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.

Development of raphe-spinal connections in the North American opossum.

The Falck-Hillarp technique, serotonin (5-HT) immunohistochemistry and the retrograde transport of horseradish peroxidase (HRP) were utilized to investigate the development of raphe-spinal connections in the pouch-young opossum. The brainstem raphe and adjacent reticular formation contain 5-HT immunoreactive neurons in the newborn opossum (12 days after conception) and processes from these cells can be visualized in the marginal zone of the spinal cord. Between eight and 15 days after birth 5-HT immunoreactive varicosities begin to grow into the presumptive deep layers of the dorsal horn, the intermediolateral cell column and the ventral horn. In the latter region some of them approximate presumed motor neurons. Between 40-50 days after birth 5-HT immunoreactive varicosities appear in presumptive laminae I and II of the dorsal horn.

The brainstem origin of monoaminergic projections to the spinal cord of the North American opossum: a study using fluorescent tracers and fluorescence histochemistry.

The retrograde transport of fluorescent markers has been combined with the glyoxylic acid and Falck-Hillarp techniques to identify the origin of monoamine axons within the spinal cord of the North American opossum. Catecholamine axons arise from neurons located within the ventrolateral medulla, dorsal to the superior olivary complex, within the dorsolateral and rostrolateral pons and within the periventricular nuclei of the hypothalamus. Such neurons are most numerous within the dorsolateral pons where they are found dorsal and lateral to the motor trigeminal nucleus, within the nucleus locus coeruleus pars alpha and adjacent reticular formation as well as within the ventral part of the nucleus locus coeruleus. Neurons containing the fluorescent marker and catecholamines were interspersed with others containing only the injected marker with the possible exception of the nucleus locus coeruleus. Spinal axons of the indoleamine type arise from neurons within the nuclei pallidus, obscurus and magnus raphe, the nucleus reticularis gigantocellularis, the nucleus reticularis gigantocellularis pars ventralis, the nucleus reticularis pontis pars ventralis and the nucleus dorsalis raphe. The latter nucleus only innervates rostral cervical levels. Most of the above areas also contain many non-indoleamine neurons which were labelled by the injected marker. This was particularly true of the nucleus magnus raphe and the adjacent nucleus reticularis points pars ventralis after injections of fluorescent markers into the superficial dorsal horn.

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 opposum. 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, '78). 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 reticularis gigantocellularis pars ventrialis labeled axons in the same areas as well as within laminae I-III. When the injection was placed within the rostral 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.

Evidence for a lack of distinct rubrospinal somatotopy in the North American opossum and for collateral innervation of the cervical and lumbar enlargements by single rubral neurons.

Studies using axonal transport techniques on the North American opossum show that rubral neurons innervating the cervical cord are not distinctly separated from those which project to lumbar levels. The absence of clear rubrospinal somatotopy contrasts with that described for the placental mammals studied to date. Use of fluorescent markers in double-labeling experiments shows that most rubral neurons in the opossum still innervate either the cervical or lumbar enlargement alone, but that some supply collaterals to both levels.

Evidence for collateral innervation of the cervical and lumbar enlargements of the spinal cord by single reticular and raphe neurons. Studies using fluorescent markers in double-labeling experiments on the North American opossum.

The use of fluorescent markers in double-labeling experiments reveals the presence of reticular and raphe neurons in the opossum's brainstem which innervate both the cervical and lumbar enlargements of the spinal cord by way of axonal collaterals. Such neurons were mixed with those innervating either the cervical or lumber enlargement alone and were found within the nuclei reticularis medullae oblongatae dorsalis and ventralis, the nucleus reticularis gigantocellularis, the nucleus reticularis gigantocellularis pars ventralis, the nucleus reticularis pontis and the nuclei obscurus and magnus raphae. In some nuclei over 50% of the neurons projecting to the cervical enlargement also innervate lumbar levels.

Spinal projections from the medullary reticular formation of the North American opossum: heterogeneity.

Retrograde and orthograde transport techniques show that the nucleus reticularis gigantocellularis pars ventralis and the nucleus reticularis gigantocellularis project the entire length of the spinal cord. Double-labelling methods show that some of the neurons in each area innervate both cervical and lumbar levels. There is evidence, however, that neurons in the lateral part of the nucleus reticularis gigantocellularis pars ventralis and the dorsal extreme of the nucleus reticularis gigantocellularis project mainly to cervical and thoracic levels. The autoradiographic method shows that the above nuclei supply direct innervation to somatic and autonomic motor columns as well as to laminae V-VIII and X. The nucleus reticularis gigantocellularis pars ventralis provides additional projections to lamina I and the outer part of lamina II. Several areas of the medullary reticular formation project mainly, and in some cases exclusively, to cervical and thoracic levels. These areas include the nucleus reticularis parvocellularis, the nucleus reticularis lateralis, the nucleus retrofacialis, the nucleus ambiguus, the nucleus lateralis reticularis, caudal parts of the nuclei reticularis medullae oblongatae dorsalis and ventralis, and the nucleus supraspinalis. Autoradiographic experiments reveal that neurons in the ventrolateral medulla, particularly rostrally (the nucleus reticularis lateralis and neurons related to the nucleus lateralis reticularis), innervate sympathetic nuclei. Our results indicate that spinal projections from bulbar areas of the reticular formation are more complicated than previously supposed. Axons from separate areas project to different spinal levels and in some cases to different nuclear targets. These data are in conformity with the evolving concept of reticular heterogeneity.

