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.     

Laminar origins of spinothalamic projections in the cat as determined by the retrograde transport of horseradish peroxidase

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the locations of cells of origin of the spinothalamic tract in the cat. Injections of from 0.2–3.0 μl of 30% HRP were made unilaterally in various regions of the somatosensory thalamus. Massive injections of the caudal thalamus in several cats showed the spinothalamic cells of origin to be located mainly in laminae I, VII and VIII in the lumbar enlargement, and in laminae I, V and VII–VIII in the cervical enlargement. Small injections of HRP were made into the three major spinothalamic terminal zones in the thalamus, to determine the laminar origin(s) of the spinal projections to each zone. Neurons in lamina I in both cervical and lumbar enlargements were found to project almost exclusively to the rostral VB-caudal VL border zone. A small number of neurons in laminae VII and VIII also project there but a larger number project to the intralaminar region. Neurons projecting to the PO regions were located mainly in laminae IV and V. This anatomical segregation of thalamic afferents probably reflects a functional segregation of input, since the functional properties of spinal neurons vary according to their laminar location. Comparison of these data with the differential projection spinothalamic neurons in the rat and monkey indicate that it is unlikely that the proposed “paleo-” and “neospinothalamic” systems would arise from anatomically separate groups of spinal neurons.     

The afferent projections to the centrum medianum of the cat as demonstrated by retrograde transport of horseradish peroxidase

The afferent projections of nucleus centrum medianum (CM) of the thalamus were studied, in the cat, by means of retrograde transport of electrophoretically ejected horseradish peroxidase. Several variations of method — survival time, fixatives, substrates, etc. — were tried to improve the amount of visible reaction product.Labeled neurons were localized primarily in two categories of nuclei in the brain. The first consisted of structures making up or closely related to the basal ganglia: the entopeduncular nucleus, the pars reticulata of the substantia nigra, and motor cortex. The second category was made up of nuclei closely related to postural and orienting functions: the deep layers of the superior colliculus ipsilaterally, and the medial and lateral vestibular nuclei bilaterally. Other nuclei containing retrogradely labeled neurons were the periaqueductal gray and locus coeruleus. Brain stem reticular projections were sparse and widely scattered. These results identify CM as an important element in the loop system linking medial thalamus and neostriatum; the probable attention and orientation related functions of this system are discussed.     

Anatomical organization of the spinocerebellar system in the cat,as studied by retrograde transport of horseradish peroxidase

The distribution of spinocerebellar tract (SCT) neurons has been studied in the entire length of the spinal cord of the cat following injections of horseradish peroxidase into the cerebellum, and whether or not the axons of the labeled neurons crossed within the spinal cord was determined in cases with injections preceded by hemisections at the cervical levels. The SCTs were classified into the following corssed and uncrossed tracts according to the cell origin and the fiber course; The crossed SCTs originate from (1) the central cervical nucleus (the CCN-SCT), (2) lamina VIII neurons of the cervical to the lumbar cord (the lamina VIII-SCT), (3) spinal border cells (the border cell-SCT), (4) neurons in the medial lamina VII of the lumbar to the caudal spinal segments (the medial lamina VII-SCT), (5) ventral horn neurons (laminae VII and VIII) of the sacral and caudal segments (the ventral horn-SCT) and (6) dorsal horn neurons (lamina V) of the sacral and the caudal segments (the dorsal horn-SCT). The uncorssed tracts originate from (1) neurons of the medial lamina VI of C2 to T1 (the medial lamina VI-SCT of the cervical cord), (2) neurons in the central part of lamina VII of C6 to T1 (the central lamina VII-SCT of the cervical enlargement), (3) lamina V neurons of the lower cervical to the lumbar cord (the lamina V-SCT), (4) Clarke's column (the Clarke's column-SCT) and (5) neurons in the medial lamina VI of L5 and L6 (the medial lamina VI-SCT of the lumbar cord). The present study suggests that the spinocerebellar system originates from more diverse laminae than has previously been known, and further refined studies on the topographic projections of each tract will yield more important and valuable information in this field.     

Dorsal column nuclei projections to the cerebellar cortex in cats as revealed by the use of the retrograde transport of horseradish peroxidase.

