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Cortical projections to the nucleus of the tractus solitarius: An HRP study in the cat
Brain Research Bulletin, Vol. 16, pp. 497-505, 1986. 0 Ankho International
0361-9230/t% $3.00 + .OO
Inc. Printed in the U.S.A.
Cortical Projections to the Nucleus of the Tractus Solitarius: An HRP Study in the Cat C. J. WILLETT,
D. G. GWYN,’
J. G. RUTHERFORD
AND R. A. LESLIE
Department of Anatomy, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received
4 March 1986
WILLETT, C. J., D. G. GWYN, J. G. RUTHERFORD AND R. A. LESLIE. Cortical projections to the nucleus ofthe tractus solitarius: An HRP study in the car. BRAIN RES BULL 16(4) 497-505, 1986.-Recent evidence suggests that
autonomic reflexes involving sensations such as olfaction and gustation may be cortically mediated via centripetal pathways to brainstem autonomic centers. A study was therefore undertaken to elucidate one of these pathways in greater detail. Lectin conjugated horseradish peroxidase was injected into the nucleus tractus solitarius. Following standard light microscopic histochemical procedures to reveal horseradish peroxidase activity, the distribution of retrogradely labeled neurons in the cortex was recorded. Retrogradely labeled somata were seen bilaterally in layer five of the orbital gyNS, anterior insular cortex and infralimbic cortex. In other cats, the same tracer was injected into the orbital gyrus or anterior insular cortex. Bilateral anterograde labeling was seen in various subnuclei throughout the rostrocaudal extent of the nucleus tractus solitarius, but was heaviest in rostral regions of the nucleus. Labeling was also seen bilaterally in the spinal trigeminal nucleus. The projection to the nucleus tractus solitarius could allow for cortical modulation of gustatory and visceral information which is conveyed to the brainstem via the facial, glossopharyngeal and vagus nerves.
Cerebral cortex
Nucleus tractus solitarius
Neuronal pathways
VISCERAL, gustatory and olfactory functions have all been associated with regions of the insular cortex and/or the orbital gyrus in several species. For example, stimulation of the insula in monkey has been shown to produce hypotension, inhibition of respiration, and decrease gastric tonus [lo]. Visceral input to the orbito-insular cortex in cat has been demonstrated physiologically by recordings of evoked potentials in the cortex following stimulation of the vagus and splanchnic nerves [13, 14, 181. In addition, it has been shown that potentials may be evoked in the orbital gyrus by stimulating the olfactory bulb 1181, and the chorda tympani nerve [3,22], indicating input to this area of cortex from both olfactory and gustatory sources. Using anatomical techniques, primary gustatory and visceral sensory fibers derived from the facial, glossopharyngeal and vagus nerves have been shown to terminate in the nucleus of the tractus solitarius (NTS) in the monkey, rat and cat [ 1, 4, 5, 7, 11, 15, 201. Gustatory fibers terminate in the rostra1 l/3 to l/2 of the nucleus [2,21], while visceral input is directed toward the caudal */3 of the NTS [8,12]. Within the visceral portion of the NTS, there is a further segregation of input, with fibers of gastric origin terminating mainly dorsomedially [8,91, while fibers of cardiovascular origin terminate in regions adjacent to the tractus solitarius 141. In the rat [23] and mouse [24] the insular cortex is the source of a projection which is distributed
Cat
Horseradish
peroxidase
throughout the entire length of the NTS. In a recent study dealing chiefly with projections from the orbital gyrus to the parabrachial nucleus in the cat, a brief reference was made to a weak projection from this region of the cortex to the NTS [29]. Since, as noted above, the orbito-insular cortex in cat, like the NTS, receives gustatory and visceral sensory input, it is of interest to obtain as much information as possible about projections from this area of the cortex to the NTS. Such a pathway could provide the morphological basis for cortical feedback and possible modification of gustatory and visceral sensory input. The purpose of this study, therefore, is to investigate in the cat the origin and termination of pathways arising from the orbito-insular cortex which project to the NTS. METHOD
Seventeen adult cats of both sexes, weighing between 2.0 and 4.0 kg, were used in this study. For all operative procedures, animals were initially anesthetized with sodium pentobarbital (Somnitol; 30 mg/kg), then maintained on a halothane-oxygen mixture. In each of six animals, four injections of 5% wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) were placed in the NTS, using stereotaxic coordinates for guidance. The first injection was made at the level of the obex. The next three injections were made at 1 mm intervals
‘Requests for reprints should be addressed to Dr. D. G. Gwyn, Department of Anatomy, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
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FIG. 1. Camera lucida drawings of transverse sections through the medulla oblongata of DC343 illustrate the extent of the HRP-WGA injection site in this case. The injection site is indicated by hatching. AP=area postrema, DCN=dorsal column nuclei, NTS=nucleus tractus solitarius, PH=nucleus prepositus hypoglossi, SPV=spinal trigeminal nucleus, TS=tractus solitarius, VC=vestibular complex, X=dorsal motor nucleus of vagus, XII=hypoglossal nucleus.
