The early development of subcortical projections to presumptive somatic sensory-motor areas of neocortex in the North American opossum.

We have studied the early development of subcortical projections to presumptive somatic sensory-motor areas of neocortex in the North American opossum Didelphis virginiana. The opossum is born in a very immature state, 12-13 days after conception, and climbs into an external pouch where it is available for experimental manipulation. Using the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, we have obtained evidence that axons from the dorsal raphe and superior central nuclei, the substantia nigra, the locus coeruleus and the parabrachial nuclei reach presumptive somatic sensory-motor areas of neocortex by at least postnatal day (PND) 10. Axons showing serotonin-like immunoreactivity, presumably from the dorsal raphe and/or superior central nuclei, and axons containing tyrosine hydroxylase immunoreactivity, presumably from the substantia nigra and/or locus coeruleus, are present in the same areas at birth or shortly thereafter. Thalamic axons do not grow into comparable areas of neocortex until after PND 10. Such axons reach the subplate region of ventrolateral neocortex first and then proceed dorsomedially; by estimated PD (EPND) 21, they are present in presumptive layers I, V and VI, but they do not innervate an identified layer IV until EPND 48. The developmental sequences suggested by our study are compared with those reported for other species and are discussed in light of their importance in the formation of major sensory and motor circuits.

Organization of forebrain projections from the medullary reticular formation in the North American opossum. Evidence for connectional heterogeneity.

We have employed axonal transport techniques to study the organization of projections from the medullary reticular formation (RF) to the forebrain of the North American opossum. The results of retrograde transport studies using large injections of horseradish peroxidase (HRP), wheat germ agglutinin conjugated to HRP (WGA-HRP), and fluorescent markers suggest that all nuclei of the medullary RF project to the forebrain although the parvocellular reticular nucleus makes only a very small contribution. When injections of 3H-leucine or WGA-HRP are centered within areas shown by the retrograde transport studies to innervate the forebrain, characteristic patterns of orthograde labeling are produced. In most cases labeled axons form a major pathway which splits into dorsal and ventral divisions. The dorsal division innervates the parafascicular and central nuclei of the thalamus as well as the pretectum. In contrast, the ventral division projects to the lateral hypothalamus, the zona incerta, the ventral and dorsal lateral geniculate nuclei, the lateral part of the ventrobasal nucleus of the thalamus, the lateral preoptic area, the septal-diagonal band region, and the cerebral cortex. When injections are centered within specific ventrolateral areas of the medullary RF, ventral division labeling is also found within the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus. Relatively small injections of HRP or WGA-HRP into specific areas of the forebrain produce labeling which suggests that some areas receive projections from all nuclei of the medullary RF, whereas others do not. Our results suggest that forebrain projections of the medullary RF, like those to the spinal cord, are connectionally heterogeneous.

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.

Serotonergic innervation of the forebrain in the North American opossum.

The forebrain distribution of axons showing serotonin-like immunoreactivity was studied in the North American opossum. Serotonergic innervation of the hypothalamus was extensive, particularly within the ventromedial nucleus, the periventricular nucleus and the rostral supraoptic nucleus. Serotonergic axons were also present within the fields of Forel and zona incerta, but they tended to avoid parts of the subthalamic nucleus. In the thalamus serotonergic innervation was dense within the midline nuclei (e.g. the central, intermediate dorsal and rhomboid nuclei) and the ventral lateral geniculate nucleus, but relatively sparse in some of the nuclei more readily associated with specific functions (e.g. the ventrobasal nucleus). Serotonergic axons innervate most areas of the rostral and dorsal forebrain. Areas containing the heaviest innervation included the interstitial nucleus of the stria terminalis and the lateral septal nucleus. Serotonergic innervation of the neocortex varied markedly from region to region and within different layers of the same regions. The retrograde transport of True Blue combined with immunofluorescence for localization of serotonin revealed that serotonergic axons within the forebrain arise mainly within the dorsal raphe and superior central nuclei, but that some originate within the midbrain and pontine reticular formation and the locus coeruleus, pars alpha. Neurons of the raphe magnus and obscurus also innervate the forebrain, but few of them are serotonergic. The use of horseradish peroxidase as a retrograde marker provided evidence that raphe projections to the forebrain are topographically organized. Our results suggest that serotonergic projections to the forebrain, like those to the spinal cord, are connectionally heterogeneous.

