Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the lateral reticular nucleus (LRN). Labeled cells were found at all spinal levels and in particular large numbers in cervical and lumbar segments. Various spinal areas gave rise to cells of origin of this tract, which appears to be more prominent than any other tract previously studied with a similar approach. Labeling common to all spinal segments was observed in (1) ventromedial parts of both intermediate zone and ventral horn (laminae VII, VIII and X), mainly contralaterally; (2) the reticular extension of the neck of the dorsal horn, partly bilateral; and (3) superficial layers of the dorsal horn and nucleus of the dorsolateral funiculus (NDLF), mainly contralateral and projecting essentially to the lateral zone of the LRN. Additional labeling was observed at cervical and lumbar levels, each with specific qualities: (1) the cervical enlargement, which displayed labeling in the central part of the ipsilateral intermediate zone (lamina VII); (2) the rostral lumbar levels, which had projections from the contralateral median portion of the neck of the dorsal horn. These latter projections appear to be specific to pathways reaching the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive (except lamina I and NDLF projections) and the differences between these afferents and those projecting to the dorsal reticular formation, i.e., the nucleus reticularis ventralis.     

Morphology of physiologically identified thalamocortical relay neurons in the rat ventrobasal thalamus

The anatomical structure of physiologically identified neurons of the rat ventrobasal thalamus was studied in order to determine if there are morphologically distinct subsets of neurons that correlate with the somatosensory submodalities processed by these cells. Intracellular recordings were used to determine the modality and receptive field of a neuron, after which horseradish peroxidase was iontophoretically injected into the cell, allowing it to be histologically visualized. Computer-assisted measurements of the labeled cells were made to quantitatively analyze the dendritic structure. Cells were divided into physiological groups stimulated by whiskers, glabrous skin, furry skin, noxious stimulation, or joint rotation. Qualitatively, all cells appeared similar, with the same types of branching patterns. Dendritic spines and long, sinuous appendages were found on all distal dendrites. Quantitatively, no statistically significant differences in dendritic structure were found between functionally defined groups with the aid of a number of parameters, including a fitted dendritic ellipse. There was a weak correlation between somal cross-sectional area and receptive field size, suggesting larger cells processed larger receptive fields. In summary, the ventrobasal thalamus of the rat, in contrast to that of higher mammals, appears to contain only one major cell type and to have a very simple neuronal circuitry.     

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.     

The structural organization of the ventrobasal complex of the rat as revealed by the analysis of physiologically characterized neurons injected intracellularly with horseradish peroxidase

The ventrobasal complex (VB) of the rat thalamus contains neurons responding to non-noxious somatic stimuli as well as neurons driven exclusively by noxious stimuli. This study presents a comparison of morphological features of these two kinds of neurons. Thirteen neurons electrophysiologically characterized were impaled with the micropipette used for the recordings and intracellularly injected with horseradish peroxidase. After revealing the marker and preparation for electron microscopic procedures, 3 out of the 13 neurons were carefully studied using both the light and the electron microscope. VB neurons are stellate cells with a central rounded cell body and 6 to 10 primary dendrites which branch rapidly, giving a 'tufted' appearance. Dendrites of all orders present various types of protrusions. At the electron microscope level, 3 main kinds of synaptic profiles were observed contacting the injected neurons: small terminals with round vesicles which make asymmetrical contacts with distal dendrites; medium-sized terminals with flattened vesicles which make symmetrical contacts with dendrites of all orders and the soma; and large terminals with round vesicles which make asymmetrical contacts with primary dendrites and the soma. This study failed to reveal obvious morphological differences between functionally different VB neurons. In addition, it showed that their synaptology was apparently equivalent.     

The inferior olivary connections to the cerebellum in the rat studied by retrograde axonal transport of horseradish peroxidase

Olivocerebellar projections were investigated in the rat using retrograde axonal transport of horseradish peroxidase. Discrete cell groups of the inferior olive were labelled, subsequent to injections in the paravermal region, the vermis, or the caudolateral hemisphere. Injections in the midrostrocaudal third of the paravermal area resulted in labelling of cells in the medial accessory olive (MAO), in cell group “b” at caudal levels, and in its lateral portion at mid-rostrocaudal levels. The rostral pole of the principal olive (PO), the dorsal accessory olive (DAO), and the dorsomedial cell column, were heavily labelled. By comparison, caudal paravermal injections resulted in labelling in the medial part of the mid-rostrocaudal levels of the MAO, but not in its caudal portion. The PO lamellae were labelled in their lateral half, excluding the lateral bend connecting them. Injections slightly lateral within this paravermal area gave no caudal MAO labelling, but did label cells in segments of both PO lamellae, medial to those in the previous case. From vermal injections, cell groups “b” and “c” of the caudal MAO were labelled, but no labelled cells were present in the PO. Subsequent to injections in the paramedian lobule, cells in the dorsal lamella of the PO were labelled. No cells of the MAO were labelled. These results are discussed in terms of specific labelling patterns and the general concepts of organization presently held for the Olivocerebellar system.     