FIG. 2 Camera lucida frontal cortex of DC343 gradely labeled neurons ILC=infralimbic cortex,
through successively more rostral levels. Total volumes of WGA-HRP injected varied between 0.6 and 1.2 ~1. With stereotaxic guidance, four injections of WGA-HRP were placed at 1 mm intervals in the orbital gyrus in each of four other cats. The injections were placed between 21 and 24 mm anterior to the interaural line. In each of four further animals, four injections of WGA-HRP were made at 1 mm intervals, extending caudally from 21 to 18 mm anterior to the interaural line. These injections were made in order to involve the anterior insular cortex. The injected volumes of the enzyme in the above cases varied from 0.12 to 0.20 ~1. Finally, because injections in the medulla oblongata involved the dorsal column nuclei, injections of WGA-HRP were placed in the pericruciate cortex in three cats as a control. Following a four day survival period, animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 11 of normal saline followed by 3 1of a fixative
consisting of a solution of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), both at room temperature. The fixative was washed out with 1 1 of cold (4°C) 10% phosphate-buffered sucrose solution. The brains were then exposed and sectioned in three stereotaxic transverse planes in order to obtain forebrain, midbrain and hindbrain tissue blocks. The blocks were stored overnight at 4°C. in the 10% phosphate-buffered sucrose solution, after which 40 pm frozen, transverse serial sections were cut through the medulla and the forebrain. Sections were washed in 0.1 M phosphate buffer, then reacted for HRP activity using the tetramethyl benzidine method of Mezulam [ 171. The sections were divided into three series, mounted on chrome-alum coated slides and air dried overnight. Two of the series were stained with Neutral Red, while the third series was left unstained. The sections were examined in the light microscope using bright- and dark-field illumination,
drawings of coronal sections through the illustrate the relative distribution of retroas indicated by dots. CG=coronal gyrus, LV=lateral ventricle.
FACING PAGE FIG. 3. (A) Brightfield photomicrograph of a transverse section through the medulla oblongata of DC343 shows the extent of the HRP-WGA injection site at the level of the area postrema (AP). NTS=nucleus tractus solitarius, bar= 1 mm. (B) Darkfield photomicrograph of a coronal section through the orbital gyrus of DC343 shows the position of retrogtadely labeled neurons. This section corresponds to the fourth level illustrated in Fig. 2. Prs=presylvian sulcus, bar=0.3 mm. (C) Brightfield photomicrograph of a coronal section through the orbital gyrus (Orb) of DC339 shows the HRP-WGA injection site. Prs=presylvian sulcus, bar=2 mm. (D) Darkfield photomicrograph of a transverse section through the medulla oblongata of DC339 near the caudal pole of the NTS shows anterograde labeling medial to the tractus solitarius (TS) in the lateral part of the commissural subnucleus. CC=central canal, Prs=presylvian sulcus, X=dorsal motor nucleus of the vagus, bar=0.2 mm.
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FIG. 4. Camera lucida drawings of coronal sections through the frontal cortex of DC339 illustrate the extent of the HRP-WGA injection site in the orbital gyms. The injection site is indicated by hatching.
and camera lucida drawings of selected levels through the injection and projection sites were prepared. RESULTS
In all six cases in which WGA-HRP was injected into the dorsomedial medulla, reaction product was found throughout most of the rostro-caudal extent of the ipsilateral NTS. However, in all these cases, the caudal portion of the contralateral NTS was invdved to varying degrees as well. In cat DC343, the spread of HRP to the side contralateral to the injection was minimal; therefore, this case will be described in detail. The injection site extended throughout the length of the NTS (Fig. 1). In the caudal medulla, the
E7’ AL.
dorsal motor nucleus of the vagus (DMV), the area postrema, part of the hypoglossal nucleus, and the dorsal column nuclei and subjacent reticular formation were also included in the injection site (Figs. 1 and 3A). At levels caudal to the obex, the enzyme spread across the midline dorsal to the central canal to involve the contralateral nucleus commissuralis and part of the DMV (Fig. t , level I). The distribution of retrogradely labeled neurons in rostra1 levels of the ipsilateral cortex of cat DC343 is illustrated in Fig 2. In the orbital gyrus, retrogradely labeled neurcns were observed bilaterally, but were more numerous on the side ipsilateral to the injection site. Labeled cells were noted in the lateral bank of the presylvian sulcus (Fig. 2, level 5), extending ventrolaterally around the tip of the gyrus, and up into the ventralmost part of the coronal gyrus (Fig. 2, level 4). Labeled cells in the orbital gyrus are illustrated in Fig. 3B. The section from which this micrograph was taken corresponds to level 4 in Fig. 2. The labeled neurons in the orbital gyrus were found within the fifth cortical layer. Neuronal labeling was observed throu~out that layer of the cortex extending for 1.6 mm rostrally from the region adjacent to the rostra1 pole of the claustrum. For the next 1.9 mm, which corresponds to the tissue between levels 1 and 2 in Fig. 2, no labeled neurons were seen. Kostral to this point, retrograde neuronal labeling extended through the orbital gyrus for a further 1.3 mm. Small numbers of labeled neurons were noted bilaterally in the rostra1 portion of the insular cortex. Again, greater numbers of labeled cells were found ipsilateral to the injection site. No labeled cells were seen in the posterior insular cortex. Retrogradely labeled neurons were found bilaterally in the infralimbic cortex (Fig. 2, level I), but the labeled cells were more commonly encountered on the side ipsitateral to the injection site. Additionally, labeled neurons were present bilaterally in the pericruciate cortex (Fig. 2, level 6), but in this region greater numbers of labeled cells were found on the side contralateral to the injection site. In the other five cases, where the WGA-HRP injection site was larger and involved the contralateral NTS to a greater extent, greater numbers of labeled neurons were seen in the cortex, both ipsi- and contr~ateral to the injection site. In these cases there was no interruption in the continuity of labeling in the orbital gyrus as was seen in DC343. Anterograde