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The organization of insular cortex projections to the amygdaloid central nucleus and autonomic regulatory nuclei of the dorsal medulla
Brain Research, 360 (1985) 355-360 Elsevier
355
BRE 21246
The organization of insular cortex projections to the amygdaloid central nucleus and autonomic regulatory nuclei of the dorsal medulla BRUCE S. KAPP 1, JAMES S. SCHWABER2 and PATRICIA A. DRISCOLL1 1Department of Psychology, University of Vermont, Burlington, VT 05405 and 2Dupont Central Research and Development, E.I. dupont de Nemours and Company, Glenolden, PA 19036 (U.S.A.) (Accepted August 6th, 1985) Key words: amygdaloid central nucleus - - insular cortex - - nucleus solitarius - - vagal motor nucleus - - retrograde labeling
Using the fluorescent dye, double retrograde-labeling tracing technique, separate populations of insular cortex neurons were demonstrated to project to the amygdaloid central nucleus and to autonomic nuclei of the dorsal medulla. Both populations were located in layer V of the agranular and granular insula with neurons projecting to the dorsal medulla demonstrating a more medial distribution. The results yield additional detail on the organization of forebrain areas involved in autonomic regulation.
A number of forebrain areas historically believed to contribute to autonomic regulation have recently been demonstrated to innervate directly autonomic regulatory nuclei of the dorsal medulla. For example, the amygdaloid central nucleus and the insular cortex have long been implicated in autonomic regulation4,6, t9 and our observations in the rabbit 14-16 as well as those in other speciesS,12,13,is have demonstrated that these regions project to the nucleus of the solitary tract (NTS) and vagal dorsal motor nucleus (DMN). We have observed that in rabbit (a) the terminal fields of the insular cortex and central nucleus projections within the NTS to some extent overlap 16 and (b) the insular cortex projects upon the medial component of the central nucleus 9, the component from which the bulk of the projections to these medullary autonomic regulatory nuclei ariseslL These observations provide neuroanatomical substrates for the contribution of the amygdaloid central nucleus and insular cortex in autonomic regulation, and suggest that they may be significant components of a forebrain system involved in such regulation. The present experiment was conducted to further delineate the anatomical organization of this system and thereby provide insights into its functional orga-
nization. The fluorescent dye, double retrograde-labeling technique was used to determine the extent to which separate populations of insular cortex neurons project to the central nucleus and NTS/DMN complex or, alternatively, the extent to which a single population of insular cortex neurons projects to these two regions via collaterals. The existence of such collaterals would indicate that the activity of insular cortex neurons has the potential to simultaneously influence both the central nucleus and the NTS/DMN complex. Forty, pure-bred, New Zealand rabbits (2.0-2.8 kg) were anesthetized with chlorpromazine hydrochloride (20 mg in 0.8 ml saline, i.v.) and Nembutal (30-75 mg, i.v.). Unilateral injections of True Blue (5%, 40-1000 nl) or Fast Blue (5%, 40-300 nl, Sigma) were aimed at the central nucleus (0.0-0.5 mm anterior to bregma; 5.5-5.7 mm lateral to the midline and 12.0-12.5 mm ventral to dura; lambda 1.5 mm below bregma). Injections were delivered via a 26-gauge needle or a glass micropipette (60~m tip diameter) cemented to a 26-gauge needle attached to a 1.0-~1 Hamilton syringe. All injections were made over a 20-40-min period. Two to four weeks later, from two to four unilateral injections of bisbenzimide ( 5 - 1 0 % , 50-300 nl, Hoechst 33258) were made
Correspondence: B.S. Kapp, Department of Psychology, University of Vermont, Burlington, VT 05405, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
356 along the rostral-caudal extent of the NTS/DMN complex (from 3.0 mm anterior to 3.0 mm posterior to obex). Injections into the NTS/DMN complex were made contralateral to the central nucleus injection since the insular cortex projection to the NTS/DMN complex is primarily contralateral while the insular cortex projection to the central nucleus is primarily ipsilateral. From 48 to 72 h following the bisbenzimide injections, the animals were sacrificed and perfused with 1000 ml of physiological saline followed by 2000 ml of a phosphate-buffered 10% formalin solution (pH = 7.4). The brains were stored for 18 h in a 20% sucrose-buffered formalin solution and alternate, 30 Bm, frozen sections through the insular cortex were collected in distilled water, mounted immediately onto uncoated slides and allowed to air-dry. Prior to viewing under fluorescent microscopy the slides were dipped for 5-10 s in a sodium citrate buffer solution