Olivocerebellar connections in sheep studied with the retrograde transport of horseradish peroxidase

We describe here the morphology of the inferior olive and the localization of labeled cells after HRP injections into various lobules of vermis and hemisphere of the cerebellum of the sheep. The medial part of the caudal half of the medial accessory olive projects to a medial zone in the anterior lobe, the simple lobule, and the lobules VII and VIII. The lateral part of the medial accessory olive projects to more lateral parts of these lobules with the exception of lobule VII. The group beta projects in a differential manner to the lateral parts of the lobules VII and VIII and the medial parts of the lobules IX and X. The dorsomedial cell column projects to lobules VIII, IX, and X; the connections of the dorsal cap are restricted to lobule X. Fibers from the caudal limb of the dorsal accessory olive terminate in the B zone, the simple lobule, and in lobule VIII. The rostral half of the medial accessory olive projects to lobule IX and to the hemisphere. The other projections of the accessory olives and the principal olive to the hemisphere are similar to those reported for the cat. An accessory cell group in the sheep, located between the principal and the dorsal accessory olive, has connections with the caudal vermis and the hemisphere.     

Muscle sensory neurons in the spinal ganglia in the rat determined by the retrograde transport of horseradish peroxidase

The method of retrograde transport of horseradish peroxidase (HRP) was used to identify muscle sensory neurons in the spinal ganglia in the rat. Experiments were conducted on 25 albino rats. Injections of 0.06 to 0.08 ml 2 to 20% Sigma type VIHRP were made unilaterally into anterior tibial muscle. Cells of origin of muscle receptors and motor endings in the same area where HRP was administered were demonstrated. The labeled cells, medium to large, were found in fourth and fifth lumbar ganglia ipsilateral to the site of injection. Simultaneously, labeled neurons were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia.     

Some claustro-cortical connections in the cat and baboon as studied by retrograde horseradish peroxidase transport.

1. The mammalian Claustrum (Cl) is a convergent multisensory structure of unknown function, and disputed ontogenetic origin. Its cortical projections, hitherto unknown, have been studied in cat and baboon by means of the horseradish peroxidase (HRP) technique. HRP was injected into the gyrus proreus (frontal eye field) of cats, and separately into the frontal eye fields, visual areas, and motor-premotor areas of the baboon cortex. 2. Differential retrograde transport to the Cl was demonstrated, such that in the cat the ipsilateral dorsal Cl was shown to be the principal origin of claustroproreate projections. In the baboon, the whole Cl projects onto area 8, while only the posteroventral part of the nucleus sends efferents to the visual cortex. The projection to the motor and premotor areas is present, but does not seem to be "essential." 3. Discussion of the physiological literature, together with anatomical evidence of reciprocal cortico-claustral projections to closely similar regions of the Cl lead to the suggestion that the Cl is concerned with the integration of messages subserving visually-directed movements. Some other functional implications are also discussed.     

Afferents to the entorhinal cortex of the rat studied by the method of retrograde transport of horseradish peroxidase

The afferents to the parahippocampal area of the rat were studied with retrograde transport of horseradish peroxidase injected into the medial entorhinal cortex, lateral entorhinal cortex, parasubiculum, presubiculum, or a large injection which stained all these structures as well as the ventral hippocampus. Control rats were injected with horseradish peroxidase into the overlying visual cortex. Labeled neurons in brains with injections into the medial entorhinal cortex and the adjacent parasubicular region were found in the ipsilateral and contralateral presubicular region, the medial septal nucleus, the thalamic nucleus reuniens, the dorsal part of the lateral nucleus of thalamus, the anterior periventricular nucleus of the thalamus, and the dorsal raphe nucleus. Brains with injections into the lateral entorhinal cortex yielded labeled neurons in the medial septal nucleus, nucleus reuniens, dorsal raphe nucleus, and nucleus locus ceruleus. Injections into the presubiculum resulted, in addition, in labeling of neurons in the lateral nucleus of the thalamus. Control injections aimed at the sensory cortex overlying the parahippocampal area yielded labeled neurons in the medial septal nucleus, the dorsal lateral geniculate nucleus, and the nucleus locus ceruleus.     

Projections of lamprey spinal neurons determined by the retrograde axonal transport of horseradish peroxidase.