Labeling of Axon Terminals in the Medulla Following Injection of WGA-HRP Into the Orbital Gyrus Pour cats received injections of WGA-HRP into the orhital gyrus. A further four animals received injections which involved the orbital and anterior insular cortex. An example of such an injection which involved only the orbital gyrus. in animal DC339, is illustrated in Fig. 3C and 4. In this animal, the injection was centered in and included most of the orbital gyrus. Dorsolaterally, the injection site spread to involve part of the coronal gyrus (Fig. 4). The insular cortex was not involved in the injection site. In all cases in which HRP was injected into the orbital gyrus, anterograde labeling was observed in the medulla. The results from cat DC339 are typical of the fiber and terminal labeling seen in such cases. Anterogradely labeled’ fibers descended into the medulla in the pyr~idal tract ipsitateral to the injection site. At between 6.5 and 10.0 mm rostra1 to the obex, Iabeled axons extended dorsolaterally from the pyramid, through the reticular formation, to reach the ipsilateral spinal trigeminal nucleus (SpV). Between these same levels, labeled fibers
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sponds to the subnucleus gelatinous (Fig. 5, level 3; Fig. 6A). Heavier labeling was present in the medial part of the NTS commencing at a level approximately 3.0 mm rostra1 to the obex and extending to the rostral pole of the nucleus (Fig. 5, levels 4 and 5; Fig. 7A and B). At the rostra1 pole of the NTS (Fig. 5, level 5; Fig. 7B), terminal labeling occupied a position medial, ventral, and ventrolateral to the solitary tract, and extended laterally to become confluent with labeling in the dorsal part of the reticular formation and the SpV. Anterograde labeling also extended throughout the length of SpV bilaterally (Fig. 5). At caudal levels (Fig. 5, levels 1 and 2), the labeling occupied the dorsomedial border of the nucleus and extended dorsolaterally along the boundary with the spinal trigeminal tract. More rostrally, beginning approximately 2.0 mm from the obex (Fig. 5, level 3), labeling was restricted to the medial border of SpV (Fig. 5, levels 3-5; Fig. 8). In the three control cats which received injections of WGA-HRP in the pericruiciate cortex, anterograde labeling was found in the contralateral dorsal column nuclei, but not in NTS. DISCUSSION
CAUDAL FIG. 5. Camera lucida drawings of transverse sections through the medulla oblongata of DC339 illustrate the relative intensity and distribution of anterograde labeling, depicted by stippling. EC=external cuneate nucleus, SNG=subnucleus gelatinosus, other abbreviations as in Fig. 1.
were also seen to cross the midline and extend to the contralateral SpV. At a level approximately 10.0 mm rostra1 to the obex and beyond the rostra1 limits of the NTS, some of the anterogradely labeled fibers extended dorsomedially from SpV on both sides, then turned caudally and came to lie adjacent to the tractus solitarius (Fig. 5, levels 2-5). These labeled fibers appeared to terminate throughout the rostrocaudal extent of the NTS. The rest of the labeled fibers which extended from the pyramids to SpV on both sides coursed rostrally to the parabrachial nuclei. The bilateral distribution of terminal labeling in the NTS is illustrated in Fig. 5. At the caudal pole of the nucleus, sparse labeling was seen in the commissural region (Fig. 3D; Fig. 5, level 1). Subjacent to the area postrema, light labeling was found in the NTS immediately surrounding the tractus solitarius, and extending into the dorsomedial regions of the nucleus (Fig. 5, levels 2 and 3; Figs. 6A and B), but little labeling was found in the extreme dorsomedial portion of the NTS which corre-
FOLLOWING
In the present study, retrogradely labeled neurons were observed bilaterally in the orbital gyrus, the anterior insular cortex and the infralimbic cortex in the cat subsequent to the injection of HRP into the dorsomedial medulla. The labeled neurons were restricted to layer five in all cortical regions described. Greater numbers of labeled cells were observed in the cortex ipsilateral to the injection site. Following injections of HRP into the orbital gyrus, labeled fibers descended through the ipsilateral pyramidal tract, then extended from the tract to the medial border of SpV both ipsi- and contralaterally. Some of the labeled fibers continued rostrally to the parabrachial complex, while others coursed dorsomedially from SpV, then descended to the NTS, possibly occupying Probst’s tract in doing so. Thus our findings indicate that there is a single pathway which descends ipsilaterally from the cortex to the medulla via the pyramidal tract and is then distributed bilaterally to the parabrachial nuclei and the NTS. Following HRP injections into the insular cortex of the rat it was reported that labeled fibers entered the medulla via two pathways [23]. One followed Probst’s tract from the parabrachial nuclei to the NTS bilaterally, while the other descended via the ipsilateral pyramidal tract, then decussated and terminated in the contralateral NTS. This latter finding may represent a species difference between rat and cat, or it may reflect differences in interpretations of the courses of these tracts. Given the ipsilateral predominance of retrogradely labeled neurons in the cortex, it was somewhat surprising to note that the density of anterograde labeling appeared to be equal bilaterally in both the NTS and SpV subsequent to
PAGES
FIG. 6. (A) Darkfield photomicrograph through the medulla oblongata of DC339 contralateral to the injection site, at a level just rostra] to the area postrema. The micrograph shows anterograde labeling in the NTS medial to the tractus solitarius (TS), but avoiding the subnucleus gelatinosus (SNG). The floor of the fourth ventricle is indicated by arrows. Bar=0.2 mm. (B) At higher magnification, the labeling adjacent to the TS is more clearly seen. X=dorsal motor nucleus of the vagus, bar=O.l mm. FIG. 7. (A) Darktield photomicrograph of a transverse section through the medulla oblongata of DC339 shows anterograde labeling in the NTS at a level corresponding to level 4 of Fig. 5. (B) This photomicrograph illustrates labeling in the NTS close to its rostra] end. Note the relatively intense anterograde label at this level, particularly medial to the tractus solitarius (TS). Labeling can be seen to extend ventrolateral to the TS. The floor of the fourth ventricle is indicated by arrows. X=dorsal motor nucleus of the vagus, bars=0.2 mm.