and allowed to air-dry. This procedure produced a yellow fluorescence within the nuclei of bisbenzimide-labeled neurons without altering the quality and intensity of the blue fluorescence within the cytoplasm of True Blue or Fast Blue-labeled neurons when using a 360-nm excitation wavelength. The additional, alternate sections were collected in 10% formalin and stained with thionin. The locations of labeled neurons were plotted with reference to various landmarks, and with the aid of the thioninstained sections, a drawing tube and Bausch and Lomb microprojector. Fig. 1 illustrates injection sites from a representative case in which a 300-nl injection of True Blue was located within the rostral portion of the amygdaloid central nucleus and three 100-nl injections of bisbenzimide were located along the rostral-caudal extent of the NTS/DMN complex. In this case, as well as in all cases in which True Blue or Fast Blue covered a portion of the central nucleus, labeled neurons were located within layers II, III and V of the insular cortex (Fig. 2). Within the more rostral insular cortex the majority of True Blue retrogradely labeled neurons were located in layer V of the dorsal agranular insular region with a few neurons also located in layer V of the ventral agranular insula and in layer V of the granular insular region lying immediately dorsal to the dorsal agranular insula (Fig. 2). At more caudal levels, numerous True Blue-labeled neurons were
observed in layer V of the posterior agranular insula, and this field of labeled neurons extended dorsally into layer V of the granular insular region. More faintly, less numerous labeled neurons were also observed in layers II and III of the posterior agranular insula. Retrogradely labeled, bisbenzimide-containing neurons were also located in layer V of the insular cortex. The distribution of these neurons differed to some extent from the distribution of True Blue-labeled neurons, thereby accounting, at least in part, for only the very rare occurrence of double-labeled neurons. In Fig. 2 it can be seen that within the more rostral insula, bisbenzimide-labeled neurons, in contrast to True Blue-labeled neurons, were more medially located and were distributed as a narrow band of neurons within the innermost region of layer V. These bisbenzimide-labeled neurons extended as an uninterrupted band from the dorsal portion of the dorsal agranular insular region through the granular insula and into the lateral precentral cortex. Labeled neurons within the lateral precentral cortex appeared most numerous when injections encroached upon the dorsal column nuclei dorsally adjacent to the NTS/DMN complex. At more caudal levels, while this medial-lateral distinction in the distribution of bisbenzimide- and True Blue-labeled neurons was still somewhat evident, there was considerable overlap of the two distributions. Greater numbers of bisbenzimide-labeled neurons, however, continued to extend more dorsally into the granular insula than True Blue-labeled neurons. Furthermore, only True Blue-labeled neurons were located within layers II and III of the agranular insular cortex, particularly at caudal levels. We have reported that the terminal distributions of projections from the insular cortex and amygdaloid central nucleus overlap within regions of the NTS/DMN complex 16. This overlap was particularly evident within the dorsomedial subnucleus of the NTS, an area recipient of baroreceptor afferents of the aortic depressor and carotid sinus nerves in rabbit3. These observations suggest that both areas may influence cardiovascular function, perhaps by modulation of the afferent or efferent side(s) of the baroreceptor reflex in this species. Indeed, additional observations in rabbit have provided evidence supporting a contribution for these two areas in cardiovascu-
357
Fig. 1. A, B: line drawings and fluorescent photomicrographs of the most rostral (A) and caudal (B) of 3 bisbenzimide injection sites (100 nl, 10%) located within the dorsomedial medulla of case TBA-18 and which covered the nucleus of the solitary tract (NTS) and vagal dorsal motor nucleus (DMN). C: line drawing and fluorescent photomicrograph of the True Blue injection site (300 nl, 5%) located in the rostral portion of the central nucleus of the amygdala of case TBA-18. The injection covered the medial component of the central nucleus and encroached upon the medial nucleus of the amygdala and the ventral globus pallidus. Abbreviations: AI, agranular insular cortex; cex, external capsule; CL, claustrum; CN, caudate nucleus; DMN, vagal dorsal motor nucleus; fx, fornix; GI, granular insular cortex; La, lateral nucleus of the amygdala; LS, lateral septal nucleus; Me, medial nucleus of the amygdala; NTS, nucleus of the solitary tract; ot, optic tract; P, putamen; PMC, posteromedial cortical nucleus of the amygdala; PP, prepiriform cortex; V, ventricle; XII, hypoglossal nucleus.