The existence of a cerebellar projection from the dorsal column nuclei (gracile and cuneate nuclei, DCN) has been proposed on electrophysiological grounds but questioned when studied with neuroanatomical techniques. The retrograde transport of horseradish peroxidase (HRP) has been used for the present study and provides anatomical evidence of a DCN-cerebellar pathway. In adult cats, 1 to 6 mul of 30% HRP were injected in pars intermedia of the anterior lobe (lobules IV-V), in paramedial lobule and in vermis of the anterior (lobules IV-V) and of the posterior lobe (lobule VII). After survival of 24 to 48 hours, all animals were perfused with a double aldehyde mixture and serial 40 mu sections through the medulla oblongata were incubated for visualization of HRP. In all cases, medullary nuclei known to project to the injected cortical regions of the cerebellum contained HRP-positive neurons mainly ipsilateral to the injection (e.g., external cuneate nucleus) or mainly contralateral to it (e.g., inferior olivary complex). Following ipsilateral injections in either the paramedian lobule or the pars intermedia, HRP-positive neurons in the cuneate nucleus were concentrated in its rostral portion where multipolar cells with radiating dendrites predominate. In contrast, none of the clusters region, in the caudal part of the cuneate nucleus, displayed HRP-positive granules. In cases in which the anterior vermis was injected a few labelled cells were present in the rostral part of the gracile nucleus but not in the clusters region of this nucleus. No labelling of DCN neurons was evident after posterior vermis injection. To compare the distribution of cells contributing to the DCN-cerebellar pathway with that of thalamic relay cells in the DCN, 0.5 to 3 mul of 30% HRP were injected in the nucleus ventralis posterolateralis of the thalamus in another series of cats. Contralateral to the thalamic injection, labelled cells were concentrated in the clusters region of the gracile and cuneate but rostrally in these nuclei they were scattered among unlabelled neurons. The preferential location in the DCN of cells which project to the cerebellum and of cells which project to the thalamus stresses the heterogeneous organization of these nuclei along the rostrocaudal axis. Further, the results indicate that regions of the DCN which have been distinguished on the basis of cytoarchitectonics (Kuypers and Tuerk, '64) and of afferents (Rustioni, '73, '74) differ also in their efferent projections.     

Regional topography within noradrenergic locus coeruleus as revealed by retrograde transport of horseradish peroxidase.

A hitherto unsuspected degree of regional topographic organization in the noradrenergic nucleus, locus coeruleus, was revealed by the use of retrograde transport of horseradish peroxidase (HRP) from terminal areas receiving noradrenergic innervation. HRP was injected into hippocampus, hypothalamus, thalamus, caudate-putamen, septum, amygdala-piriform cortex, cerebellum and cortex. Successful transport was obtained from all areas, including the caudate-putamen and cerebral cortex. The pattern of HRP positive cells in the ipsilateral locus coeruleus was markedly different depending on the location of the HRP injection. Thus, hippocampal injections labeled cells in the dorsal locus coeruleus but not at all in the ventral tip. Injections of HRP into caudate-putamen or cerebellum labeled the ventral tip along with the rest of the dorsal portion. HRP injections into the septum labeled cells only in the dorsal half of the dorsal locus coeruleus. There thus exists a three tier division of locus coeruleus into the ventral one third, dorsal one third and intermediate one third. A further division was seen in the anterior-posterior plane with HRP injections into the thalamus labeling the posterior pole of locus very intensely but with little transport to more anterior levels; conversely HRP injection into the hypothalamus resulted in intense labeling only in the anterior pole of locus coeruleus. Amygdala-piriform cortex HRP injections revealed a further pattern with very intensely reactive cells scattered sparsely throughout the nucleus. Cortical HRP injections yielded weaker labeling also in occasional, scattered cells. All HRP transport to locus coeruleus was shown to be noradrenergic by degeneration with 6-hydroxydopamine and due to terminal, rather than fiber of passage, uptake by control injection into the dorsal NA bundle. It is concluded that the locus coeruleus is not an homogenous nucleus with respect to the origin of the noradrenergic projections to sundry forebrain, spinal and cerebellar areas but is comprised of distinct subdivisions of noradrenergic neurons.     