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FIG. 8. Darktield photomicrograph of a transverse section through the medulla oblongata of DC339 showing anterograde labeling (arrow) along the medial border of the spinal trigeminal nucleus (SpV) extending dorsomedially toward the tractus solitarius (TS). SpVT=spinal trigeminal tract, bar=0.2 mm. _ cortical injections of HRP. A recent study designed to examine cortical projections to the parabrachial region of the cat described, but did not illustrate, sparse terminal labeling in
the NTS following injections into the orbital gyrus [29]. Although it is diflicult to make such a comparison, the cortical projections to NTS described in the present report appear to be heavier than those described by these workers. We found anterograde labeling to be heaviest in the rostromedial portion of the nucleus, an area known to receive first order gustatory fibers 1211 from the chorda tympani nerve [2,19]. In the caudal NTS, a very small projection to lateral parts of the commissural region and a moderate projection in the region immediately surrounding the solitary tract were observed. These are areas which receive first order cardiovascular fibers [4] via the vagus or glossopharyngeal nerves. There was, however, no labeling present within the subnucleus gelatinosus, a region of the NTS which is known to receive vagal fibers from the gastrointestinal tract [8,9]. The insular cortex in the mouse [24] has been shown to project to the NTS, while in the rat, both the insular [23, 27, 281 and medial frontal or infralimbic cortices [26,28] have been reported to project to the NTS. In these species, the insular projection ‘was described as being topographically distributed, with rostral regions of the insular cortex projecting to rostral areas of the NTS, and caudal portions projecting to the caudal NTS. The projection from the insular cortex to the NTS in the mouse is bilateral with an ipsilateral pre-
dominance [24], while the projection from both infralimbic and insular cortices in the rat is bilateral with a contralateral predominance [23,26]. In our study, the predominance of terminal labeling in the rostra1 NTS and its relative paucity at caudal levels of that nucleus is in contrast to the projection from the insular cortex to the NTS in the rat [23] and mouse [24]; in these rodents this projection is described as terminating more evenly throughout the entire length of the NTS. These differences notwithstanding, the orbital gyrus of the cat is similar to the insular cortex of the mouse and rat in that it has been shown to receive olfactory [18] as well as gustatory [3] and visceral input [13,14]. The convergence of these modalities within the orbital gyrus of cat may be indicative of an integrative role similar to that hypothesized for the insular cortex of mouse [25]. If this is the case, then the descending projection described in the present report could provide a pathway for cerebral cortical modulation of gustatory and visceral information conveyed to NTS by cranial nerves VII, IX and X. The present study also describes a projection from the orbital gyNS which courses through portions of SpV and, in a band-like fashion, through the region between SpV and the NTS. Should this represent a terminal projection, it would end within a region of the brainstem implicated in mediating complex reflexes such as swallowing and emesis [6,16]. Thus gustatory information reaching the orbital gyrus might, through this projection, influence autonomic activity.
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REFERENCES 1. Beckstead, R. M. and R. Norgren. An automdiographic examination of the central distribution of the trigeminal, facial, glossopharyngeal and vagal nerves in the monkey. J Comp New01 184: 455-472, 1979. 2. Blomquist, A. J. and A. Antem. Localization of the terminals of the tongue afferents in the nucleus of the solitary tract. J Cornp Neurol 124: 127-130, 1965. 3. Burton, H. and F. Earls. Cortical representation of the ipsilatera1 chorda tympani nerve in the cat. Brain Res 16: 520-523, 1%9. 4. Ciriello, J., A. Hrychshyn and F. R. Calaresu. Horseradish peroxidase study of brain stem projections of carotid sinus and aortic depressor nerves in the cat. J Auto Nerv Syst 4: 43-61, 1981. and F. R. Calaresu. Glos5. Ciriello, J., A. Hrychshyn sopharyngeal and vagal afferent projections to the brain stem of the cat: A horseradish peroxidase study. J Auto Nerv Syst 4: 63-79, 1981. 6. Doty, R. W. Neural organization of deglutition. In: Handbook of Physiology. Vol IV. Alimentary Canal, edited by W. Heidel. Washington, DC: American Physiological Society, 1968, pp. 1861-1902. 7. Gwyn, D. G. and R. A. Leslie. A projection of vagus nerve to the area subpostrema in the cat. Brain Res 161: 335-341, 1979. 8. Gwyn, D. G., R. A. Leslie and D. A. Hopkins. Gastric afferents to the nucleus of the solitary tract in the cat. Neurosci Lett 14: 13-17, 1979. 9. Gwyn, D. G., R. A. Leslie and D. A. Hopkins. Observations on the afferent and efferent organization of the vagus nerve and the innervation of the stomach in the squirrel monkey. J Camp Neurol 239: 163-175, 1985. 10. Hoffman, B. L. and T. Rasmussen. Stimulation studies of insular cortex of the Macaca mulatta. J Neurophysiol 16: 343-350, 1953.
11. Kalia, M. and M. Mesulam. Brain stem projections of sensory and motor components of the vagus complex in the cat: I. The cervical vagw and nodose ganglion. J Comp Neurol 193: 435466, 1980. 12. Kalia, M. and M. Mesulam. Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac and gastrointestinal branches. J Comp Neurol 193: 467-508, 1980. 13. Horn, H. Splanchnic projection to the orbital cortex of the cat. Brain Res 16: 23-38, 1969. 14. Kom, H., R. Wendt and D. Albe-Fessard. Somatic projection to the orbital cortex of the cat. Electroencephalogr Clin Neurophysiol 21: 209-226, 1966.