The spinal cords of larval sea lampreys (Petromyzon marinus) and adult river lampreys (Ichthyomyzon unicuspis) were injected with horseradish peroxidase through a transection 1 cm caudal to the last gill. Some animals also had a spinal hemisection 1 cm caudal to the injection. After recovery periods of 1 to 52 days, the spinal cords were treated with diaminobenzidene and hydrogen peroxide, and the projections of various cell types determined in wholemount slides. From these observations the following conclusions were drawn. Most dorsal cells (primary sensory cells) are bipolar with a long rostral projection and a short caudal projection of no more than 5-10 mm. Both processes travel in the ipsilateral dorsal column. Their peripheral processes enter the dorsal roots as branches of their central axons. Some dorsal cells send processes out three or more dorsal roots both rostral and caudal to the cell body. Myotomal motoneurons have characteristic locations in the medial gray column and send prominent transversely oriented dendrites into the lateral columns. A few motoneurons are unusually large. In addition to giant interneurons the majority of smaller rostrally projecting interneurons also have decussating axons. A recently described cell type, the oblique bipolar cell, appears to have an exclusively crossed rostral projection. Although most edge cells project rostrally, as many as 20% may have a caudal projection or both rostral and caudal projections. Edge cells project equally to the ipsilateral and contralateral spinal hemicord, but their processes do not extend more than about 18 mm in sea lamprey larvae and 37 mm in adult river lampreys. Lateral cells project exclusively to the ipsilateral caudal hemicord. A few cells which resemble lateral cells in location and in possessing large lateral dendrites, project rostrally. However, these have atypical morphologic features which probably distinguish them from true lateral cells. Thus far, regardless of cell type, all decussating axons seem to pass ventral to the central canal, while decussating medial dendrites pass dorsally.     

Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase

Horseradish peroxidase (HRP) injected into rat hippocampus was transported to the perikarya of neurons which project to the hippocampus. HRP-labeled cells were present in both medial and lateral entorhinal cortex; cells of the medial entorhinal cortex appeared to be topographically organized. The mediaal septal nucleus contained stained cells; its mediaal aspect was labeled after dorsal hippocampal injections, while ventral hippocampal injections resulted in the labeling of more laterally located cells. Stained cells were also observed in the ipsilateral nucleus locus coeruleus, dorsal and median raphe nuclei and areas CA3–4 of the contralateral hippocampus. In additions, cells in the supramammillary region, an area not previously recognized to project to the hippocampus, were labeled. Finally, the mossy fiber terminal zone and the CA3–4 terminal zone in the dentate molecular layer of the ipsilateral hippocampus demonstrated HRP activity, presumably the result of orthograde axonal transport from the injection site.     

Spinal afferents to lamina IX of the cervical enlargement in the rat studied by the retrograde transport of horseradish peroxidase

Spinal neurons that project to the ventrolateral, dorsolateral and ventromedial portions of lamina IX of the cervical enlargement in the rat were investigated by means of horseradish peroxidase retrograde transport. In the cervical and upper thoracic segments, labeled neurons were observed ipsilaterally in laminae V–VIII and contralaterally in laminae VII–VIII. In the lower thoracic and upper lumbar segments, labeled neurons were seen after HRP injection into the ventrolateral part of lamina IX, and were distributed mainly in the lateral parts of the ipsilateral laminae V–VI.     

The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase.

The distribution of labeled cells in the inferior olive of the cat has been mapped following injections of small amounts of horseradish perosidase in the paramedian lobule of the cerebellum. The distribution of labeled cells was plotted in drawings of approximately serial transverse sections. The findings in each case were transferred to a standard diagram of the olive to facilitate comparison of cases. Previous studies of the distribution of retrograde cell loss in the inferior olive following cerebellar lesions (Brodal, '40b) showed that fibers ending in the paramedian lobule come from the caudal part of the ventral lamella of the principla olive. This was confirmed with the peroxidase method, but in addition three other separate and well circumscribed area of the olive showed labeling: one in the dorsal accessory olive, another in the rostral part of the medial accessory olive, a third in the caudal part of the dorsal lamella of the principal olive (fig. 7). There is some degree of topical arrangement within the projection of each of these olivary areas to the paramedian lobule. It is particularly striking that the projection areas of the caudal one-third of the lobule are different from and overlap only little with those of the orstral two-thirds. On account of diffusion of the injected perosidase solution in the folia it could not be decided whether the different olivary areas project to particular longitudinal zones in the paramedian lobule. The main findings can be correlated with the physiological observations of Armstrong et al. ('74). Some of the "paramedian" olivary areas are labeled also following peroxidase injections in other cerebellar parts, among them the nuclei interpositus anterior and posterior. The findings are compatible with the notion that olivocerebellar fibers branch to supply more than one cerebellar region. It is confirmed that the olivocerebellar projection, including that of the nuclei, is almost completely crossed. In the discussion it is emphasized that afferents from several sources converge on all four olivary regions projecting onto the paramedian lobule. The olivocerebellar projection obviously allows for divergence as well as convergence of impulses from the olive to the cerebellum. For further insight into the anatomical organization of the inferior olive, the entire olivocerebellar projection has to be mapped with the peroxidase methods, and further studies of the afferents to the olive are needed. In such studies, as well as in physiological ones, it is essential that findings are described with meticulous reference to the topography of the olivary subdivisions.     