358 A
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Fig. 2. Line drawings of representative coronal sections through the rostral (A) to caudal (E) extent of the insular cortex of case T B A 18 illustrating the location of neurons labeled with True Blue transported from the central nucleus (open circles) or bisbenzimide transported from the dorsomedial medulla (closed circles). Each circle represents a single neuron from that section alone. No doublelabeled neurons were observed. (For abbreviations, see Fig. 1).
359 lar regulation. For example, electrical stimulation of the central nucleus produced bradycardia and depressor responsesS, and has recently been demonstrated to excite NTS neurons excited by stimulation of the aortic nerve 17. Stimulation of the insular cortex also results in bradycardia and depressor responses1,10, while lesions of either the central nucleus 7 or insular cortex ~0 attenuate conditioned bradycardia during pavlovian fear conditioning. These observations are consistent with the existence of direct projections from these two regions to the NTS/DMN complex and suggest that they may share (a) common function(s) in cardiovascular and autonomic regulation as components within a larger forebrain autonomic regulatory system. The existence in rabbit of a projection from the insular cortex to the NTS/DMN complex 16, as well as the projection from the insular cortex to the central nucleus% provides both a direct and indirect route by which .the insular cortex may influence the N T S / D M N complex and, in turn, autonomic function. The observations that (a) rarely a double-labeled neuron was observed within the insular cortex following injections of different dyes into the central nucleus and N T S / D M N complex and (b) insular cortex neurons projecting to the central nucleus and to the NTS/DMN complex demonstrated somewhat different spatial distributions indicate that separate pop-
1 Buchanan, S.L., Poweli, D.A. and Valentine, J., Cardiovascular adjustments elicited by electrical stimulation of frontal cortex in conscious rabbits, Neurosci. Abstr., 10 (1984) 614. 2 Gallagher, M., Kapp, B.S., Frysinger, R.C. and Rapp, P.R., fl-Adrenergic manipulation in amygdala central n. alters rabbit heart rate conditioning, Pharmacol. Biochem. Behav., 12 (1980) 419-426. 3 Higgins, G.A. and Schwaber, J.S., Afferent organization of the nucleus tractus solitarius of the rabbit: evidence for overlap of forebrain and primary afferent inputs, Anat. Rec., 199 (1981) 144a. 4 Hilton, S.M. and Zbrozyna, A.W., Amygdaloid region for defense reactions and its efferent pathway to the brain stem, J. Physiol. (London), 165 (1963) 160-173. 5 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 6 Kaada, B.R., Somato-motor, autonomic and electrocorticographic responses to electrical stimulation of 'rhinencephalic' and other structures in primates, cat and dog. A study of responses from the limbic, subcallosal, orbito-insular, piriform and temporal cortex, hippocampus-fornix and
ulations of neurons within the insular cortex innervate the central nucleus and the N T S / D M N complex. The results, therefore, do not support the notion that the activity of single neurons within the insular cortex may simultaneously influence neurons of the central nucleus and NTS/DMN complex via collateral innervation of these two regions. Rather, the results are not inconsistent with the hypothesis that these separate populations of neurons might relay different types of information to the central nucleus and NTS/DMN complex, a hypothesis which is consistent with the varied functions in which the insular cortex has been implicated, including respiratory and cardiovascular function1,6,10 as well as taste 20. Additional neurophysiological experiments in which insular cortex neurons projecting to the central nucleus and NTS/DMN complex are identified and characterized with respect to response profiles will aid in determining the validity of this hypothesis. The results of the present study further detailing the neuroanatomical organization and interactions of forebrain areas implicated in autonomic regulation should significantly enhance the design of physiological experiments designed to elucidate the functional contribution of these areas in such regulation. Supported by U S P H S Grant NS 16107.
