion. When the hemisection was made at PD15, a response was present in the ventral and ventrolateral funiculi, but not in that part of the lateral funiculus that contains rubrospinal axons. As expected from previous studies, rubral neurons were labeled contralateral to the lesion, suggesting that their axons had grown around it. When the lesion was made at PD21, the glial response was still limited to the white matter, but it extended into that part of the lateral funiculus that contains rubral axons. In spite of the presence of a glial response, rubral neurons were still labeled contralateral to the lesion. When the lesion was made at PD26, hypertrophied astrocytes were present at the gray/white matter junction and small cavities were noted at the lesion site. In such cases, there was no evidence for rubrospinal plasticity. An astrocytic response was not observed in the gray matter of the dorsal horn, an area used by rubral axons to grow around a lesion of their pathway, until well after the end of the critical period. We conclude that the initial development of a glial scar in the white matter after lesioning does not determine the end of the critical period for rubrospinal plasticity. Loss of rubrospinal plasticity correlates most closely with the appearance of hypertrophied astrocytes at the gray/white matter junction and the formation of cystic cavities.     

Retinal ganglion cells in the South American opossum (Didelphis aurita)

By using the Golgi technique, the authors investigated the morphology of ganglion cells in the retinas of South American opossums. In flat-mount preparations of the retinas, cell bodies, entire dendritic fields, and the stratification level of ganglion cells were studied. Fractal dimensions of dendritic trees, an objective quantitative measure of morphological complexity, were included as a morphological parameter of classification. Based on these characteristics, nineteen types of ganglion cells were described. A great number of opossum ganglion cell types had dendrites stratifying in both sublaminae of the inner plexiform layer (IPL) in five different ways (S1-S3 [G9], S1-S4 [G17 and G22], S2/S3 [G19], S2-S4 [G15, G16, G21 and G221, and S2-S5 [G61), and only two types (G8, and G10) showed narrow field dendritic trees ramifying in S4 only. Morphological types of opossum ganglion cells were compared to their counterparts in cat retina. The distribution pattern of large cell bodies on the ganglion cell layer was analyzed employing the Nissl staining method, immunocytochemistry for neurofilaments, and the reduced silver neurofibrillar staining method. The results showed a random pattern of distribution.