Wheat Germ Agglutinin-conjugated Horseradish Peroxidase Injections Research Articles

Anatomical evidence for the re-crossing of lateral corticospinal fibers via the posterior gray commissure in the cat spinal cord

This study demonstrated the projections of the corticospinal tract (CST) by using the anterograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the pre- and post-cruciate cortices (somatosensory area) on one side, labeled fibers were found in the spinal cord both in the lateral and in the anterior CSTs; both crossed, and uncrossed. The most conspicuous labeling was found in the crossed lateral CST. In addition, labeled lateral CST fibers were seen to recross via the posterior gray commissure. These re-crossed lateral CST fibers were most frequently observed in the cervical spinal segments.

Projections of the intermediate subdivision of the central amygdaloid nucleus to the bed nucleus of the stria terminalis and medial diencephalon

The forebrain projections of the intermediate subdivision of the central amygdaloid nucleus (CI) were studied using the horseradish peroxidase (HRP) retrograde transport technique. Injections of wheatgerm agglutinin-conjugated HRP into the bed nucleus of the stria terminalis and medial thalamus resulted in moderate retrograde labeling in CI while injections into the medial hypothalamus produced very dense cell labeling in CI. The dorsal hypothalamic area appears to be the main target of the efferents to the hypothalamus. Although previous histochemical studies have suggested that CI may be part of the lateral subdivision of the central nucleus (CL), the projection of CI to the medial diencephalon clearly distinguishes CI from CL.

Non-cholinergic basal forebrain neurons project to the contralateral basal forebrain in the rat.

Following injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or the fluorescent tracer fluoro-gold into the magnocellular preoptic area and the horizontal limb of the diagonal band, retrogradely labelled neurons were found in the homotopic region of the contralateral basal forebrain. Labelled fibers apparently arising from these neurons travelled in the stria medullaris and the habenular commissure to terminate in the contralateral basal forebrain. Although the neurons retrogradely labelled with fluoro-gold in the contralateral basal forebrain were similar in size to choline acetyltransferase (ChAT)-immunoreactive neurons, and were intermingled with them, none was ChAT-positive. WGA-HRP injections into the nucleus basalis magnocellularis did not result in retrograde labelling in the contralateral basal forebrain. These findings suggest that non-cholinergic neurons may serve as a direct link between the two sides of selective magnocellular basal forebrain regions.

Cerebro-cerebellar projections from the lateral suprasylvian visual area in the cat.

1. A projection from the medial bank of the lateral suprasylvian visual area, one of the targets of the cerebello-cerebral projection, back to the cerebellar cortex was demonstrated electrophysiologically in the cat. The anatomical pathways underlying this projection were investigated using orthograde and retrograde transport of wheatgerm-agglutinin-conjugated horseradish peroxidase (WGA-HRP). 2. Responses were recorded in the cerebellar cortex on stimulation of the medial bank of the lateral suprasylvian area, and were compared with those evoked by stimulation of the motor cortex and the crown part of the parietal association cortex. 3. Responses induced by stimulation of the lateral suprasylvian area were shown to consist of early mossy and late climbing fibre responses. The mossy fibre response was evoked, at a latency of 2-3 ms, predominantly in the lateral part of the contralateral cerebellar cortex (mainly, crus I, crus II, dorsal paraflocculus and paramedian lobule) and the posterior part of the vermis (mainly, lobules VII and VIII). Climbing fibre responses were elicited with the preceding mossy fibre responses and were elicited at a much longer latency than the motor cortex-induced climbing fibre response. 4. The orthograde and retrograde HRP studies suggested that the mossy fibre response is mediated by the pontine grey whereas the climbing fibre response is conveyed indirectly to the inferior olive which sends the climbing fibres to the cerebellar cortex. After WGA-HRP injections into both the medial bank of the lateral suprasylvian area and the cerebellar responsive area, orthogradely labelled terminals of cortico-pontine projection fibres and retrogradely labelled ponto-cerebellar neurones were found in the pontine grey, where distributions of the two kinds of labelling overlapped. On the other hand, retrograde neuronal labelling alone was found in the inferior olive, implying that the climbing fibre responses evoked from the lateral suprasylvian area were relayed via indirect cortico-olivary pathways.

The GABAergic neurons and axon terminals in the lateralis medialis-suprageniculate nuclear complex of the cat: GABA-immunocytochemical and WGA-HRP studies by light and electron microscopy.