amygdala, Acta Physiol. Scand., 24 Suppl. 83 (1951) 1-285. 7 Kapp, B.S., Frysinger, R.C., Gallagher, M. and Haselton, J., Amygdala central nucleus lesions: effects on heart rate conditioning in the rabbit, Physiol. Behav., 23 (1979) 1109-1117. 8 Kapp, B.S., Gallagher, M., Underwood, M.D., McNall, C.L. and Whitehorn, D., Cardiovascular responses elicited by electrical stimulation of the amygdala central nucleus in the rabbit, Brain Research, 234 (1982) 251-262. 9 Kapp, B.S., Schwaber, J.S. and Driscoll, P.A., Frontal cortex projections to the amygdaloid central nucleus in the rabbit, Neuroscience, 15 (1985) 327-346. 10 Markgraf, C.G., Contributions of the insular cortex to cardiovascular regulation during aversive Pavlovian conditioning in the rabbit, Unpublished Master's Thesis, University
of Vermont, 1984. 11 Pascoe, J.P. and Kapp, B.S., Electrophysiological characteristics of amygdaloid central nucleus neurons during differential pavlovian fear conditioning in the rabbit, Behav. Brain Res., 16 (1985) 117-133. 12 Price, J.L. and Amaral. D.G., An autoradiographic study of the projections of the central nucleus of the monkey
3611 amygdala, J. Neurosci., I (1981) 1242-1259. 13 Saper, C.B., Convergence of autonomic and limbic connections in the insular cortex of the rat, J. Comp. Neurol., 210 (1982) 163-173. 14 Schwaber, J.S., Kapp, B.S. and Higgins, G.A.. The origin and extent of direct amygdala projections to the region of the dorsal motor nucleus of the vagus and the nucleus of the solitary tract, Neurosci. Lett., 20 (1980) 15-20. 15 Schwaber. J.S., Kapp, B.S., Higgins, G.A. and Rapp, P . R , Amygdaloid and basal forebrain direct connections with the nucleus of the solitary tract and the dorsal motor nucleus, J. Neurosci., 2 (1982) 1424-1438. 16 Schwaber, J.S., Kapp, B.S., Higgins, G.A. and DriscollMendes, P.A., Comparison of the topographic distribution of insular cortical and central amygdaloid inputs to the nu-
17
18
19
20
cleus solitarius and dorsal motor nucleus in the rabbit, Neurosci. Abstr., 9 (1983) 113. Schwaber, J.S., Turner. S.A.. Moruzzi, P. and Spyer, K.M., Amygdaloid influences on brainstem neurons in the rabbit, Neurosci. Abstr., 10 (1984) 615. Shipley, M.T., Insular cortex projections to the nucleus of the solitary tract and brainstem visceromotor regions in the mouse, Brain Res. Bull., 8 (1982) 139-148. Ursin, H. and Kaada, B.R., Functional localization within the amygdaloid complex in the cat, Electroencephalogr. Clin. Neurophysiol., 12 (1960) 1-20. Yamamoto, T., Matsuo, R. and Kawamura, Y., Localization of cortical gustatory area in rats and its role in taste discrimination, J. Neurophysiol.. 44 (1980) 440-455.