Development of catecholaminergic projections to the spinal cord in the North American opossum, Didelphis virginiana

The intent of our study was to determine when catecholaminergic axons grow into each of their adult targets in the spinal cord of the North American opossum (Didelphis virginiana) and to identify the origin of catecholaminergic axons in the lumbosacral cord at different stages of development. Tyrosine hydroxylase-like immunoreactive axons, presumed to be catecholaminergic, were demonstrated at different stages of development by the indirect antibody peroxidase-antiperoxidase technique of Sternberger. The neurons giving rise to such axons in the lumbosacral cord were identified by using the retrograde transport of Fast Blue and immunofluorescence for tyrosine hydroxylase-like immunoreactive neurons. At birth, 12-13 days after conception, tyrosine hydroxylase-like immunoreactive axons are present in the marginal zone throughout the length of the spinal cord. Such axons are particularly numerous in the dorsolateral marginal zone, the region containing most of them in adult animals. By postnatal day 3, a few immunoreactive axons are present in the intermediate (mantle) zone of the spinal cord; and by postnatal day 8, they are most concentrated in the presumptive intermediolateral cell column. Laminae I and II of the dorsal horn are not innervated by such axons until approximately postnatal day 15. By postnatal day 44, the distribution of tyrosine hydroxylase-like immunoreactive axons in the spinal cord resembles that in adult animals, although some areas may be hyperinnervated. At birth, tyrosine hydroxylase-like immunoreactive cell bodies are present in all of the brainstem areas providing catecholaminergic projections to the spinal cord in adult animals (Pindzola et al.: Brain Behav. Evol. 32:281-292, '88); and by at least postnatal day 5, lumbosacral injections of Fast Blue retrogradely label tyrosine hydroxylase-like immunoreactive neurons in all such areas. Retrogradely labeled immunoreactive neurons were also found in areas that do not contain them in adult animals. Such areas include the dorsal part of the nucleus coeruleus and certain areas of the reticular formation. During development, spinally projecting tyrosine hydroxylase-like immunoreactive neurons are numerous medial to the nucleus ventralis lemnisci lateralis (the paralemniscal region), whereas only a few are present in the same location in adult animals. Our results suggest that catecholaminergic axons grow into the spinal cord prenatally, that they innervate their adult targets postnatally and over an extended time period, and that during some stages of development they originate from areas that do not supply them in the adult animal.     

Afferents to the flocculus of the cerebellum in the rhesus macaque as revealed by retrograde transport of horseradish peroxidase

To investigate the afferent projections to the flocculus in a nonhuman primate, we injected horseradish peroxidase into one flocculus of six rhesus macaques (Macaca mulatta) and processed their brains according to the tetramethylbenzidine protocol to reveal retrogradely labeled neurons. Labeled neurons were found in a large set of nuclei within the rostral medulla and the pons. The greatest numbers of labeled neurons were in the vestibular complex and the nucleus prepositus hypoglossi. There were neurons labeled bilaterally throughout all the vestibular nuclei except the lateral vestibular nucleus, but most of the labeled neurons were in the caudal parts of the medial and inferior vestibular nuclei and in the central part of the superior vestibular nucleus; the nucleus prepositus was also labeled bilaterally, primarily caudally. Modest numbers of labeled neurons were found in the y-group, most ipsilaterally, and many neurons were labeled in the interstitial nucleus of the vestibular nerve. No labeled neurons were found in the vestibular ganglion following a large injection into the flocculus. A second large source of afferents to the flocculus was the medial, paramedial, and raphe reticular formation. Dense aggregates of labeled neurons were located in several pararaphe nuclei of the rostral medulla and the rostral pons and in the nucleus reticularis paramedianus of the medulla and several component nuclei of the nucleus reticularis tegmenti pontis bilaterally. Several groups of cells within and abutting upon the medial and rostral aspects of the abducens nucleus were labeled bilaterally. There was a modest projection from two parts of the pontine nuclei. Both a dorsal midline nucleus ventral to the nucleus reticularis tegmenti pontis and a collection of nuclei in a laminar region adjacent to the contralateral middle cerebellar peduncle contained labeled neurons whose numbers, while modest, were large compared to the projections to the flocculus in other animals. This generic difference may be due to the greater development of the smooth pursuit system in monkeys and the consequent need for a more substantial input from the cerebral cortex. As in other genera, the inferior olive projected to the flocculus via the dorsal cap of Kooy and the contiguous ventrolateral outgrowth. The projection was completely crossed and large injections labeled virtually every neuron in the dorsal cap, suggesting that the dorsal cap is the principal source of climbing fiber afferents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spinocerebellar projections to lobules I and II of the anterior lobe in the cat, as studied by retrograde transport of horseradish peroxidase