In order to get more detailed information on the neural circuit of the lateralis medialis-suprageniculate nuclear (LM-Sg) complex of the cat, the GABAergic innervation of this complex was studied by GABA immunohistochemical techniques. Small immunoreactive cells were found throughout the LM-Sg complex. On the basis of their ultrastructural features, these GABAergic cells were identified as Golgi type II interneurons. The neuropil of this nucleus displayed a conspicuous granular immunoreactivity. Ultrastructurally, the immunoreactive neural profiles in the neuropil were identified as the presynaptic dendrites of interneurons, myelinated axons, or axon terminals. The GABAergic dendritic profiles, containing pleomorphic synaptic vesicles, were involved in synaptic glomeruli. Additionally, GABAergic axon terminals containing pleomorphic synaptic vesicles formed symmetric axodendritic synaptic contacts mainly in the extraglomerular neuropil. They appeared to correspond to either axon terminals from the thalamic reticular nucleus (TRN) or the axon terminals of interneurons. The projections from the TRN to the LM-Sg complex were studied by using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the LM-Sg complex, a number of retrogradely labeled cells were observed in the TRN. The connections between the TRN and the LM-Sg complex appeared to be topographically organized, the dorsal TRN being connected mainly with the dorsomedial portion of the LM-Sg complex, and the ventral TRN being connected chiefly with the ventrolateral portion of the LM-Sg complex. Following injection of the tracer into the TRN, ultrastructural examination of anterograde labeling in the LM-Sg complex revealed that labeled terminals contain pleomorphic vesicles and make symmetric synaptic contacts mainly with small to medium-sized dendrites. The labeled terminals were not involved in synaptic glomeruli. The present results provide anatomic support for the contention that the projection cells of the LM-Sg complex may be inhibited by both the TRN axons and interneurons, probably through the mediation of GABA.

The terminal distribution pattern of spinocerebellar fibers

The terminal fields of spinocerebellar fibers from different levels (cervical, thoracic and lumbar) of the spinal cord in the chick were determined by using wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), an anterograde labelling technique. More terminals were found in the anterior lobe than in the posterior lobe. Following injections in the lumbar spinal cord, mossy fiber terminals were found mostly in the anterior lobe (lobules I-V). The highest density of labelled terminals was found in lobule I, and labelled terminals were found in all parts of the lobule. However in lobules II-IV, labelled fibers were mediolaterally arranged in three longitudinal strips on both sides of the midline, and formed a distinct zonal distribution pattern. Labelled terminals were distributed evenly from the apical to basal regions of lobules I-V. A large number of mossy fiber terminals originating from the thoracic spinal cord were located in the anterior lobe and lobule VI; some labelled terminals were found in lobule IX. Three less distinct longitudinal strips, compared to those following WGA-HRP injections in the lumbar spinal cord, were recognized on each side of the midline. Labelled mossy terminals were observed in lobules II-IX following WGA-HRP injections in the cervical spinal cord. In transverse sections two longitudinal strips were found at the apical parts of lobules II and III, whereas four thin longitudinal strips were located in lobules IV and V. The present study showed that the terminal fields of spinocerebellar fibers from each level of the spinal cord have different distribution patterns.

Bulbar neurones with axonal projections to the trigeminal motor nucleus in the cat

The location of bulbar neurones with axons projecting to the ipsi- and contralateral trigeminal motor nucleus were investigated in cats anaesthetized with sodium pentobarbital. Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected in amounts of 5-24 nl. A volume-calibrated microelectrode was used for recording of evoked potentials and pressure injection of WGA-HRP. The injection site was guided by the position where a maximal antidromic response was evoked by electrical stimulation of the masseteric nerve. The survival time was 19-22 h. In preparations with the depot located in the masseteric subnucleus retrogradely stained neurones were found bilaterally in the borderzone of the trigeminal motor nucleus. Dense populations of stained neurones were observed ipsi- and contralaterally in the dorsal division of the main sensory trigeminal nucleus and the subnucleus-gamma of the oral nucleus of the spinal trigeminal tract. Clusters of WGA-HRP-neurones were observed bilaterally in the lateral tegmental field at the level of the subnucleus-beta of the oral nucleus of the spinal trigeminal tract, bilaterally dorsal to the facial nucleus and contralaterally adjacent to the hypoglossal nucleus. No stained neurones were found in the gigantocellular reticular nucleus. A group of stained neurones was located in the marginal nucleus of brachium conjunctivum and some were found in the raphé nuclei near obex. Cell profiles were of two types: medium-sized neurones with a triangular profile and 30-40 micron diameter, and fusiform neurones 10 X 50-70 micron. Convergence of descending cortical and trigeminal afferent inputs on interneurones located in the lateral borderzone of the trigeminal motor nucleus, i.e. the intertrigeminal area, is reported in the preceding paper.

On the development of the pyramidal tract in the rat. II. An anterograde tracer study of the outgrowth of the corticospinal fibers.