Projections from the brain stem reticular formation to laminae I and II of the spinal cord. Studies using light and electron microscopic techniques in the North American opossum.

The horseradish peroxidase and autoradiographic methods show that laminae I and outer II are innervated by the nucleus reticularis gigantocellularis pars ventralis, and the nucleus reticularis pontis pars ventralis. Both areas contain neurons of the indolamine type and probably account for the indolamine-like varicosities which are present within laminae I and II. Degeneration materiom the above nuclei end on small dendritic shafts and spines as well as on vesicle-filled proflies. The terminals identified formed asymmetrical contacts and contained clear as well as dense-cored vesicles. No terminals were present within glomeruli. A projection to laminae I and outer II also arises within the dorsolateral pons and several ines of evidence suggest that it is catecholaminergic. The electron microscope revealed that axons from the dorsolateral pons are fairly numerous within laminae I and II, but that terminal contacts are relatively rare. Those present are asymmetrical and alternate with intermediate-sized dendrites. They contain clumps of clear, spherical vesicles as well as larger vesicles with a variety of dense cores.

Spinal projections from the mesencephalic and pontine reticular formation in the North American Opossum: a study using axonal transport techniques.

The results from several experimental approaches lead to the following conclusions. The nucleus cuneiformis projects to at least lumbar levels of the spinal cord. Its axons course through the ipsilateral sulcomarginal and ventral funiculi to distribute within lamina VIII and adjacent portions of lamina VII. Neurons within the nucleus reticularis pontis (RP), particularly within more medial parts of the nucleus, project through comparable routes to the same laminae. In addition, however, neurons within the lateral and dorsolateral RP relay through the lateral and dorsolateral funiculi, ipsilaterally, and the dorsolateral funiculus, contralaterally. Axons could be traced from the dorsolateral tracts to laminae IV through VII, lamina X and, in some instances, to laminae I and II. Injections of the dorsolateral pons also label the intermediolateral cell column and an area presumed to be the sacral parasympathetic nucleus. Many of the neurons which contribute to the contralateral bundle are located adjacent to the ventral nucleus of the lateral lemniscus. The nucleus reticularis gigantocellularis projects mainly via the sulcomarginal, ventral and lateral funiculi to laminae VIII and adjacent portions of lamina VII. The nucleus reticularis gigantocellularis pars ventralis innervates the same laminae; but, in addition, projects heavily to laminae I and II, to lateral portions of laminae IV through VII; to laminae IX and X and to the intermediolateral cell column. Axons destined for laminae I and II, as well as IV through VII and X, traverse the dorsolateral funiculi as described for the cat by Basbaum et al. ('78). Neurons within the nucleus reticularis parvocellularis project to cervical levels, mainly through the ventral funiculi. In general our results show that reticulospinal projections are more complex than suggested by degeneration methods and that laminae I, II. lateral parts of laminae IV-VII, laminae IX and X, as well as the intermediolateral cell column and sacral parasympathetic nucleus are targets of axons from specific areas.

The development of monoaminergic brainstem-spinal systems in the North American opossum.

Evidence is presented for an early appearance of monoaminergic neurites within the spinal cord of the developing opossum. They are present within the marginal zone before hindlimb movements begin (stage I) and they start to grow into the intermediate zone by the time hindlimb movements are first observed (stage II). Monoaminergic neurites grow first into the dorsolateral intermediate zone and the intermediolateral cell column where they can be found by the beginning of stage II. Shortly thereafter, fluorescent varicosities can be traced into the area dorsal to the central canal presumed to become lamina X. Fluorescent processes extend in to the ventral intermediate zone (ventral horn) somewhat later in development. Monoaminergic axons have grown into all of the areas they occupy in the adult animal, except for laminae I and II, by the time immature hindlimb movements can be altered by cutting all brainstem projections to the lumbosacral cord (stage III). Monoaminergic innervation of laminae I and II is the last to develope, but it is present by the time thoracic transection produces complete spinal shock.

Observations on the development of brainstem-spinal systems in the North American oppossum.

The North American oppossum is born 12 to 13 days after conception and 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 state 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 micrometer 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.