Gracilo-diencephalic relay cells: A quantitative study in the cat using retrograde transport of horseradish peroxidase

In a quantitative study in the cat, gracilo-diencephalic relay cells were labeled by the use of retrograde axonal transport of horseradish peroxidase injected into the ventroposterolateral nucleus of the thalamus. An initial series comprising 22 animals with survival periods varying between 2 h and 4 days showed maximal labeling in the gracile nucleus after 24–48 h. The earliest appearing peroxidase-positive neurons were found after only 6 h, implying a transport rate in the medial lemniscus of at least 100 mm/day. In a second series of five cats, which were killed 24 h after injection, serial sections from the gracile nucleus were embedded in Epon following peroxidase processing, and cut at 2 μm. By stratified random sampling, about 2,000 cells were selected for light microscopic examination. The total number of nerve cells in a single gracile nucleus was calculated to be about 50,000, out of which about 15,000 were retrogradely labeled. In agreement with previous reports, rostrocaudal differences were observed. Thus, less than a third of the neurons in the rostral part of the nucleus were peroxidase-positive, whereas about half of those in the middle and caudal regions were retrogradely labeled. The total number of labeled neurons was, however, about the same in the rostral as in the middle-caudal part of the nucleus. The size of the nerve cell bodies, measured as the cross-sectional area in the nucleolar plane, differed significantly between labeled and unlabeled neurons. The estimated average diameter of the former was 22 μm and of the latter 17 μm. There was a considerable overlap between the two groups, however, and labeled cells as small as 12 μm in diameter were found. Mainly because of the uneven distribution of labeled neurons, cells in the rostral region were, on the average, significantly smaller than those in the middle and caudal regions; these mean diameters were calculated to be 18, 23, and 21 μm, respectively. The results of this study support the idea of a heterogeneous organization of the gracile nucleus. However, a much larger proportion of the gracilo-diencephalic relay cells is situated in the rostral part of the gracile nucleus than has previously been thought. The concept that only medium-sized neurons project to the diencephalon also seems to need revision. It is concluded that although gracilo-diencephalic relay cells are, on the average, larger than the nonlabeled neurons, single nerve cells cannot be identified on the basis of size alone. The function of the unlabeled neurons is discussed. Although many of these might relay to various extra-diencephalic sites, it is suggested that a large number are internuncial neurons.     

Dendritic orientation of thalamocortical projection neurons in the ventrobasal complex of macaques

This study describes the orientation of dendritic arbors from intracellularly labeled thalamocortical projection (TCP) neurons of Macaca fascicularis or Macaca mulatta. All neurons were located in the ventrobasal complex and responded to non-noxious stimuli. Each neuron was composed of dendrites that varied considerably in size and each dendrite tended to occupy a particular region of the perisomatic space with minimal overlap with other dendrites. Quantitative and qualitative analysis of the dendritic arbors of the labeled neurons showed they had an asymmetric distribution so that some regions of the perisomatic space contained more of the dendritic tree than others. Eleven of the thirteen reconstructed neurons had a larger percentage of the dendritic tree projecting into the medial portion of the perisomatic space. These results show that the dendritic arbors of macaque TCP neurons are not organized in a radially symmetric pattern as previously described and the asymmetric distribution of dendrites may be related to synaptic input.     

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.     

Identification of the trochlear motoneurons by retrograde transport of horseradish peroxidase

A common technique for demonstrating projections in the brain is to electrically stimulate one part of the brain and record mass or field potentials from another part. We showed in the visual system of the cat, where connections between retina, lateral geniculate nucleus, and superior colliculus are very well known, that the recording of field potentials is not at all sufficient to demonstrate connections. The most prominent potential after electrical stimulation of the optic tract is the field potential created by the Y-ganglion cell fibers of the optic nerve. We recorded this potential in the optic nerve head of the eye, in the lateral geniculate nucleus, and in the superior colliculus. To our surprise, we also could record this potential 7 mm in front of the retina, with the electrode in the vitreous, and 5 mm above the lateral geniculate nucleus and the superior colliculus, where there are no direct inputs from the optic tract. These results show quite clearly that field potentials can “stray” much farther than the underlying anatomical structure projects.