An anterograde tracer study has been made of the developing corticospinal tract (CST) in the rat using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Analysis of normal Rager stained material revealed that corticospinal axons reach upper cervical spinal cord levels at the day of birth (PO). Postnatal rats ranging in age from one (P1) to fourteen (P14) days received multiple WGA-HRP injections into the cortex of their left hemisphere and were allowed to survive for 24 h. The first labeled CST fibers caudally extend into the third thoracic spinal cord segment at P1; into the eighth thoracic segment at P3; into the first or second lumbar segment at P7 and into the second to third sacral segment at Pg. Thus the outgrowth of the leading 'pioneer' fibers of the CST is completed at P9 but later developing axons are continuously added even beyond P9. Quantitative analysis of the amount of label along the length of the outgrowing CST revealed a characteristic pattern of labeling varying with age. The most striking features of that pattern are: the formation of two standing peaks at the level of the cervical and lumbar enlargements respectively and the transient presence of a smaller running peak which moves caudally with the front of the outgrowing bundle. The standing peaks are ascribed to the branching of the axon terminals at both intumescences, whereas the running peak probably arises by the accumulation of tracer within the growth cones at the tips of the outgrowing CST axons. Factors such as the number of axons, the varying axon diameters, the branching collaterals, the presence of varicosities, the transport rate of the tracer, the uptake of the tracer at the injection site, which possibly may affect the amount of label present in both the entire bundle and in the individual axons are discussed. Current research is focused upon an analysis of the relation between the site of injection within the cortex and the pattern of labeling of the CST. A delay of two days was found between the arrival of the CST axons at a particular spinal cord level and their outgrowth into the adjacent spinal gray. However, combined HRP and electronmicroscopic experiments are necessary to determine the factors behind the maturation of the CST as well as the maturation of the spinal gray.

Evidence for a direct projection from the superior temporal gyrus to the entorhinal cortex in the monkey

During the course of a larger study of the afferent and efferent connections of the entorhinal cortex in the macaque monkey we have found evidence for a hitherto undescribed projection to the entorhinal cortex from the superior temporal gyrus. The evidence is derived principally from experiments in which small volumes of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) were injected into different parts of the entorhinal cortex, but has been confirmed by 3H-amino acid autoradiography. After WGA-HRP injections into the entorhinal cortex, retrogradely labeled neurons have been mainly in layer III, but also to some extent in layer VI, throughout much of the superior temporal gyrus. The projection appears to be topographically organized in the sense that the ventral insular cortex and the adjoining temporal operculum have been found to project to the periamygdaloid cortex and the lateral division of the entorhinal cortex; the convexity of the superior temporal gyrus and the cortex along the dorsal bank of the superior temporal gyrus project further caudally to the medial division of the entorhinal cortex; and the cortex surrounding the fundus of the superior temporal sulcus projects to the perirhinal cortex. Following an injection of 3H-amino acids into the convexity of the superior temporal gyrus, terminal labeling has been seen over layers I and II of the entorhinal cortex and over layer I in the most lateral portion of the presubiculum. While the distribution of retrogradely labeled cells in our WGA-HRP experiments encompasses several cytoarchitectonically distinguishable areas in the superior temporal gyrus, the most heavily labeled field appears to coincide with what Gross and his colleagues have termed the ‘superior temporal polysensory area’ on the dorsal bank of the superior temporal sulcus.

Crossed connections of the substantia nigra in the rat.

The existence of crossed multisynaptic pathways that allow for the interdependent control of activity in one substantia nigra and its contralateral counterpart has been inferred from a number of recent biochemical and neurophysiological investigations. This prompted a reexamination of the connections of the substantia nigra with an emphasis on crossed inputs to and crossed projections from that nucleus. Male albino rats received 20-50-nl pressure injections of a 1% wheat germ agglutinin-conjugated horse-radish peroxidase (WGA-HRP) solution into the substantia nigra or into surrounding areas as controls. Following a 24-hour survival period the animals were processed according to the tetramethylbenzidine protocol for the visualization of HRP. The pattern of anterograde transport of WGA-HRP after substantia nigra injections, confirming for the most part previous reports, demonstrated ipsilateral nigral efferent projections to the striatum; globus pallidus; subthalamic nucleus; the lateral dorsal, paralamellar mediodorsal, ventromedial, and parafascicular thalamic nuclei; central gray, midbrain reticular formation; superior colliculus; and peribrachial area, including the pedunculopontine nucleus. Additionally, the nigral projections to the paralamellar mediodorsal and ventromedial thalamic nuclei and to the superior colliculus were demonstrated to be bilateral. Most of these connections were confirmed by the complementary retrograde experiment. In accordance with previous reports, intranigral WGA-HRP injections retrogradely labeled neurons located in the ipsilateral prefontal cortex, motor cortex, striatum, globus pallidus, central nucleus of the amygdala, anterior hypothalamic area, subthalamic nucleus, and dorsal raphe. Additionally, labeled perikarya were observed in the ipsilateral parafascicular thalamic nucleus, in the contralateral posterior lateral hypothalamic area, and in the ipsilateral and contralateral peribrachial-pedunculopontine area. These latter nigral afferents were confirmed with complementary WGA-HRP injections into each of the regions of origin. While bilateral peribrachial-pedunculopontine innervation of the substantia nigra has been reported in the cat there has been no previous demonstration of a crossed nigral afferent system from the contralateral posterior lateral hypothalamic area. The results are discussed with reference to the pathways that may mediate the interdependent control of the activity of neurons in the left and right substantia nigra. Additionally, the association of the substantia nigra with a variety of neuronal circuits, including the cerebellofugal, tectothalamic, thalamocortical, thalamostriatal, and basal ganglia pathways, are discussed.