Spinocerebellar tract (SCT) neurons projecting to lobules I and II of the cerebellar anterior lobe were identified by the retrograde horseradish peroxidase technique in the cat. Instead of a conventional stereotaxic approach, we removed ventral parts of the vermis of the posterior lobe and approached the posterior aspect of lobule I through the fourth ventricle. Under direct visual guidance, discrete injections were made into lobule I or II with a glass micropipette. Neurons projecting to lobule I were located mainly in the central cervical nucleus (CCN), the medial part of lamina VII of L6 to the causal segments, and in lamina VIII of S2 to the caudal segments (with crossed ascending axons). The latter two groups correspond to medial lamina VII group of the lumbar to the caudal segments and the ventral horn group of the sacral-caudal segments of our previous studies. A small number of Clarke column neurons (with uncrossed ascending axons) also projected to lobule I. All of these neuronal groups projected to lobule II. In addition, large neurons in lamina V and the border between laminae IV and V from S2 to the caudal segments projected to sublobule IIA, and more numerously to sublobule IIB (with crossed ascending axons). They belong to the dorsal horn group of the sacral-caudal segments of our previous studies. Spinal border cells (with crossed ascending axons) projected to sublobule IIB, and a small number, to sublobule IIA. It was suggested that the CCN neurons project more densely to the median region whereas Clark column neurons project to the lateral part of these lobules.     

Spinocerebellar projections to lobules III to V of the anterior lobe in the cat,as studied by retrograde transport of horseradish peroxidase

Spinocerebellar tract (SCT) neurons projecting to lobules III to V of the cerebellar anterior lobe were identified by the retrograde horseradish peroxidase technique. SCT neurons projecting to lobule III with crossed ascending axons were located mainly in the central cervical nucleus (CCN), the medial part of lamina VII of L6 to the caudal segments, and the dorsal horn (lamina V) and ventral horn (lamina VIII) of the sacral-caudal segments. Spinal border cells with crossed ascending axons also projected to lobule III. SCT neurons projecting to this lobule with uncrossed ascending axons were located in the medial part of lamina VI of the cervical segments and the middle part of lamina VII of C6 to T1, lamina V of the lower cervical, thoracic and the lumbar segments, Clarke's column including marginal neurons, and the medial part of lamina VI of L5 and L6. These neuronal groups also projected to lobule IV, except for those present caudal to L6 (in the medial part of lamina VII, and laminae V and VIII of the sacral-caudal segments). A far smaller number of similar neurons projected to lobule V. Injections of HRP restricted to the vermal region labeled mainly neurons in the CCN and Clarke's column while restricted injections to the intermediate-lateral regions labeled ipsilaterally spinal border cells, lamina V neurons, and Clarke column neurons, especially of the lumbar segments as well as marginal neurons of this column.     

Nigrostriatal projections in the rat as demonstrated by retrograde transport of horseradish peroxidase. I. Projections to the rostral striatum

The distribution of nigral neurons projecting to the rostral part of the striatum was studied in 12 rats using the horseradish peroxidase or the wheatgerm lectin bound horseradish peroxidase labelling techniques. Labelled neurons localized in the substantia nigra pars compacta (SNc) were demonstrated throughout the anteroposterior extent of the nucleus. Most of the labelled neurons were localized in the medial half of the SNc, predominantly in its basal part. Labelled neurons localized in the substantia nigra pars reticulata (SNr) predominated in the caudal half of the nucleus where they were found in its medial, central and lateral parts. A quantitative evaluation of the shape and size of the labelled neurons showed no statistically significant differences between the SNc and SNr as to the shape of the labelled perikarya. In contrast, nigrostriatal neurons in the SNc were found to have larger perikarya than their counterparts in the SNr.     