355
BRE 21246
The organization of insular cortex projections to the amygdaloid central nucleus and autonomic regulatory nuclei of the dorsal medulla BRUCE S. KAPP 1, JAMES S. SCHWABER2 and PATRICIA A. DRISCOLL1 1Department of Psychology, University of Vermont, Burlington, VT 05405 and 2Dupont Central Research and Development, E.I. dupont de Nemours and Company, Glenolden, PA 19036 (U.S.A.) (Accepted August 6th, 1985) Key words: amygdaloid central nucleus - - insular cortex - - nucleus solitarius - - vagal motor nucleus - - retrograde labeling
Using the fluorescent dye, double retrograde-labeling tracing technique, separate populations of insular cortex neurons were demonstrated to project to the amygdaloid central nucleus and to autonomic nuclei of the dorsal medulla. Both populations were located in layer V of the agranular and granular insula with neurons projecting to the dorsal medulla demonstrating a more medial distribution. The results yield additional detail on the organization of forebrain areas involved in autonomic regulation.
A number of forebrain areas historically believed to contribute to autonomic regulation have recently been demonstrated to innervate directly autonomic regulatory nuclei of the dorsal medulla. For example, the amygdaloid central nucleus and the insular cortex have long been implicated in autonomic regulation4,6, t9 and our observations in the rabbit 14-16 as well as those in other speciesS,12,13,is have demonstrated that these regions project to the nucleus of the solitary tract (NTS) and vagal dorsal motor nucleus (DMN). We have observed that in rabbit (a) the terminal fields of the insular cortex and central nucleus projections within the NTS to some extent overlap 16 and (b) the insular cortex projects upon the medial component of the central nucleus 9, the component from which the bulk of the projections to these medullary autonomic regulatory nuclei ariseslL These observations provide neuroanatomical substrates for the contribution of the amygdaloid central nucleus and insular cortex in autonomic regulation, and suggest that they may be significant components of a forebrain system involved in such regulation. The present experiment was conducted to further delineate the anatomical organization of this system and thereby provide insights into its functional orga-
nization. The fluorescent dye, double retrograde-labeling technique was used to determine the extent to which separate populations of insular cortex neurons project to the central nucleus and NTS/DMN complex or, alternatively, the extent to which a single population of insular cortex neurons projects to these two regions via collaterals. The existence of such collaterals would indicate that the activity of insular cortex neurons has the potential to simultaneously influence both the central nucleus and the NTS/DMN complex. Forty, pure-bred, New Zealand rabbits (2.0-2.8 kg) were anesthetized with chlorpromazine hydrochloride (20 mg in 0.8 ml saline, i.v.) and Nembutal (30-75 mg, i.v.). Unilateral injections of True Blue (5%, 40-1000 nl) or Fast Blue (5%, 40-300 nl, Sigma) were aimed at the central nucleus (0.0-0.5 mm anterior to bregma; 5.5-5.7 mm lateral to the midline and 12.0-12.5 mm ventral to dura; lambda 1.5 mm below bregma). Injections were delivered via a 26-gauge needle or a glass micropipette (60~m tip diameter) cemented to a 26-gauge needle attached to a 1.0-~1 Hamilton syringe. All injections were made over a 20-40-min period. Two to four weeks later, from two to four unilateral injections of bisbenzimide ( 5 - 1 0 % , 50-300 nl, Hoechst 33258) were made
Correspondence: B.S. Kapp, Department of Psychology, University of Vermont, Burlington, VT 05405, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
356 along the rostral-caudal extent of the NTS/DMN complex (from 3.0 mm anterior to 3.0 mm posterior to obex). Injections into the NTS/DMN complex were made contralateral to the central nucleus injection since the insular cortex projection to the NTS/DMN complex is primarily contralateral while the insular cortex projection to the central nucleus is primarily ipsilateral. From 48 to 72 h following the bisbenzimide injections, the animals were sacrificed and perfused with 1000 ml of physiological saline followed by 2000 ml of a phosphate-buffered 10% formalin solution (pH = 7.4). The brains were stored for 18 h in a 20% sucrose-buffered formalin solution and alternate, 30 Bm, frozen sections through the insular cortex were collected in distilled water, mounted immediately onto uncoated slides and allowed to air-dry. Prior to viewing under fluorescent microscopy the slides were dipped for 5-10 s in a sodium citrate buffer solution and allowed to air-dry. This procedure produced a yellow fluorescence within the nuclei of bisbenzimide-labeled