15. Leslie, R. A., D. G. Gwyn and D. A. Hopkins. The central distribution of cervical vagus nerve and gastric aBerent and efferent projections in the rat. Brain Res Bull 8: 37-43, 1982. 16. Mehler, W. R. Observations on the connectivity of the parvicellular reticular formation with respect to a vomiting center. Brain Behav Evol23: 63-80, 1983. 17. Mesulam, M.-M. Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: A non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26: 106-117, 1978. 18. Motokizawa, F. and Y. Ino. A search for olfactory receiving areas in the cerebral cortex of cats. Neuroscience 6: 39-46, 1981. 19. Nomura, S. and N. Mizuno. Central distribution
20. 21.
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of afferent and efferent components of the chorda tympani in the cat as revealed by the horseradish peroxidase method. Brain Res 214: 229-237, 1981. Nomura, S. and N. Mizuno. Central distribution of aIferent and efferent components of the glossopharyngeal nerve: An HRP study in the cat. Brain Res 236: l-14, 1982. Norgren, R. and C. M. Leonard. Ascending central gustatory pathways. J Comp Neurol 150: 217-238, 1973. Patton, H. D. and V. E. Amassian. Cortical projection zone of chorda tympani nerve in cat. J Neurophysiol 15: 245-250, 1952. Saper, C. B. Convergence of autonomic and limbic connections in the insular cortex of the rat. J Comp Neurol 210: 163-173,
1982. 24. Shipley, M. T. Insular cortex projection
solitary tract and brainstem visceromotor
to the nucleus of the regions in the mouse.
Brain Res Bull 8: 139-148, 1982. 25. Shipley, M. T. and Y. Geinisman.
Anatomical evidence for convergence of olfactory, gustatory, and visceral afferent pathways in mouse cerebral cortex. Brain Res Bull 12: 221-226, 1984. 26. Terreberry, R. R. and E. J. Neafsey. Rat medial frontal cortex: A visceral motor region with a direct projection to the solitary nucleus. Brain Res 278: 245-249, 1983. 27. Van der Kooy, D., J. F. McGinty, L. Y. Koda, C. R. Gerfen and F. E. Bloom. Visceral cortex: A direct connection from prefrontal cortex to the solitary nucleus in rat. Neurosci Lett 33: 123-127, 1982. 28. Van der Kooy, D., L. Y. Koda, J. F. McGinty, C. R. Gerfen
and F. E. Bloom. The organization of projections from tex, amygdala, and hypothalamus to the nucleus of the tract in rat. J Comp Neurol 224: l-24, 1984. 29. Yasui, Y., K. Itoh, M. Takada, A. Mitani, T. Kaneko Mizuno. Direct cortical projections to the parabrachial in the cat. J Comp Neurol 234: 77-86, 1985.
the corsolitary and N. nucleus
0361-9230/t% $3.00 + .OO
Inc. Printed in the U.S.A.
Cortical Projections to the Nucleus of the Tractus Solitarius: An HRP Study in the Cat C. J. WILLETT,
D. G. GWYN,’
J. G. RUTHERFORD
AND R. A. LESLIE
Department of Anatomy, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received
4 March 1986
WILLETT, C. J., D. G. GWYN, J. G. RUTHERFORD AND R. A. LESLIE. Cortical projections to the nucleus ofthe tractus solitarius: An HRP study in the car. BRAIN RES BULL 16(4) 497-505, 1986.-Recent evidence suggests that
autonomic reflexes involving sensations such as olfaction and gustation may be cortically mediated via centripetal pathways to brainstem autonomic centers. A study was therefore undertaken to elucidate one of these pathways in greater detail. Lectin conjugated horseradish peroxidase was injected into the nucleus tractus solitarius. Following standard light microscopic histochemical procedures to reveal horseradish peroxidase activity, the distribution of retrogradely labeled neurons in the cortex was recorded. Retrogradely labeled somata were seen bilaterally in layer five of the orbital gyNS, anterior insular cortex and infralimbic cortex. In other cats, the same tracer was injected into the orbital gyrus or anterior insular cortex. Bilateral anterograde labeling was seen in various subnuclei throughout the rostrocaudal extent of the nucleus tractus solitarius, but was heaviest in rostral regions of the nucleus. Labeling was also seen bilaterally in the spinal trigeminal nucleus. The projection to the nucleus tractus solitarius could allow for cortical modulation of gustatory and visceral information which is conveyed to the brainstem via the facial, glossopharyngeal and vagus nerves.