Thalamic projections to the cortical gustatory area in the cat studied by retrograde axonal transport of horseradish peroxidase

The thalamic projections to the cortical gustatory area in the cat were studied using the horseradish peroxidase (HRP) method. The gustatory area extends from the lateral lip of the presylvian sulcus (posterior two-thirds) to the posterior part of the orbital gyrus. It is bounded anteriorly by area 6a beta, laterally by the first somatosensory area, medially by the fundus and medial bank of the presylvian sulcus (prefrontal area), and posteriorly by the insular area. The cortical gustatory area receives fibers mainly from the medial smaller-celled part of the posteromedial ventral nucleus (VPMM). Cortical projections of the VPMM are organized topically; the anterior part of the gustatory cortex receives fibers from the anterodorsal and posteroventral portions of the anterior two-thirds of the VPMM, whereas the posterior gustatory cortex receives fibers from the anteroventral, posterodorsal and posterior portions of the posterior two-thirds of the VPMM. In addition, there appears to be a mediolateral organization of the cortical projections of the VPMM to the gustatory area. The cortical gustatory area receives a few projections from the ventral lateral, ventral medial, submedial, paracentral, lateral central, parafascicular and medial central nuclei.     

The inferior olivary nucleus of the opossum (Didelphis marsupialis virginiana), its organization and connections

Although the inferior olivary nucleus of the opossum is small, sections stained either for Nissl substance, normal axons or cholinesterase activity reveal distinct medial, dorsal and principal nuclei. The medial nucleus contains three major subdivisions (labelled a, b, c after Bowman and Sladek, 1973) and a group of neurons which is comparable to the cap of Kooy. In contrast to the cat and monkey, the major portion of the “medial” nucleus (subgroup a) lies lateral to the principal nucleus in rostral sections. The dorsal nucleus can also be subdivided, as can the principal nucleus which contains distinct dorsal and ventral lamellae. A small area is identified which based on position and connections may conform to the dorsal medial cell group. The experimental portion of the study provides evidence for an olivary projection from the motor-sensory cortex and a massive input from the midbrain (red nucleus, pretectum, midbrain tegmentum). In addition, the opossum inferior olive receives fibers from the deep cerebellar nuclei (cerebellar feedback loops), the spinal cord and the dorsal column nuclei. Of particular interest is the finding that fibers from the nucleus cuneatus and nucleus gracilis have distinctly different olivary targets and that those from the nucleus gracilis, but not the cuneate nucleus, overlap (in part, at least) with the direct spinal fibers. Other examples of overlapping fields of terminal degeneration are present and are discussed. In general our results reveal that although certain relationships between the nuclear divisions are different, the opossum olive conforms well to that of placental mammals and provides a basic mammalian model for future experimental electron microscopic and physiological studies.     

Striatal afferent connections in the turtle (Chrysemys picta) as revealed by retrograde axonal transport of horseradish peroxidase.

Horseradish peroxidase (HRP, 30% solution, 0.1-0.3 mul, 72 h) was injected unilaterally into the basal striatum (STR) and the dorsal ventricular ridge (DVR) of adult turtles (Chrysemys picta) in order to demonstrate the cells of origin of some afferents to these telencephalic structures. After selective STR injection, HRP-labeled cells were visualized in the dorsal thalamus and midbrain tegmentum, ipsilaterally. At thalamic level, HRP-positive neurons were located around nucleus rotundus, i.e., mainly within nuclei dorsomedialis anterior, dorsolateralis anterior and less abundantly in nuclei ventralis and reuniens. At midbrain level, a large population of labeled neurons was disclosed within the ventrolateral portion of rostral tegmentum. Other HRP-positive neuronal somata were found scattered throughout the lateral portion of the caudal midbrain tegmentum. In addition, labeled axons were visualized in both peduncles of the lateral forebrain bundle (LFB) after STR injection. The HRP-positive fibers of the dorsal peduncle of the LFB were followed up to the ipsilateral labeled thalamic cells where they appear to arise, whereas the HRP-containing axons of the ventral peduncle were traced down to the lateral midbrain tegmentum where they appear to arborize. Most of the HRP injections into the DVR were confined to the mediodorsal quadrant of the rostral half of the DVR. In such a case, a very large number of HRP-positive cells were disclosed within all thalamic nuclei surrounding nucleus rotundus, ipsilaterally. In addition, numerous labeled neurons were also found in nucleus rotundus itself and within nucleus reuniens. No HRP-positive cells were disclosed caudally to the meso-diencephalic junction after DVR injection.