neurons without altering the quality and intensity of the blue fluorescence within the cytoplasm of True Blue or Fast Blue-labeled neurons when using a 360-nm excitation wavelength. The additional, alternate sections were collected in 10% formalin and stained with thionin. The locations of labeled neurons were plotted with reference to various landmarks, and with the aid of the thioninstained sections, a drawing tube and Bausch and Lomb microprojector. Fig. 1 illustrates injection sites from a representative case in which a 300-nl injection of True Blue was located within the rostral portion of the amygdaloid central nucleus and three 100-nl injections of bisbenzimide were located along the rostral-caudal extent of the NTS/DMN complex. In this case, as well as in all cases in which True Blue or Fast Blue covered a portion of the central nucleus, labeled neurons were located within layers II, III and V of the insular cortex (Fig. 2). Within the more rostral insular cortex the majority of True Blue retrogradely labeled neurons were located in layer V of the dorsal agranular insular region with a few neurons also located in layer V of the ventral agranular insula and in layer V of the granular insular region lying immediately dorsal to the dorsal agranular insula (Fig. 2). At more caudal levels, numerous True Blue-labeled neurons were
observed in layer V of the posterior agranular insula, and this field of labeled neurons extended dorsally into layer V of the granular insular region. More faintly, less numerous labeled neurons were also observed in layers II and III of the posterior agranular insula. Retrogradely labeled, bisbenzimide-containing neurons were also located in layer V of the insular cortex. The distribution of these neurons differed to some extent from the distribution of True Blue-labeled neurons, thereby accounting, at least in part, for only the very rare occurrence of double-labeled neurons. In Fig. 2 it can be seen that within the more rostral insula, bisbenzimide-labeled neurons, in contrast to True Blue-labeled neurons, were more medially located and were distributed as a narrow band of neurons within the innermost region of layer V. These bisbenzimide-labeled neurons extended as an uninterrupted band from the dorsal portion of the dorsal agranular insular region through the granular insula and into the lateral precentral cortex. Labeled neurons within the lateral precentral cortex appeared most numerous when injections encroached upon the dorsal column nuclei dorsally adjacent to the NTS/DMN complex. At more caudal levels, while this medial-lateral distinction in the distribution of bisbenzimide- and True Blue-labeled neurons was still somewhat evident, there was considerable overlap of the two distributions. Greater numbers of bisbenzimide-labeled neurons, however, continued to extend more dorsally into the granular insula than True Blue-labeled neurons. Furthermore, only True Blue-labeled neurons were located within layers II and III of the agranular insular cortex, particularly at caudal levels. We have reported that the terminal distributions of projections from the insular cortex and amygdaloid central nucleus overlap within regions of the NTS/DMN complex 16. This overlap was particularly evident within the dorsomedial subnucleus of the NTS, an area recipient of baroreceptor afferents of the aortic depressor and carotid sinus nerves in rabbit3. These observations suggest that both areas may influence cardiovascular function, perhaps by modulation of the afferent or efferent side(s) of the baroreceptor reflex in this species. Indeed, additional observations in rabbit have provided evidence supporting a contribution for these two areas in cardiovascu-
357
Fig. 1. A, B: line drawings and fluorescent photomicrographs of the most rostral (A) and caudal (B) of 3 bisbenzimide injection sites (100 nl, 10%) located within the dorsomedial medulla of case TBA-18 and which covered the nucleus of the solitary tract (NTS) and vagal dorsal motor nucleus (DMN). C: line drawing and fluorescent photomicrograph of the True Blue injection site (300 nl, 5%) located in the rostral portion of the central nucleus of the amygdala of case TBA-18. The injection covered the medial component of the central nucleus and encroached upon the medial nucleus of the amygdala and the ventral globus pallidus. Abbreviations: AI, agranular insular cortex; cex, external capsule; CL, claustrum; CN, caudate nucleus; DMN, vagal dorsal motor nucleus; fx, fornix; GI, granular insular cortex; La, lateral nucleus of the amygdala; LS, lateral septal nucleus; Me, medial nucleus of the amygdala; NTS, nucleus of the solitary tract; ot, optic tract; P, putamen; PMC, posteromedial cortical nucleus of the amygdala; PP, prepiriform cortex; V, ventricle; XII, hypoglossal nucleus.
