Cerebellar afferents in the frogs,Rana esculenta and Rana temporaria

Summary Afferents to the cerebellum in frogs (Rana esculenta, Rana temporaria) were studied by use of retrograde transport of horseradish peroxidase. Following injections restricted to the molecular layer of the cerebellum cell labelling was found in the contralateral inferior olive and the ventral portion of the caudal medullary raphe. Injections involving the granular layer resulted in labelling in the ventral horn of the cervical spinal cord, the caudal spinal trigeminal nucleus, the nucleus caudalis and the medial portion of the nucleus ventralis of the vestibular nerve, the inferior reticular nucleus and the nucleus of the fasciculus longitudinalis medialis. Following larger injections, which may have spread significantly into the cerebellar, secondary gustatory, trigeminal or vestibular nuclei, labelled cell bodies were also found in the nucleus ruber, nucleus solitarius, the rostral spinal trigeminal nucleus and the rostral rhombencephalic reticular formation. It is unclear whether the fibers from these latter areas innervate the cerebellum of the frog, as they do in mammals, or only reach the underlying areas. This situation emphasizes a limitation of the HRP technique when applied to small structures as is often the case in lower vertebrates.Supported by Grant Gr 276 to U. G.-C. from the Deutsche Forschungsgemeinschaft.     

The vestibular nuclei in the domestic hen (Gallus domesticus). VII.--Afferents from the spinal cord

Lesions of different parts of the spinal cord at different levels in the hen have been made and the resulting degeneration in the vestibular complex has been studied in silver impregnated sections. Spinovestibular fibres originate from cervical as well as lumbosacral levels of the cord and run in the dorsal part of the lateral funiculus. The spinovestibular fibres from all levels of the spinal cord terminate ipsilaterally in the nucleus Deiters ventralis, the nucleus Deiters dorsalis, the medial nucleus and rostrally in the descending nucleus. The spinovestibular fibres terminating in the above nuclei are few in number while spinovestibular fibres terminating bilaterally in the caudal part of the descending nucleus are much more abundant. In a few cases HRP injections in the vestibular complex resulted in labelled cells in upper cervical segments of the spinal cord localized in lamina VII. The findings are discussed in the light of data concerning the spinovestibular pathway in mammals.     

Distribution of trigeminothalamic and spinothalamic lamina I terminations in the cat

The distribution in the thalamus of terminal projections from lamina I neurons of the trigeminal, cervical, and lumbosacral dorsal horn was investigated with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in the cat. Iontophoretic injections were guided by single- and multi-unit physiological recordings. The injections in particular cases were essentially restricted to lamina I, whereas in others they spread across laminae I-III or laminae I-V. The trigemino- and spinothalamic (TSTT) terminations were identified immunohistochemically. In all cases, regardless of the level of the injections, terminal fibers were consistently distributed in three main locations: the submedial nucleus; the ventral aspect of the basal ventral medial nucleus and ventral posterior nuclei; and, the dorsomedial aspect of the ventral posterior medial nucleus. The terminal fields in the submedial nucleus and the ventral aspect of the ventral posterior group were topographically organized. Terminations along the ventral aspect of the ventral posterior group extended posterolaterally into the caudal part of the posterior nucleus and anteromedially into the ventromedial part of the ventral lateral nucleus. In several cases with trigeminal lamina I injections, a terminal labeling patch was observed within the core of the ventral posterior medial nucleus. In cases with spinal lamina I injections, terminations were also consistently found in the lateral habenula, the parafascicular nucleus, and the nucleus reuniens. Isolated terminal fibers were occasionally seen in the zona incerta, the dorsomedial hypothalamus, and other locations. These anatomical observations extend prior studies of TSTT projections and identify lamina I projection targets that are important for nociceptive, thermoreceptive, and homeostatic processing in the cat. The findings are consistent with evidence from physiological (single-unit and antidromic mapping) and behavioral studies. The novel identification of spinal lamina I input to the lateral habenula could be significant for homeostatic behaviors.     

