Brain stem localization of neurons of the subdiaphragmatic vagus nerve fibres in the ferret (Mustela Putorius Furo): a WGA-HRP neurohistochemical study

The brain stem localization of neurons of nerve fibres in the ventral and dorsal abdominal vagal trunks were studied in the ferret. A total of 14 adult ferrets (six experimental and eight controls) were used for the study. Following anesthesia with pentobarbitone sodium, an upper midline laparotomy was done to expose the abdominal trunks of the vagus nerve. After dissecting the trunks clear of the abdominal oesophagus and the cardia of the stomach the nerve trunks were cut and WGA-HRP was applied to the proximal stump of the cut trunks. Control ferrets were divided into four groups of two ferrets. In the first group normal saline instead of the tracer was applied to the proximal stump of the vagal trunks. The second group was treated in a similar manner as the experimental animal except that the application of tracer was preceded by bilateral cervical vagotomy. In the third group of controls 0.1 ml of WGA-HRP was injected into the abdominal cavity and the fourth group had tracer injection into the hepatic portal vein. All animals were allowed a survival period of 48-72 hours after tracer injection following which each animal was perfused with normal saline, fixative and buffered sucrose. The brain stem was extracted and cut in transverse section (40 µm with the freezing microtome. Sections were then processed for WGA-HRP neurohistochemistry and subsequently viewed and analyzed under light/dark-field illuminations. In the experimental ferrets labeled cells were seen bilaterally in the dorsal motor nucleus of the vagus nerve (DMNV), the nucleus dorsomedialis (nDm), the nucleus ambiguus (nA) and the nucleus retroambiguus (nrA). The DMNV was the most intensely labeled nucleus. Sporadic distribution of labeled cells was also observed in the reticular formation (rf) between the nA and the DMNV. Labeled neurons were not seen in any of the control experiments.

Origins of colonic preganglionic parasympathetic motoneurons from the central nervous system: a wheatgerm agglutinin-horseradish peroxidase study in the ferret (Mustela Putorius Furo).

The Wheat germ Agglutinin-Horseradish Peroxidase (WGA-HRP) transneuronal nerve tracing technique was used to study the localization of colonic preganglionic parasympathetic neurons in the central nervous system of the ferret. The entire colon, from the iliocecal junction to the colorectal junction was subdivided into four segments and the muscular wall of each segment injected separately with the tracer. The ferrets used as controls were also subdivided into four groups. The first group was injected with normal saline, the second group with the tracer following bilateral trunkal vagotomy, the third group intraperitoneally and the fourth group had tracer injection into the hepatic portal vein. The experimental as well as the control ferrets were allowed to survive for 24 to 96 hours after which they were anaesthetized and perfused sequentially with normal saline, buffered fixative and buffered sucrose. Serial transverse frozen sections were taken from the brainstem and the sacral segments of the spinal cord of the ferrets. These were then processed for WGA-HRP neurohistochemistry and analyzed under light and dark-field illuminations. The results of the study show that the dorsal motor nucleus of the vagus nerve (DMX) supplies the anterior 3 segments of the colon while the sacral segment of the spinal cord supplies the post 2 segments of the colon.

Morphological evidence for secondary vestibular afferent connections to the dorsal cochlear nucleus in the rabbit.

An analysis of central afferent projections to the dorsal cochlear nucleus (dCo), one of the three target nuclei of the auditory nerve, was made using retrograde axonal tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the rabbit. The findings showed that, in addition to its afferents from the brainstem auditory nuclei (they are not described herein), the dCo received sparse bilateral connections from the caudal three quarters of the vestibular nuclear complex (VNC). Following selective iontophoretic injections of WGA-HRP into the dCo, a small number of labelled neurones (from 2 to 28 per case) was found in the rostral and caudal portions of the medial vestibular nucleus and in the inferior vestibular nucleus. These neurones were observed mainly in the lateral regions of the nuclei. No labelling appeared in other nuclei which belonged to the VNC. The secondary vestibulocochlear connections have not been reported before in any species. With the anatomical method used, however, their functional role is difficult to explain. Further study is necessary to identify the type of neurotransmitter as well as the physiological properties of vestibular neurones projecting to the dCo, in terms of their responses to a change of the head position and to sound.

Axonal growth of newly formed vomeronasal receptor neurons after nerve transection.