358 A
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Fig. 2. Line drawings of representative coronal sections through the rostral (A) to caudal (E) extent of the insular cortex of case T B A 18 illustrating the location of neurons labeled with True Blue transported from the central nucleus (open circles) or bisbenzimide transported from the dorsomedial medulla (closed circles). Each circle represents a single neuron from that section alone. No doublelabeled neurons were observed. (For abbreviations, see Fig. 1).
359 lar regulation. For example, electrical stimulation of the central nucleus produced bradycardia and depressor responsesS, and has recently been demonstrated to excite NTS neurons excited by stimulation of the aortic nerve 17. Stimulation of the insular cortex also results in bradycardia and depressor responses1,10, while lesions of either the central nucleus 7 or insular cortex ~0 attenuate conditioned bradycardia during pavlovian fear conditioning. These observations are consistent with the existence of direct projections from these two regions to the NTS/DMN complex and suggest that they may share (a) common function(s) in cardiovascular and autonomic regulation as components within a larger forebrain autonomic regulatory system. The existence in rabbit of a projection from the insular cortex to the NTS/DMN complex 16, as well as the projection from the insular cortex to the central nucleus% provides both a direct and indirect route by which .the insular cortex may influence the N T S / D M N complex and, in turn, autonomic function. The observations that (a) rarely a double-labeled neuron was observed within the insular cortex following injections of different dyes into the central nucleus and N T S / D M N complex and (b) insular cortex neurons projecting to the central nucleus and to the NTS/DMN complex demonstrated somewhat different spatial distributions indicate that separate pop-
1 Buchanan, S.L., Poweli, D.A. and Valentine, J., Cardiovascular adjustments elicited by electrical stimulation of frontal cortex in conscious rabbits, Neurosci. Abstr., 10 (1984) 614. 2 Gallagher, M., Kapp, B.S., Frysinger, R.C. and Rapp, P.R., fl-Adrenergic manipulation in amygdala central n. alters rabbit heart rate conditioning, Pharmacol. Biochem. Behav., 12 (1980) 419-426. 3 Higgins, G.A. and Schwaber, J.S., Afferent organization of the nucleus tractus solitarius of the rabbit: evidence for overlap of forebrain and primary afferent inputs, Anat. Rec., 199 (1981) 144a. 4 Hilton, S.M. and Zbrozyna, A.W., Amygdaloid region for defense reactions and its efferent pathway to the brain stem, J. Physiol. (London), 165 (1963) 160-173. 5 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 6 Kaada, B.R., Somato-motor, autonomic and electrocorticographic responses to electrical stimulation of 'rhinencephalic' and other structures in primates, cat and dog. A study of responses from the limbic, subcallosal, orbito-insular, piriform and temporal cortex, hippocampus-fornix and
ulations of neurons within the insular cortex innervate the central nucleus and the N T S / D M N complex. The results, therefore, do not support the notion that the activity of single neurons within the insular cortex may simultaneously influence neurons of the central nucleus and NTS/DMN complex via collateral innervation of these two regions. Rather, the results are not inconsistent with the hypothesis that these separate populations of neurons might relay different types of information to the central nucleus and NTS/DMN complex, a hypothesis which is consistent with the varied functions in which the insular cortex has been implicated, including respiratory and cardiovascular function1,6,10 as well as taste 20. Additional neurophysiological experiments in which insular cortex neurons projecting to the central nucleus and NTS/DMN complex are identified and characterized with respect to response profiles will aid in determining the validity of this hypothesis. The results of the present study further detailing the neuroanatomical organization and interactions of forebrain areas implicated in autonomic regulation should significantly enhance the design of physiological experiments designed to elucidate the functional contribution of these areas in such regulation. Supported by U S P H S Grant NS 16107.