Distribution of trigeminothalamic and spinothalamic lamina I terminations in the cat

The distribution in the thalamus of terminal projections from lamina I neurons of the trigeminal, cervical, and lumbosacral dorsal horn was investigated with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in the cat. Iontophoretic injections were guided by single- and multi-unit physiological recordings. The injections in particular cases were essentially restricted to lamina I, whereas in others they spread across laminae I–III or laminae I–V. The trigemino- and spinothalamic (TSTT) terminations were identified immunohistochemically. In all cases, regardless of the level of the injections, terminal fibers were consistently distributed in three main locations: the submedial nucleus; the ventral aspect of the basal ventral medial nucleus and ventral posterior nuclei; and, the dorsomedial aspect of the ventral posterior medial nucleus. The terminal fields in the submedial nucleus and the ventral aspect of the ventral posterior group were topographically organized. Terminations along the ventral aspect of the ventral posterior group extended posterolaterally into the caudal part of the posterior nucleus and anteromedially into the ventromedial part of the ventral lateral nucleus. In several cases with trigeminal lamina I injections, a terminal labeling patch was observed within the core of the ventral posterior medial nucleus. In cases with spinal lamina I injections, terminations were also consistently found in the lateral habenula, the parafascicular nucleus, and the nucleus reuniens. Isolated terminal fibers were occasionally seen in the zona incerta, the dorsomedial hypothalamus, and other locations. These anatomical observations extend prior studies of TSTT projections and identify lamina I projection targets that are important for nociceptive, thermoreceptive, and homeostatic processing in the cat. The findings are consistent with evidence from physiological (single-unit and antidromic mapping) and behavioral studies. The novel identification of spinal lamina I input to the lateral habenula could be significant for homeostatic behaviors.     

Neck muscle and trigeminal input to the upper cervical cord and lower medulla of the cat.

Experiments on chloralose-anaesthetized cats have shown that low-threshold neck muscle afferents project to laminae IV and V in the dorsal horn of the upper cervical cord, to lamina VI including the region which encompasses the central cervical nucleus, as well as to extensive regions of the ventral horn. At posterior medullary levels projections also exist to laminae IV, V, and VI of the spinal nucleus of V (although those to lamina IV are circumscribed), to the deep layers and lateral margin of the cuneate nucleus, and to the inferior olive. These projections are both from low- and high-threshold afferents. Evidence of a functional relationship between the trigeminal and neck muscle afferent system was found both in the upper cervical cord and lower medulla. About 40% of units in both regions receive a convergent input and when convergence could not be demonstrated, prior stimulation of one modality in some instances affected the responsiveness of the unit to the other modality. A motor role was found for some trigeminal afferent projections to the upper cervical cord. Trigeminal afferents consistently activated antidromically identified motoneurons of splenius, biventer cervicis, and complexus.     

Insulin-like immunoreactivity in the monkey spinal cord.

Insulin-like immunoreactive neurons were localized in the cervical, thoracic, lumbar and sacral segments of the monkey spinal cord. Both dorsal and ventral horn cells were labelled. Insulin-like reaction product was localized in the cell nucleus and cytoplasm. Both inner and outer nuclear membranes were labelled. Reaction product appeared to be scattered throughout the nucleoplasm but not within the nucleolus. In the cytoplasm, labelling was mainly localized in the cisternae of rER and saccules of Golgi apparatus. Both proximal and distal dendrites were labelled, the reaction product was closely associated with the parallel arrays of neurotubules. Most of the distal dendrites were postsynaptic to non-labelled axon terminals; however, some were postsynaptic to lightly labelled axon terminals. A labelled dendrite often formed the central element of a synaptic glomerulus with several nonlabelled axon terminals. It is hypothesized that insulin-like substance(s) may be modulating nuclear activities as well as neurotransmission at the synapse.     

Spinal projections from the rhombencephalon in the toad

Rhombencephalic cell groups projecting to the spinal cord are demonstrated following single pressure injections and/or iontophoretic ejections of HRP solution in either cervical or lumbar enlargements of the toad spinal cord. A group uptake and transport of HRP were obtained with both application techniques, when sufficiently long survival times (8-11 days) were used. Following injections in the cervical cord labeled cells are located mostly in the ventral nucleus of the VIIIth nerve and in the medial zone of the rhombencephalic reticular formation, i.e. the nucleus reticularis inferior, medius and superior. Following injections in the lumbar enlargement the majority of labeled cells are situated in the caudalmost portion of the ventral nucleus of the VIIIth nerve and in the nucleus reticularis inferior. These observations indicate that in the toad the main supraspinal descending pathways from the rhombencephalon originate in the ventral nucleus of the VIIIth nerve and the medial zone of the reticular formation, and that both these pathways are somatotopically organized.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.     

Neurons of the medulla oblongata and hypothalamus projecting on the sympathetic neurons of the ventral horn of the spinal cord]

Connections of the neurons of the spinal cord ventral horn with the structures, situating above have been investigated. After injection of uranyl acetate into the TIII segment of the spinal cord, labelled neurons are found in various reticular nuclei of the medulla oblongata. At the level of the roots of the XII pair of the cranial nerves they are revealed in the reticular paramedian, ventral, parvocellular and lateral nuclei. The formations mentioned participate in regulation of the cardio-vascular system. More rostral (2 and 4 mm relatively to the roots of the XII pair of the cranial nerves) the neurons are observed in the reticular giant cellular nucleus, in nuclei of the raphe and in the group of the P-substance reactive neurons. Besides, labelled neurons are revealed in the posterior, lateral fields and in the dorso- and ventromedial nuclei of the hypothalamus.     