Cerebral cortex
Nucleus tractus solitarius
Neuronal pathways
VISCERAL, gustatory and olfactory functions have all been associated with regions of the insular cortex and/or the orbital gyrus in several species. For example, stimulation of the insula in monkey has been shown to produce hypotension, inhibition of respiration, and decrease gastric tonus [lo]. Visceral input to the orbito-insular cortex in cat has been demonstrated physiologically by recordings of evoked potentials in the cortex following stimulation of the vagus and splanchnic nerves [13, 14, 181. In addition, it has been shown that potentials may be evoked in the orbital gyrus by stimulating the olfactory bulb 1181, and the chorda tympani nerve [3,22], indicating input to this area of cortex from both olfactory and gustatory sources. Using anatomical techniques, primary gustatory and visceral sensory fibers derived from the facial, glossopharyngeal and vagus nerves have been shown to terminate in the nucleus of the tractus solitarius (NTS) in the monkey, rat and cat [ 1, 4, 5, 7, 11, 15, 201. Gustatory fibers terminate in the rostra1 l/3 to l/2 of the nucleus [2,21], while visceral input is directed toward the caudal */3 of the NTS [8,12]. Within the visceral portion of the NTS, there is a further segregation of input, with fibers of gastric origin terminating mainly dorsomedially [8,91, while fibers of cardiovascular origin terminate in regions adjacent to the tractus solitarius 141. In the rat [23] and mouse [24] the insular cortex is the source of a projection which is distributed
Cat
Horseradish
peroxidase
throughout the entire length of the NTS. In a recent study dealing chiefly with projections from the orbital gyrus to the parabrachial nucleus in the cat, a brief reference was made to a weak projection from this region of the cortex to the NTS [29]. Since, as noted above, the orbito-insular cortex in cat, like the NTS, receives gustatory and visceral sensory input, it is of interest to obtain as much information as possible about projections from this area of the cortex to the NTS. Such a pathway could provide the morphological basis for cortical feedback and possible modification of gustatory and visceral sensory input. The purpose of this study, therefore, is to investigate in the cat the origin and termination of pathways arising from the orbito-insular cortex which project to the NTS. METHOD
Seventeen adult cats of both sexes, weighing between 2.0 and 4.0 kg, were used in this study. For all operative procedures, animals were initially anesthetized with sodium pentobarbital (Somnitol; 30 mg/kg), then maintained on a halothane-oxygen mixture. In each of six animals, four injections of 5% wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) were placed in the NTS, using stereotaxic coordinates for guidance. The first injection was made at the level of the obex. The next three injections were made at 1 mm intervals
‘Requests for reprints should be addressed to Dr. D. G. Gwyn, Department of Anatomy, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
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FIG. 1. Camera lucida drawings of transverse sections through the medulla oblongata of DC343 illustrate the extent of the HRP-WGA injection site in this case. The injection site is indicated by hatching. AP=area postrema, DCN=dorsal column nuclei, NTS=nucleus tractus solitarius, PH=nucleus prepositus hypoglossi, SPV=spinal trigeminal nucleus, TS=tractus solitarius, VC=vestibular complex, X=dorsal motor nucleus of vagus, XII=hypoglossal nucleus.
FIG. 2 Camera lucida frontal cortex of DC343 gradely labeled neurons ILC=infralimbic cortex,
through successively more rostral levels. Total volumes of WGA-HRP injected varied between 0.6 and 1.2 ~1. With stereotaxic guidance, four injections of WGA-HRP were placed at 1 mm intervals in the orbital gyrus in each of four other cats. The injections were placed between 21 and 24 mm anterior to the interaural line. In each of four further animals, four injections of WGA-HRP were made at 1 mm intervals, extending caudally from 21 to 18 mm anterior to the interaural line. These injections were made in order to involve the anterior insular cortex. The injected volumes of the enzyme in the above cases varied from 0.12 to 0.20 ~1. Finally, because injections in the medulla oblongata involved the dorsal column nuclei, injections of WGA-HRP were placed in the pericruciate cortex in three cats as a control. Following a four day survival period, animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 11 of normal saline followed by 3 1of a fixative
consisting of a solution of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), both at room temperature. The fixative was washed out with 1 1 of cold (4°C) 10% phosphate-buffered sucrose solution. The brains were then exposed and sectioned in three stereotaxic transverse planes in order to obtain forebrain, midbrain and hindbrain tissue blocks. The blocks were stored overnight at 4°C. in the 10% phosphate-buffered sucrose solution, after which 40 pm frozen, transverse serial sections were cut through the medulla and the forebrain. Sections were washed in 0.1 M phosphate buffer, then reacted for HRP activity using the tetramethyl benzidine method of Mezulam [ 171. The sections were divided into three series, mounted on chrome-alum coated slides and air dried overnight. Two of the series were stained with Neutral Red, while the third series was left unstained. The sections were examined in the light microscope using bright- and dark-field illumination,
drawings of coronal sections through the illustrate the relative distribution of retroas indicated by dots. CG=coronal gyrus, LV=lateral ventricle.
FACING PAGE FIG. 3. (A) Brightfield photomicrograph of a transverse section through the medulla oblongata of DC343 shows the extent of the HRP-WGA injection site at the level of the area postrema (AP). NTS=nucleus tractus solitarius, bar= 1 mm. (B) Darkfield photomicrograph of a coronal section through the orbital gyrus of DC343 shows the position of retrogtadely labeled neurons. This section corresponds to the fourth level illustrated in Fig. 2. Prs=presylvian sulcus, bar=0.3 mm. (C) Brightfield photomicrograph of a coronal section through the orbital gyrus (Orb) of DC339 shows the HRP-WGA injection site. Prs=presylvian sulcus, bar=2 mm. (D) Darkfield photomicrograph of a transverse section through the medulla oblongata of DC339 near the caudal pole of the NTS shows anterograde labeling medial to the tractus solitarius (TS) in the lateral part of the commissural subnucleus. CC=central canal, Prs=presylvian sulcus, X=dorsal motor nucleus of the vagus, bar=0.2 mm.