amygdala, Acta Physiol. Scand., 24 Suppl. 83 (1951) 1-285. 7 Kapp, B.S., Frysinger, R.C., Gallagher, M. and Haselton, J., Amygdala central nucleus lesions: effects on heart rate conditioning in the rabbit, Physiol. Behav., 23 (1979) 1109-1117. 8 Kapp, B.S., Gallagher, M., Underwood, M.D., McNall, C.L. and Whitehorn, D., Cardiovascular responses elicited by electrical stimulation of the amygdala central nucleus in the rabbit, Brain Research, 234 (1982) 251-262. 9 Kapp, B.S., Schwaber, J.S. and Driscoll, P.A., Frontal cortex projections to the amygdaloid central nucleus in the rabbit, Neuroscience, 15 (1985) 327-346. 10 Markgraf, C.G., Contributions of the insular cortex to cardiovascular regulation during aversive Pavlovian conditioning in the rabbit, Unpublished Master's Thesis, University
of Vermont, 1984. 11 Pascoe, J.P. and Kapp, B.S., Electrophysiological characteristics of amygdaloid central nucleus neurons during differential pavlovian fear conditioning in the rabbit, Behav. Brain Res., 16 (1985) 117-133. 12 Price, J.L. and Amaral. D.G., An autoradiographic study of the projections of the central nucleus of the monkey
3611 amygdala, J. Neurosci., I (1981) 1242-1259. 13 Saper, C.B., Convergence of autonomic and limbic connections in the insular cortex of the rat, J. Comp. Neurol., 210 (1982) 163-173. 14 Schwaber, J.S., Kapp, B.S. and Higgins, G.A.. The origin and extent of direct amygdala projections to the region of the dorsal motor nucleus of the vagus and the nucleus of the solitary tract, Neurosci. Lett., 20 (1980) 15-20. 15 Schwaber. J.S., Kapp, B.S., Higgins, G.A. and Rapp, P . R , Amygdaloid and basal forebrain direct connections with the nucleus of the solitary tract and the dorsal motor nucleus, J. Neurosci., 2 (1982) 1424-1438. 16 Schwaber, J.S., Kapp, B.S., Higgins, G.A. and DriscollMendes, P.A., Comparison of the topographic distribution of insular cortical and central amygdaloid inputs to the nu-
17
18
19
20
cleus solitarius and dorsal motor nucleus in the rabbit, Neurosci. Abstr., 9 (1983) 113. Schwaber, J.S., Turner. S.A.. Moruzzi, P. and Spyer, K.M., Amygdaloid influences on brainstem neurons in the rabbit, Neurosci. Abstr., 10 (1984) 615. Shipley, M.T., Insular cortex projections to the nucleus of the solitary tract and brainstem visceromotor regions in the mouse, Brain Res. Bull., 8 (1982) 139-148. Ursin, H. and Kaada, B.R., Functional localization within the amygdaloid complex in the cat, Electroencephalogr. Clin. Neurophysiol., 12 (1960) 1-20. Yamamoto, T., Matsuo, R. and Kawamura, Y., Localization of cortical gustatory area in rats and its role in taste discrimination, J. Neurophysiol.. 44 (1980) 440-455.