Projections to the midbrain tectum in Salamandra salamandra L.

Following unilateral iontophoretic application of HRP into the optic tectum of Salamandra salamandra, retrogradely HRP-filled cells were found bilaterally in the pretectum, tegmentum isthmi, the reticular formation, pars medialis, and in the nucleus vestibularis magnocellularis. The area octavo-lateralis projects only to the caudal part of the tectum. Ipsilateral projections were noted from the dorsal gray columns of the cervical spinal cord, the dorsal tegmentum, the thalamus dorsalis pars medialis, thalamus dorsalis, pars anterior (to the rostral one-third of the tectum), the thalamus ventralis (in its entire rostro-caudal extent), and the preoptico-hypothalamic complex. Retrogradely filled cells were identified in deeper layers of the contralateral tectum. There are two telencephalic nuclei projecting ipsilaterally to the tectum via the lateral forebrain: the ventral part of the lateral pallium, and the posterior strioamygdalar complex.     

Tectal and Related Target Areas of Spinal and Dorsal Column Nuclear Projections in Hedgehog Tenrecs

The terminal distributions of spinal and dorsal column nuclear projections to tectum, pretectum, and central gray of hedgehog tenrecs (Echinops telfairi and Setifer setosus) were investigated using anterograde axonal flow and various tracer substances. In the inferior colliculus, the densest and most extensive mesencephalic projections were found within the pericentral regions. One target area, referred to as the external portion of the inferior colliculus, was represented as a semicircle of grain patches lateral and caudal to the central nucleus. This region received somesthetic afferents from the dorsal column nuclei and from spinal segments at various levels. In contrast, after high cervical injections, the pericentral portion dorsomedial to the rostral half of the central nucleus was labeled almost exclusively. This area of labeling was distinct from the labeling in the central gray and might be best compared with the intercollicular zone in other species. The superior colliculus received projections predominantly from the high cervical cord; minor projections also arose from lumbar spinal segments and the dorsal column nuclei. The terminal field covered roughly the caudal half of the colliculus and involved the stratum griseum intermediale in a patch-like fashion. Some labeling was also found in the stratum griseum profundum and in the stratum griseum superficiale. Other than in the colliculi, weak pretectal projections were observed following dorsal column nuclear injections, while the nucleus of Darkschewitsch was labeled best following lumbosacral injections. All mesencephalic target areas were labeled consistently on the contralateral side, while their ipsilateral side was involved to a varying degree: The relatively most prominent ipsilateral labeling was seen in the central gray, being roughly similar on both sides; scarcely any labeling was noted in the ipsilateral superior colliculus. Tectal injections of retrograde tracer, in addition, revealed a considerable number of labeled neurons in a relatively cell-poor region immediately ventral to the high cervial dorsal horn. This region might correspond to the lateral cervical nucleus, an aggregation of neurons that so far has only been demonstrated in higher mammals.     

Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system

With the use of an antiserum generated in rabbits against synthetic human calcitonin gene-related peptide (CGRP) the distribution of CGRP-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system. A detailed stereotaxic atlas of CGRP-like neurons was prepared. CGRP-like immunoreactivity was widely distributed in the rat central nervous system. CGRP positive cell bodies were observed in the preoptic area and hypothalamus (medial preoptic, periventricular, anterior hypothalamic nuclei, perifornical area, medial forebrain bundle), premamillary nucleus, amygdala medialis, hippocampus and dentate gyrus, central gray and the ventromedial nucleus of the thalamus. In the midbrain a large cluster of cells was contained in the peripeduncular area ventral to the medial geniculate body. In the hindbrain cholinergic motor nuclei (III, IV, V, VI, VII XII) contained CGRP-immunoreactivity. Cell bodies were also observed in the ventral tegmental nucleus, the parabrachial nuclei, superior olive and nucleus ambiguus. The ventral horn cells of the spinal cord, the trigeminal and dorsal root ganglia also contained CGRP-immunoreactivity. Dense accumulations of fibers were observed in the amydala centralis, caudal portion of the caudate putamen, sensory trigeminal area, substantia gelatinosa, dorsal horn of the spinal cord (laminae I and II). Other areas containing CGRP-immunoreactive fibers are the septal area, nucleus of the stria terminalis, preoptic and hypothalamic nuclei (e.g., medial preoptic, periventricular, dorsomedial, median eminence), medial forebrain bundle, central gray, medial geniculate body, peripeduncular area, interpeduncular nucleus, cochlear nucleus, parabrachial nuclei, superior olive, nucleus tractus solitarii, and in the confines of clusters of cell bodies. Some fibers were also noted in the anterior and posterior pituitary and the sensory ganglia. As with other newly described brain neuropeptides it can only be conjectured that CGRP has a neuroregulatory action on a variety of functions throughout the brain and spinal cord.     