Chemosensory neurons in the vomeronasal epithelium (vomeronasal neurons) regenerate following experimentally induced degeneration. Transection of the vomeronasal nerves leads to retrograde degeneration of vomeronasal neurons followed by replacement of the cell population. The projection of the axons of regenerated vomeronasal neurons was examined by horseradish peroxidase(HRP) histochemistry and electron microscopy. HRP-wheat germ agglutinin (WGA) was placed on the surface of the vomeronasal organ of the rat. Dense distribution of HRP-labeled fibers was observed in the vomeronasal nerve and glomerular layers in the accessory olfactory bulb (AOB) of the intact rat. At one week after transection, HRP-labeled fibers were not found in the AOB, and no labeled fibers could be observed on the medial surface of the olfactory bulb where the vomeronasal nerve traversed. Three weeks after transection, labeled fiber bundles were observed on the medial surface of the olfactory bulb in all animals. No labeled fibers were detected in the AOB. From 12 to 32 weeks after transection, projection of HRP-labeled fibers was identified in the AOB in 8 out of 26 rats (the incidence of projection was 30%). But the number of projection fibers on the operated side was much smaller than on the control side. Electron microscopy confirmed that the HRP-labeled terminals make synaptic contacts with neurons in the AOB.

A visuo-somatomotor pathway through superior parietal cortex in the macaque monkey: cortical connections of areas V6 and V6A.

This report addresses the connectivity of the cortex occupying middle to dorsal levels of the anterior bank of the parieto-occipital sulcus in the macaque monkey. We have previously referred to this territory, whose perimeter is roughly circumscribed by the distribution of interhemispheric callosal fibres, as area V6, or the 'V6 complex'. Following injections of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA-HRP) into this region, we examined the laminar organization of labelled cells and axonal terminals to attain indications of relative hierarchical status among the network of connected areas. A notable transition in the laminar patterns of the local, intrinsic connections prompted a sub-designation of the V6 complex itself into two separate areas, V6 and V6A, with area V6A lying dorsal, or dorsomedial to V6 proper. V6 receives ascending input from V2 and V3, ranks equal to V3A and V5, and provides an ascending input to V6A at the level above. V6A is not connected to area V2 and in general is less heavily linked to the earliest visual areas; in other respects, the two parts of the V6 complex share similar spheres of connectivity. These include regions of peripheral representation in prestriate areas V3, V3A and V5, parietal visual areas V5A/MST and 7a, other regions of visuo-somatosensory association cortex within the intraparietal sulcus and on the medial surface of the hemisphere, and the premotor cortex. Subcortical connections include the medial and lateral pulvinar, caudate nucleus, claustrum, middle and deep layers of the superior colliculus and pontine nuclei. From this pattern of connections, it is clear that the V6 complex is heavily engaged in sensory-motor integration. The specific somatotopic locations within sensorimotor cortex that receive this input suggest a role in controlling the trunk and limbs, and outward reaching arm movements. There is a secondary contribution to the brain's complex oculomotor circuitry. That the medial region of the cortex is devoted to tightly interconnected representations of the sensory periphery, both visual and somatotopic-which are routinely stimulated in concert-would appear to be an aspect of the global organization of the cortex which must facilitate multimodal integration.

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study.

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.

Projections from the caudal spinal trigeminal nucleus to commissural interneurons in the supratrigeminal region: an electron microscope study in the rat.

Electron microscopic double-labeling study in the rat indicated that projection fibers from the caudal spinal trigeminal nucleus (Vc) were distributed ipsilaterally within the supratrigeminal region (STR) capping the trigeminal motor nucleus (Tm) and made synaptic contact with neurons projecting to the contralateral Tm. Nociceptive inputs to the Vc may reflexly control, via interneurons in the STR, the activities of Tm neurons innervating the masticatory, tensor tympani, and/or tensor veli palatine muscles.

Reduced vagal sensory innervation of the small intestinal myenteric plexus following capsaicin treatment of adult rats.

To determine whether capsaicin treatment damages small intestinal vagal sensory nerve endings, we made intra-nodose injections of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) to label peripheral and central vagal endings in control and capsaicin-treated rats. Labeled intraganglionic laminar endings (IGLEs), characteristic of vagal sensory endings of the myenteric plexus, were counted. In controls IGLEs were numerous in the duodenum, less numerous in the jejunum and scarce in the ileum. In capsaicin-treated rats, IGLEs were significantly diminished in all areas of the small intestine. Capsaicin also reduced WGA-HRP activity in the medial and commissural nucleus of the solitary tract. Systemic capsaicin produces a long-lasting loss of vagal IGLEs in the small intestinal myenteric plexus. Such loss is consistent with capsaicin-induced impairment of intestinal reflexes and controls of food intake.