A. W. Hrycyshyn

Horseradish peroxidase study of brain stem projections of carotid sinus and aortic depressor nerves in the cat.

Abstract The connections of carotid sinus (CSN) and aortic depressor (ADN) afferent fibers in the brain stem of the cat were studied using horseradish peroxidase (HRP). Crystalline HRP was applied to the proximal cut end of either the CSN or ADN for 4–10.5 h and after a survival period of 24–120 h the animals were perfused and frozen sections of brain stem, nodose and petrosal ganglia were processed according to the tetramethyl-benzidine method. CSN labeled axons were found to project ipsilaterally to several nuclei of the solitary complex: the dorsal aspect of the medial (Sm), the lateral (Slt), the ventrolateral (Svl), the commissural (Com), the intermediate (Int) and the dorsomedial aspect of the parvocellular (Spc) solitary nuclei. These nuclei, except for the Svl, also received a less intense contralateral projection. Additional terminal labeling was observed on the ipsilateral side along the dorsal border of the dorsal motor nucleus of the vagus (DMV), in the reticular formation ventral to the solitary complex, and in the ventrolateral external cuneate nucleus, and labeled fibers were found in the dorsolateral spinal trigeminal tract. On the other hand, ADN labeled fibers were found to project only to the solitary complex, bilaterally. Terminal labeling was found primarily in the Sm, Slt, Com and dorsal Spc. After HRP application to the CSN few cells were found labeled in the petrosal ganglion; in addition clusters of labelled neurons were found bilaterally in the region of the rostral nucleus ambiguus, retrofacial and facial nuclei, and in the ipsilateral rostral dorsomedial reticular formation. ADN labeled ganglion cells were identified in clusters in the medial aspect of the nodose ganglion primarily near the entry of the superior laryngeal nerve; additional clusters of labeled smaller neurons were found in the ventromedial portion of the ganglion and intermingled with vagal fibers just caudal to the ganglion. No HRP-positive cells were identified in the brain stem after ADN labeling. These data demonstrate that different regions of the solitary complex receive direct inputs from either one or both buffer nerves, suggesting a degree of separation of central pathways carrying CSN and ADN afferent information. Furthermore, the finding of labeled cell bodies in the medulla after CSN labeling suggests a possible route by which the central nervous system may alter activity of receptors in the carotid sinus and body.

Efferent connections of the A1 noradrenergic cell group: A DBH immunohistochemical and PHA-L anterograde tracing study

Immunohistochemical localization of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) was employed to reveal the anatomical organization of the A1 noradrenergic cell group in the caudal ventrolateral medulla oblongata of the rat. Subsequently, the supraspinal efferent axonal projections of A1 were investigated with a view to elucidating the anatomical substrates underlying its postulated function in central fluid and cardiovascular homeostasis. Within the caudal medulla, DBH-positive/PNMT-negative (noradrenergic) neurons were observed extending bilaterally through the ventrolateral medullary reticular formation from upper cervical spinal cord levels to the level of the area postrema. At the rostral pole of A1, its neurons intermingled with PNMT-immunoreactive perikarya of the more rostrally situated C1 adrenergic cell group. Discrete injections of the anterogradely transported plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into A1 resulted in terminal labeling in a number of presumptive efferent target sites including the nucleus of the solitary tract, rostral ventrolateral medulla, dorsal parabrachial nucleus, Kolliker-Fuse nucleus, central grey, dorsomedial nucleus of the hypothalamus, perifornical region, zona incerta, lateral hypothalamus, paraventricular nucleus of the hypothalamus, supraoptic nucleus, bed nucleus of the stria terminalis, and organum vasculosum of the lamina terminalis. Tissue sections adjacent to those reacted for PHA-L were processed immunohistochemically for DBH to determine if anterogradely labeled terminals were localized in regions that demonstrated appropriate immunoreactivity. The majority of regions in which PHA-L terminal labeling was present also exhibited moderate to intense DBH activity. These experiments provide neuroanatomical evidence for direct efferent pathways from the A1 noradrenergic cell group to a number of supraspinal sites that have been reliably implicated in the neural circuitry underlying the central regulation of fluid and cardiovascular homeostasis. Furthermore, the results suggest a selective anatomical interrelation between A1 and sites in the basal forebrain and hypothalamus in which vasopressinergic neurons have been previously demonstrated. It is postulated that the noradrenergic A1 projections observed in this investigation represent the morphological substrate through which A1 exerts a significant influence on cardiovascular regulatory mechanisms.