Spinal projections of brainstem respiratory related neurons in the cat as revealed by retrograde fluorescent markers

By means of retrograde axonal transport of fluorescent tracers, connections between brainstem respiratory related regions and the spinal cord has been studied in the cat. Neurons at the pneumotaxic center project bilaterally (90% ipsi-, 10% contra-) to cervical and lumbar spinal cord and ipsilaterally to thoracic levels. The ventrolateral nucleus of the tractus solitarius project mainly contralaterally (85%) to cervical levels and only contralaterally to thoracic levels; no efferent projections were found to lumbar levels. The ventral respiratory group showed a great number of neurons projecting to the spinal cord especially from the nucleus retroambiguus. Both nuclei, ambiguus and retroambiguus, project mainly contralaterally (70%) to the spinal cord. The B?tzinger complex showed rather scarce bilateral projections to cervical and only ipsilateral projections to lower cervical, thoracic and lumber levels.     

Areal Distributions of Cortical Neurons Projecting to Different Levels of the Caudal Brain Stem and Spinal Cord in Rats

Distributions of corticospinal and corticobulbar neurons were revealed by tetramethylbenzidine (TMB) processing after injections of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) into the cervical or lumbar enlargements of the spinal cord, or medullary or pontine levels of the brain stem. Sections reacted for cytochrome oxidase (CO) allowed patterns of labeled neurons to be related to the details of the body surface map in the first somatosensory cortical area (SI). The results indicate that a number of cortical areas project to these subcortical levels: (1) Projection neurons in granular SI formed a clear somatotopic pattern. The hindpaw region projected to the lumbar enlargement, the forepaw region to the cervical enlargement, the whisker pad field to the lower medulla, and the more rostral face region to more rostral brain stem levels. (2) Each zone of labeled neurons in SI extended into adjacent dysgranular somatosensory cortex, forming a second somatotopic pattern of projection neurons. (3) A somatotopic pattern of projection neurons in primary motor cortex (MI) paralleled SI in mediolateral sequence corresponding to the hindlimb, forelimb, and face. (4) A weak somatotopic pattern of projection neurons was suggested in medial agranular cortex (Ag_m), indicating a premotor field with a rostromedial-to-caudolateral representation of hindlimb, forelimb, and face. (5) A somatotopic pattern of projection neurons representing the foot to face in a mediolateral sequence was observed in medial parietal cortex (PM) located between SI and area 17. (6) In the second somatosensory cortical area (SII), neurons projecting to the brain stem were immediately adjacent caudolaterally to the barrel field of SI, whereas neurons projecting to the upper spinal cord were more lateral. No projection neurons in this region were labeled by the injections in the lower spinal cord. (7) Other foci of projection neurons for the face and forelimb were located rostral to SII, providing evidence for a parietal ventral area (PV) in perirhinal cortex (PR) lateral to SI, and in cortex between SII and PM. None of these regions, which may be higher-order somatosensory areas, contained labeled neurons after injections in the lower spinal cord. Thus, more cortical fields directly influence brain stem and spinal cord levels related to sensory and motor functions of the face and forepaw than the hindlimb.

The termination patterns of corticospinal and corticobulbar projections were studied in other rats with injections of WGA:HRP in SI. Injections in lateral SI representing the face produced dense terminal label in the contralateral trigeminal complex. Injections in cortex devoted to the forelimb and forepaw labeled the contralateral cuneate nucleus and parts of the dorsal horn of the spinal cord. The cortical injections also demonstrated interconnections of parts of SI with some of the other regions of cortex with projections to the spinal cord, and provided further evidence for the existence of PV in rats.     

Somatotopic organization of the vestibulospinal tract in the toad

The origin of the vestibulospinal projection in the toad has been investigated by using the method of the retrograde axonal transport of HRP injected at various levels of the spinal cord. The vestibulospinal projection, in this species, was found to be somatotopically organized, since neurons projecting to the cervical segments of the spinal cord were located within the rostromedial part of the ventral vestibular nucleus and those neurons projecting to the lumbosacral segments of the spinal cord were located within the caudolateral part of that nucleus. This pattern of organization of the vestibulospinal projection in amphibia is similar to that described in mammals and birds.     