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FIG. 4. Camera lucida drawings of coronal sections through the frontal cortex of DC339 illustrate the extent of the HRP-WGA injection site in the orbital gyms. The injection site is indicated by hatching.
and camera lucida drawings of selected levels through the injection and projection sites were prepared. RESULTS
In all six cases in which WGA-HRP was injected into the dorsomedial medulla, reaction product was found throughout most of the rostro-caudal extent of the ipsilateral NTS. However, in all these cases, the caudal portion of the contralateral NTS was invdved to varying degrees as well. In cat DC343, the spread of HRP to the side contralateral to the injection was minimal; therefore, this case will be described in detail. The injection site extended throughout the length of the NTS (Fig. 1). In the caudal medulla, the
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dorsal motor nucleus of the vagus (DMV), the area postrema, part of the hypoglossal nucleus, and the dorsal column nuclei and subjacent reticular formation were also included in the injection site (Figs. 1 and 3A). At levels caudal to the obex, the enzyme spread across the midline dorsal to the central canal to involve the contralateral nucleus commissuralis and part of the DMV (Fig. t , level I). The distribution of retrogradely labeled neurons in rostra1 levels of the ipsilateral cortex of cat DC343 is illustrated in Fig 2. In the orbital gyrus, retrogradely labeled neurcns were observed bilaterally, but were more numerous on the side ipsilateral to the injection site. Labeled cells were noted in the lateral bank of the presylvian sulcus (Fig. 2, level 5), extending ventrolaterally around the tip of the gyrus, and up into the ventralmost part of the coronal gyrus (Fig. 2, level 4). Labeled cells in the orbital gyrus are illustrated in Fig. 3B. The section from which this micrograph was taken corresponds to level 4 in Fig. 2. The labeled neurons in the orbital gyrus were found within the fifth cortical layer. Neuronal labeling was observed throu~out that layer of the cortex extending for 1.6 mm rostrally from the region adjacent to the rostra1 pole of the claustrum. For the next 1.9 mm, which corresponds to the tissue between levels 1 and 2 in Fig. 2, no labeled neurons were seen. Kostral to this point, retrograde neuronal labeling extended through the orbital gyrus for a further 1.3 mm. Small numbers of labeled neurons were noted bilaterally in the rostra1 portion of the insular cortex. Again, greater numbers of labeled cells were found ipsilateral to the injection site. No labeled cells were seen in the posterior insular cortex. Retrogradely labeled neurons were found bilaterally in the infralimbic cortex (Fig. 2, level I), but the labeled cells were more commonly encountered on the side ipsitateral to the injection site. Additionally, labeled neurons were present bilaterally in the pericruciate cortex (Fig. 2, level 6), but in this region greater numbers of labeled cells were found on the side contralateral to the injection site. In the other five cases, where the WGA-HRP injection site was larger and involved the contralateral NTS to a greater extent, greater numbers of labeled neurons were seen in the cortex, both ipsi- and contr~ateral to the injection site. In these cases there was no interruption in the continuity of labeling in the orbital gyrus as was seen in DC343. Anterograde Labeling of Axon Terminals in the Medulla Following Injection of WGA-HRP Into the Orbital Gyrus Pour cats received injections of WGA-HRP into the orhital gyrus. A further four animals received injections which involved the orbital and anterior insular cortex. An example of such an injection which involved only the orbital gyrus. in animal DC339, is illustrated in Fig. 3C and 4. In this animal, the injection was centered in and included most of the orbital gyrus. Dorsolaterally, the injection site spread to involve part of the coronal gyrus (Fig. 4). The insular cortex was not involved in the injection site. In all cases in which HRP was injected into the orbital gyrus, anterograde labeling was observed in the medulla. The results from cat DC339 are typical of the fiber and terminal labeling seen in such cases. Anterogradely labeled’ fibers descended into the medulla in the pyr~idal tract ipsitateral to the injection site. At between 6.5 and 10.0 mm rostra1 to the obex, Iabeled axons extended dorsolaterally from the pyramid, through the reticular formation, to reach the ipsilateral spinal trigeminal nucleus (SpV). Between these same levels, labeled fibers
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sponds to the subnucleus gelatinous (Fig. 5, level 3; Fig. 6A). Heavier labeling was present in the medial part of the NTS commencing at a level approximately 3.0 mm rostra1 to the obex and extending to the rostral pole of the nucleus (Fig. 5, levels 4 and 5; Fig. 7A and B). At the rostra1 pole of the NTS (Fig. 5, level 5; Fig. 7B), terminal labeling occupied a position medial, ventral, and ventrolateral to the solitary tract, and extended laterally to become confluent with labeling in the dorsal part of the reticular formation and the SpV. Anterograde labeling also extended throughout the length of SpV bilaterally (Fig. 5). At caudal levels (Fig. 5, levels 1 and 2), the labeling occupied the dorsomedial border of the nucleus and extended dorsolaterally along the boundary with the spinal trigeminal tract. More rostrally, beginning approximately 2.0 mm from the obex (Fig. 5, level 3), labeling was restricted to the medial border of SpV (Fig. 5, levels 3-5; Fig. 8). In the three control cats which received injections of WGA-HRP in the pericruiciate cortex, anterograde labeling was found in the contralateral dorsal column nuclei, but not in NTS. DISCUSSION
CAUDAL FIG. 5. Camera lucida drawings of transverse sections through the medulla oblongata of DC339 illustrate the relative intensity and distribution of anterograde labeling, depicted by stippling. EC=external cuneate nucleus, SNG=subnucleus gelatinosus, other abbreviations as in Fig. 1.