Glossopharyngeal and vagal afferent projections to the brain stem of the cat: A horseradish peroxidase study

Brain stem projections of the glossopharyngeal and vagus nerves in the cat were studied using the anterograde transport of horseradish peroxidase (HRP). Crystalline HRP was applied to the proximal cut ends of the nerves for a period of 4-10.5 h, and after a survival time of 24-120 h, transverse and horizontal sections of the brain stem were processed according to the tetramethylbenzidine method. Labeled fibers from both nerves were found to project bilaterally to the solitary complex, and ipsilaterally to the ventral region of the external cuneate nucleus and to the medial region of the nucleus praepositus hypoglossi, just dorsolateral to the medial longitudinal fasciculus. Within the solitary complex terminal labeling was found in the parvocellular, ventrolateral, lateral, medial and commissural solitary nuclei. Exclusive glossopharyngeal nerve projections were found ipsilaterally in the rostral dorsal motor nucleus of the vagus, the ventrolateral portion of the medial cuneate nucleus, the dorsal part of the nuclei caudalis and interpolaris of the trigeminal complex, the nuclei insulae cuneati lateralis, and the dorsolateral aspect of the nucleus medullae oblongata centralis. Finally, in the area postrema a bilateral projection of vagal and an ipsilateral projection of glossopharyngeal fibers were found. These findings demonstrate that the glossopharyngeal nerve has more widely distributed brain stem projections that the vagus nerve and provide essential information on projection sites of visceral and taste inputs to the central nervous system.

Collateral axonal projection from the A1 noradrenergic cell group to the paraventricular nucleus and bed nucleus of the stria terminalis in the rat

The A1 noradrenergic cell group in the caudal ventrolateral medullary reticular formation of the rat sends efferent projections to a number of regions in the basal forebrain and hypothalamus, but the extent to which these projections represent collateral branches of individual axons is not known. Immunohistochemical labeling of medullary neurons containing the catecholamine biosynthetic enzymes tyrosine hydroxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase was used to reveal the anatomical location of A1 noradrenergic neurons within the ventrolateral medulla. Subsequently, the retrograde fluorescence double-labeling technique was employed to investigate the collateralization of ascending A1 efferent axons. The subcommissural bed nucleus of the stria terminalis (BST) was injected with rhodamine-fluorescent latex microspheres and the ipsilateral left paraventricular nucleus of the hypothalamus (PVN) was injected with Fast blue. Within the ventrolateral medulla, single- and double-labeled neurons were identified in a distribution corresponding to that demonstrated for A1 noradrenergic perikarya. The results indicate that some ascending axons from cells within the A1 region collateralize to effect a simultaneous innervation of the BST and PVN. The innervation of multiple efferent targets by single neurons within the A1 region may have important implications with respect to A1s postulated role in central cardiovascular regulation.

An anterograde HRP-WGA study of aberrant corticorubral projections following neonatal lesions of the rat sensorimotor cortex

SummaryAnterograde transport of horseradish peroxidase — wheat germ agglutinin (HRP-WGA) was used to examine the effect of unilateral neonatal ablation of the sensorimotor cortex on the remaining corticofugal projections to the midbrain in the rat. In unlesioned animals, the sensorimotor cortical efferents to the midbrain were entirely ipsilateral, terminal labeling being evident in the red nucleus, the midbrain reticular formation, the periaqueductal gray, the intermediate gray layer of the superior colliculus, the nucleus parafascicularis prerubralis and the perilemniscal area. Corticorubral fibers were seen to reach the midbrain through the thalamus or the cerebral peduncle. In the red nucleus, terminal labeling was essentially restricted to the parvocellular region. In neonatally lesioned adults, aberrant corticofugal fibers crossed the midline to terminate in the contralateral red nucleus, the midbrain reticular formation, the periaqueductal gray, the nucleus parafascicularis prerubralis and the intermediate gray layer of the superior colliculus. The aberrant projections maintained the topographic specificity of the normal ipsilateral projections. This was most evident in the corticorubral projection, where the aberrant contralateral fibers terminated in the parvocellular area of the red nucleus.

Topographic specificity of aberrant cerebellorubral projections following neonatal hemicerebellectomy in the rat.

Anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) was used to examine the topographic specificity of ascending cerebellar efferent projections in adult rats which were hemicerebellectomized at birth. The results were compared to similar cerebellar projections in unlesioned adults. HRP-WGA placement in the nucleus interpositus of control rats resulted in a dense projection of labeled fibers which decussated in the midbrain, caudal to the red nucleus. In the red nucleus, dense terminal labeling was confined to the magnocellular region, while retrogradely labeled rubrocerebellar neurons were present throughout both parvo- and magnocellular areas. Similar HRP-WGA placements in the nucleus lateralis gave rise to fewer labeled fibers which terminated in the parvocellular red nucleus. In addition to the cerebellorubral projection, other areas of terminal labeling included the mid-brain reticular formation, nucleus parafascicularis prerubralis, zona incerta, fields of Forel and ventral thalamus. In neonatally lesioned adults, aberrant cerebellorubral and cerebellothalamic projections were observed deflecting ipsilaterally at the decussation of the normal contralateral projection. Topographic specificity of the aberrant ipsilateral cerebellorubral projection mirrored that of the normal contralateral fibers. In addition, an ipsilateral projection from the cerebellum could be followed rostral to the red nucleus, to terminate in the ipsilateral ventral thalamus. Lesioned animals also demonstrated marked cell loss in the red nucleus contralateral to the hemicerebellectomy.