Avian pancreatic polypeptide (APP) immunoreactive neurons in the spinal cord and spinal trigeminal nucleus

Using indirect immunofluorescence technique, avian pancreatic polypeptide (APP) immunoreactive cell bodies and fibres have been observed in the superficial laminae of the dorsal horn of the spinal cord and of the spinal trigeminal nucleus. Fibres were also seen in the ventral horns, in low numbers at the cervical and thoracic levels and in high numbers at the lower lumbar and upper sacral levels. Neither total cord transection, nor dorsal rhizotomy, nor capsaicin treatment seemed to affect the APP systems described above. The present findings suggest that an APP-like peptide may be involved in processing of sensory information at the level of the first relay station.     

The hypothalamo-cerebellar projection in the rat: origin and transmitter.

Hypothalamic neurons projecting to cerebellum were identified by retrograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the rat. Selective D-[3H]aspartate labelling was used to investigate whether any of these connections may use excitatory amino acids as transmitters. The WGA-HRP experiments revealed that the hypothalamo-cerebellar fibers have their main origins in the lateral, dorsal and posterior hypothalamic areas, and the tubero-mammillary nucleus, while smaller numbers of cells were observed in tuber cinereum, the anterior hypothalamic area, and the periventricular and paraventricular nuclei. After injections of D-[3H]aspartate into the cerebellar cortex, intense labelling of the olivocerebellar climbing fiber system was observed, but hypothalamic cells were not retrogradely labelled with this selective tracer. The absence of D-[3H]aspartate labelling indicates that hypothalamo-cerebellar neurons lack specific uptake mechanisms for excitatory amino acids, but it does not entirely preclude the possibility that some of these hypothalamic neurons may use such transmitters. Many cerebellar projecting cells were located in the tubero-mammillary nucleus, which is known to contain histaminergic and GABAergic neurons, and it was concluded that part of the hypothalamo-cerebellar pathways may use histamine and/or GABA as transmitters. The transmitter remains unknown for other parts of the hypothalamo-cerebellar pathways.     

Projections of the central cerebellar nuclei to the intralaminar thalamic nuclei in cats

Projections of the central cerebellar nuclei to the intralaminar thalamic nuclei were studied in cats with the use of light and electron microscopy. Almost all intralaminar nuclei were shown to obtain cerebello-thalamic projections. The entire complex of the central cerebellar nuclei serves as a source of such projections; yet, involvement of different nuclei is dissimilar. Destruction of the central and, especially, caudal regions of the fastigial nucleus evoked in the intralaminar thalamic nuclei degenerative changes in the nerve fibers (from swelling and development of varicosities up to total fragmentation). Pathological phenomena could be noticed in the most caudal regions of the above thalamic nuclear group, including the medial dorsal nucleus. Projections of the cerebellar interpositus nucleus were directed toward nearly the same regions of the intralaminar nuclei; degeneration was more intensive (covered thecentrum medianum) when posterior regions of the interpositus nucleus were destroyed. Destruction of the lateral cerebellar nucleus evoked a similar pattern of pathological changes, but degeneration was also observed in some structures of the ventral and anterior nuclear groups of the thalamus. Electron microscopic examination showed that degeneration of dark and light types developed in the fiber preterminals and terminals. It can be concluded that the central cerebellar nuclei project not only to the ventral complex of the thalamic nuclei, but also to the anterior, medial, and intralaminar nuclear groups (rostral and caudal portions).     

Distribution of dipeptidyl peptidase II (Dpp II) in rat spinal cord

The histochemical localization of dipeptidyl peptidase II (Dpp II; E.C. 3.4.14.2) activity was demonstrated at the light microscope level in the rat spinal cord. Prominent staining was observed in motoneurons of the ventral horn and in medium to large neurons in the deep laminae of the dorsal horn, the intermediate gray, and in lamina X surrounding the spinal canal. Within neurons, Dpp II was localized largely in cell perikarya and large primary dendrites with no staining observed in cell nuclei. Neurons in the superficial dorsal horn lack Dpp II enzyme activity. Nonneuronal elements which also stained prominently were pericytes associated with blood vessels and ependymal cells lining the lumen of the spinal canal. A few oligodendrocytes and astrocytes were also stained, but they represented a minor component of the total amount of Dpp II activity. Following ventral root injury, Dpp-II-containing motoneurons degenerate; some glial cells in the region of degenerating neurons become Dpp II positive. The localized distribution of Dpp II in spinal cord neurons suggests that this proteolytic enzyme may play a role in the metabolism of an unidentified neuropeptide.