were also seen to cross the midline and extend to the contralateral SpV. At a level approximately 10.0 mm rostra1 to the obex and beyond the rostra1 limits of the NTS, some of the anterogradely labeled fibers extended dorsomedially from SpV on both sides, then turned caudally and came to lie adjacent to the tractus solitarius (Fig. 5, levels 2-5). These labeled fibers appeared to terminate throughout the rostrocaudal extent of the NTS. The rest of the labeled fibers which extended from the pyramids to SpV on both sides coursed rostrally to the parabrachial nuclei. The bilateral distribution of terminal labeling in the NTS is illustrated in Fig. 5. At the caudal pole of the nucleus, sparse labeling was seen in the commissural region (Fig. 3D; Fig. 5, level 1). Subjacent to the area postrema, light labeling was found in the NTS immediately surrounding the tractus solitarius, and extending into the dorsomedial regions of the nucleus (Fig. 5, levels 2 and 3; Figs. 6A and B), but little labeling was found in the extreme dorsomedial portion of the NTS which corre-
FOLLOWING
In the present study, retrogradely labeled neurons were observed bilaterally in the orbital gyrus, the anterior insular cortex and the infralimbic cortex in the cat subsequent to the injection of HRP into the dorsomedial medulla. The labeled neurons were restricted to layer five in all cortical regions described. Greater numbers of labeled cells were observed in the cortex ipsilateral to the injection site. Following injections of HRP into the orbital gyrus, labeled fibers descended through the ipsilateral pyramidal tract, then extended from the tract to the medial border of SpV both ipsi- and contralaterally. Some of the labeled fibers continued rostrally to the parabrachial complex, while others coursed dorsomedially from SpV, then descended to the NTS, possibly occupying Probst’s tract in doing so. Thus our findings indicate that there is a single pathway which descends ipsilaterally from the cortex to the medulla via the pyramidal tract and is then distributed bilaterally to the parabrachial nuclei and the NTS. Following HRP injections into the insular cortex of the rat it was reported that labeled fibers entered the medulla via two pathways [23]. One followed Probst’s tract from the parabrachial nuclei to the NTS bilaterally, while the other descended via the ipsilateral pyramidal tract, then decussated and terminated in the contralateral NTS. This latter finding may represent a species difference between rat and cat, or it may reflect differences in interpretations of the courses of these tracts. Given the ipsilateral predominance of retrogradely labeled neurons in the cortex, it was somewhat surprising to note that the density of anterograde labeling appeared to be equal bilaterally in both the NTS and SpV subsequent to
PAGES
FIG. 6. (A) Darkfield photomicrograph through the medulla oblongata of DC339 contralateral to the injection site, at a level just rostra] to the area postrema. The micrograph shows anterograde labeling in the NTS medial to the tractus solitarius (TS), but avoiding the subnucleus gelatinosus (SNG). The floor of the fourth ventricle is indicated by arrows. Bar=0.2 mm. (B) At higher magnification, the labeling adjacent to the TS is more clearly seen. X=dorsal motor nucleus of the vagus, bar=O.l mm. FIG. 7. (A) Darktield photomicrograph of a transverse section through the medulla oblongata of DC339 shows anterograde labeling in the NTS at a level corresponding to level 4 of Fig. 5. (B) This photomicrograph illustrates labeling in the NTS close to its rostra] end. Note the relatively intense anterograde label at this level, particularly medial to the tractus solitarius (TS). Labeling can be seen to extend ventrolateral to the TS. The floor of the fourth ventricle is indicated by arrows. X=dorsal motor nucleus of the vagus, bars=0.2 mm.
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FIG. 8. Darktield photomicrograph of a transverse section through the medulla oblongata of DC339 showing anterograde labeling (arrow) along the medial border of the spinal trigeminal nucleus (SpV) extending dorsomedially toward the tractus solitarius (TS). SpVT=spinal trigeminal tract, bar=0.2 mm. _ cortical injections of HRP. A recent study designed to examine cortical projections to the parabrachial region of the cat described, but did not illustrate, sparse terminal labeling in
the NTS following injections into the orbital gyrus [29]. Although it is diflicult to make such a comparison, the cortical projections to NTS described in the present report appear to be heavier than those described by these workers. We found anterograde labeling to be heaviest in the rostromedial portion of the nucleus, an area known to receive first order gustatory fibers 1211 from the chorda tympani nerve [2,19]. In the caudal NTS, a very small projection to lateral parts of the commissural region and a moderate projection in the region immediately surrounding the solitary tract were observed. These are areas which receive first order cardiovascular fibers [4] via the vagus or glossopharyngeal nerves. There was, however, no labeling present within the subnucleus gelatinosus, a region of the NTS which is known to receive vagal fibers from the gastrointestinal tract [8,9]. The insular cortex in the mouse [24] has been shown to project to the NTS, while in the rat, both the insular [23, 27, 281 and medial frontal or infralimbic cortices [26,28] have been reported to project to the NTS. In these species, the insular projection ‘was described as being topographically distributed, with rostral regions of the insular cortex projecting to rostral areas of the NTS, and caudal portions projecting to the caudal NTS. The projection from the insular cortex to the NTS in the mouse is bilateral with an ipsilateral pre-
dominance [24], while the projection from both infralimbic and insular cortices in the rat is bilateral with a contralateral predominance [23,26]. In our study, the predominance of terminal labeling in the rostra1 NTS and its relative paucity at caudal levels of that nucleus is in contrast to the projection from the insular cortex to the NTS in the rat [23] and mouse [24]; in these rodents this projection is described as terminating more evenly throughout the entire length of the NTS. These differences notwithstanding, the orbital gyrus of the cat is similar to the insular cortex of the mouse and rat in that it has been shown to receive olfactory [18] as well as gustatory [3] and visceral input [13,14]. The convergence of these modalities within the orbital gyrus of cat may be indicative of an integrative role similar to that hypothesized for the insular cortex of mouse [25]. If this is the case, then the descending projection described in the present report could provide a pathway for cerebral cortical modulation of gustatory and visceral information conveyed to NTS by cranial nerves VII, IX and X. The present study also describes a projection from the orbital gyNS which courses through portions of SpV and, in a band-like fashion, through the region between SpV and the NTS. Should this represent a terminal projection, it would end within a region of the brainstem implicated in mediating complex reflexes such as swallowing and emesis [6,16]. Thus gustatory information reaching the orbital gyrus might, through this projection, influence autonomic activity.
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the corsolitary and N. nucleus