On the diffusion of horseradish peroxidase into muscles and the “spurious” labeling of motoneurons

There is some evidence to suggest that the fascial sheaths of muscles are important in preventing the labeling of motoneurons that occurs, apparently, as a result of the diffusion of horseradish peroxidase (HRP) into the muscles. To test this possibility, Gelfoam soaked in HRP was implanted over flexor and extensor muscles in the proximal forelimb of the rat. When the fascial sheaths were damaged, labeled neurons were found in the motoneuronal pools of the exposed muscles; if intact, virtually no labeling of motoneurons was observed. These results suggest that, if intramuscular injections are to be used as a method for identifying motoneuronal pools, care should be taken when exposing the muscle to be injected, to ensure that surrounding muscles and their fascial sheaths are not damaged.

Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projection

Unilateral neonatal cortical ablation induces the development of a bilateral corticorubral projection from the remaining sensorimotor cortex. The retrograde fluorescent tracers Fast blue (FB) and Nuclear yellow (NY) were used to determine if the aberrant contralateral projection arises from axon collaterals of the normal uncrossed projection. Six to 8 weeks after unilateral cortical ablation in neonatal rats, the red nuclei were injected with FB on one side and NY on the other to study the source of the normal and aberrant afferents from the cerebral cortex. In control animals, many neurons in layer V of the sensorimotor cortex were retrogradely labeled with the tracer that had been injected into the ipsilateral red nucleus. In animals with unilateral ablations, many neurons throughout the remaining sensorimotor cortex were retrogradely labeled with FB or NY. No cortical neurons were doubly labeled. In addition to demonstrating the bilaterality of the corticorubral projection in animals which had received neonatal lesions, these results indicate that the aberrant contralateral corticorubral projection does not consist of axon collaterals of the normal ipsilateral fibers.

Role of the mesolimbic cholinergic projection to the septum in the production of 22 kHz alarm calls in rats

The role of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) to septum in the production of 22 kHz ultrasonic vocalization was studied in adult rats, using behavioral-pharmacological and anatomical tracing methods. Direct application of carbachol, a muscarinic agonist, into the lateral septal region induced species-typical 22 kHz alarm calls. The septum receives cholinergic input from LDT, thus, activation with glutamate of predominantly cholinergic neurons of the LDT induced comparable 22 kHz alarm calls in the same animals. This glutamate-induced response from LDT was significantly reduced when the lateral septum was pretreated with scopolamine, a cholinergic antagonist. To investigate the localization of the cell groups projecting to septum, the fluorescent retrograde tracer, fluorogold, was pressure injected into the lateral septum and sections from these brains were also immunostained against choline acetyltransferase (ChAT) to visualize cholinergic cell bodies. Several ChAT-fluorogold double-labeled cells within the boundaries of the LDT were found, while other fluorogold-labeled regions did not contain double-labeled cells. These results provide both direct and indirect evidence that at least a part of the mesolimbic ascending cholinergic projection from LDT to septum is involved in the initiation of the 22 kHz vocalization. It is concluded that the septum is an integral part of the medial cholinoceptive vocalization strip and the 22 kHz alarm vocalization is triggered from septum by the cholinergic input from the LDT.

Subpallidal outputs to the nucleus accumbens and the ventral tegmental area: anatomical and electrophysiological studies.

The goal of this study was to investigate the functional organization of the subpallidal-->accumbens direct and indirect feedback loops by both anatomical and electrophysiological methods. The results of the dextran-conjugated rhodamine injections into the subpallidal area has shown three distinct projections: (1) a substantial pathway from the subpallidal area to the ventral tegmental area, (2) a more diffuse rostral projection from the subpallidal area to the core area of the nucleus accumbens, and (3) a sparse pathway projecting rostrodorsally from the subpallidal area toward the thalamic regions. Electrical or chemical stimulation of the subpallidal region, which was studied by the axonal tracer, evoked inhibitory responses in the majority (60 and 80%, respectively) of the accumbens and ventral tegmental area neurons in a standard extracellular recording study. Less than 1/3 of the accumbens or ventral tegmental area cells showed an increase in the mean firing rate. The majority (77.5%) of all responded neurons had a latency of less than 10 ms. Furthermore, injection of glutamate into the subpallidal area not only altered the firing pattern of the accumbens neurons, but also attenuated their excitatory responses elicited by the electrical stimulation of the ventral subiculum. Our results indicate that the subpallidal area plays a predominantly inhibitory role in the ventral tegmental area-accumbens-subpallidal circuitry, presumably by its GABAergic projections, and may also modulate subicular input into the nucleus accumbens.