Developmental sequence in the origin of descending spinal pathways. Studies using retrograde transport techniques in the North American opossum (Didelphis virginiana).

The origin of descending pathways to thoracic and cervical levels of the spinal cord has been investigated with retrograde tracing techniques in a series of pouch young and adult opossums. The opossum was chosen because it is born in a very immature state, 12-13 days after conception, and has a protracted development in an external pouch. A few neurons in the pontine reticular formation and nucleus coeruleus were labeled by horseradish peroxidase (HRP) injections of the thoracic cord as early as postnatal day (PND) 3. By PND 5, similar injections labeled neurons in the same areas as well as in the medullary reticular formation, the raphe nuclei of the caudal pons and medulla, the spinal trigeminal nuclei, the vestibular complex, the accessory oculomotor nuclei and the interstitial nucleus of Cajal. When Nuclear Yellow (NY) was employed, neurons were also labeled in the red nucleus, the hypothalamus and possibly in the nucleus of the solitary tract. Regardless of the technique employed, neurons in the dorsal column nuclei were not labeled by thoracic injections until at least PND 14. Axons from the nucleus ambiguus, the fastigial and interposed nuclei of the cerebellum as well as the intermediate and deep layers of the superior colliculus reach cervical levels of the cord, where they are specifically targeted, by at least PND 17. They do not significantly overgrow those levels during development. Corticospinal axons are the last of the major descending pathways to innervate the spinal cord. Cortical neurons cannot be labeled by cervical injections of either HRP or NY until at least PND 30. Evidence for transient brainstem-spinal and corticospinal projections was obtained.

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.

The origin of brain stem-spinal projections at different stages of development in the North American opossum.

The origin of brain stem neurons giving rise to axons innervating the thoracic cord has been determined in a developmental series of pouch-young opossums. The first spinal axons identified (17 days after conception, postnatal day 5) arise from the medullary and pontine reticular formation, certain raphe nuclei, the vestibular nuclei and the coeruleus complex. The contribution of the red nucleus before postnatal day 10 is not certain. The brain stem sensory relay nuclei and the hypothalamus do not project spinalwards until much later in development.

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.

Corticobulbar fibres in the North American opossum (Didelphis marsupialis virginiana) with notes on the Tasmanian brust-tailed possum (Trichosurus vulpecula) and other marsupials.

Corticobulbar projections have been studied in the American opossum by both degeneration and autoradiographic methods and, for the most part, the results confirm our earlier observations (Martin & West, 1967; Martin, 1968). However, we have obtained evidence for certain connexions not previously described and have delineated the origin(s) of several connexions more precisely by paying particular attention to the degeneration present at thalamic levels in all cases and by the use of autoradiography. When our results are collated and correlated with new somatosensory cortical maps arrived at by microelectrode techniques (Pubols et al. 1975), it is obvious that corticolbulbar connexions in the North American opossum are remarkably similar to those in the monkey and differ mainly in quantity, relative origins and distribution and in the fact that some of them arise from spatially co-extensive motor-sensory areas (Lende, 1963a, b). In the light of our findings on the American opossum we have examined a large collection of brush-tailed possum material (as well as some from the potoroo and Tasmanian native cat) and have been able to extend our previous findings (Martin et al. 1971; Martin & Megirian, 1972) to a more precise evaluation of the origin of projections from the limb, face motor-sensory cortex. Differences between these representatives of the marsupial radiation, as well as features which are common to all, are described.