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Somatostatinergic neurons in the insular cortex project to the spinal cord: combined retrograde axonal transport and immunohistochemical study
Brain Research, 326 (1985) 197-200 Elsevier
197
BRE 20596
Somatostatinergic neurons in the insular cortex project to the spinal cord: combined retrograde axonal transport and immunohistochemical study SHOICHI SHIMADA. SADAO SHIOSAKA. KENJI TAKAMI, MARIKO YAMANO and MASAYA TOHYAMA Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan)
(Accepted September 4th, 1984) Key words: somatostatin - - combination - - retrograde axonal transport - - biotin-HRP - immunohistochemistry - - insular cortex - - spinal cord
A double-labeling method combining immunohistochemistry and a retrograde tracer technique using biotin-horseradish peroxidase (B-HRP) was employed to identify a descending somatostatinergic fiber system from the insular cortex to the spinal cord. Injection of B-HRP into the spinal cord at cervical or lumbar levels resulted in the labeling of a number of neurons in the insular cortex. Simultaneous immunostaining revealed the existence of double-labeled neurons in the insular cortex. The result provides direct evidence for the presence of a descending somatostatinergic pathway from the insular cortex to lumbar levels of the spinal cord. Retrograde tracing methods have revealed many afferent fiber pathways to the insular cortex from visceral relay centers and limbic areas such as the solitary nucleus, parabrachial nuclei, and hypothalamus 10,11. Efferents from the insular cortex to the caudate nucleus, thalamus, parabrachial nucleus, solitary tract nucleus, and spinal cord have been found using the anterograde tracing technique16.19. As a result of these studies, it is speculated that the insular cortex is one of the important integrative centers of the autonomic nervous system converging visceral and limbic inputs 1°,16,19. On the other hand, the insular cortex is known to contain various peptidergic neurons, such as somatostatin (SOM) 5.13,14,vasoactive intestinal polypeptide (VIp)8, cholecystokinin-8 (CCK-8) 9 and corticotropin releasing factor (CRF) 17. For elucidating the function of these peptides, it is necessary to reveal their neural circuits. However, such studies have been hampered by experimental techniques lacking the necessary sensitivity and specificity for evaluating peptidergic fiber connections. The present study has attempted to reveal somatostatinergic neuron projections from cortex to spinal cord, using the newly developed sensitive combination method of biotin
conjugated horseradish peroxidase (B-HRP) as the tracer and immunohistochemistry using Texas red as the fluorochrome ( B H T I method)12,15. Details of the procedures have been described elsewhere 12,15. Briefly, 10 adult Wistar rats (50-120 g) were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg, i.p.). Then, 1 kti of 5% of B - H R P solubilized in 0.05 M Tris-HC1 buffer, pH 7.5 (Vector Lab. Inc., CA, U.S.A.) was injected into the upper cervical ( C 3 - C 5 ) or lumbar cord ( L 3 - L 6 ) of the rats using a 10-~tl Hamilton's microsyringe. Twenty-four to 48 h later, animals were anesthetized as above and perfused intracardially with 4.0% paraformaldehyde-0.1 M sodium phosphate buffer (pH 7.4). Brains were removed immediately and immersed in the same fixative for 12 h followed by immersion for 24 h in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Next, the brain was sectioned at 20 ktm in a cryostat and rinsed in 0.02 M sodium phosphate-buffered saline (PBS), pH 7.4. The sections were first incubated for 24 h with SOM antiserum diluted with PBS (1:1000) and avidin-fluorescein isothiocyanate (FITC): (1:250, E Y Laboratory, U . S . A . ) PBS solution at 15 °C. Next, they were washed 3 times for 10
Correspondence: S. Shimada, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka (530), Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V.
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B Fig. 1. Distribution of neurons labeled retrogradely by B-HRP after injection into lumbar spinal cord (open circles), somatostatin-positive cells (open triangles), and double-labeled cells (black dots) (A and B) and (C) a precise illustration of cortical region of level (A). Abbreviations: AH, anterior hypothalamic nucleus; CA, anterior commissura; CC; corpus catlosum; CL, claustrum; CO, optic chiasma; E, endopyriform nucleus; IC, insular cortex; PC, pyriform cortex; RS, rhinal sulcus; I, II, III, IV, V and VI, layers of insular cortex.
min each with PBS at 4 °C and incubated with Texas red conjugated donkey anti-rabbit IgG (Amersham, U.K.) at a dilution of 1:100 with PBS for 12 h at 15 °C. The sections were then washed 3 times for 10 min with PBS at 4 °C and mounted in a glycerin-PBS (1:1) mixture. For observation of B - H R P reacted with avidin-FITC, a Nikon EF microscope equipped with a B dichroic mirror filter and IF520-545 interference filter (Nikon Co, Japan) was used. For antigen reacted with Texas red conjugated anti-rabbit IgG the same microscope with G dichroic mirror filter and 580 nm absorption filter was used. Texas red has a strong red fluorescence of 615 nm length emission upon 596 nm length excitation, while FITC has a 517 nm length emission upon 492 nm length excitation. Thus, these two substances were easily discriminated without overlap of fluorescein emission 18. The antiserum to SOM was obtained after immunization of a rabbit with a synthetic SOMj~-bovine se-
rum albumin conjugate. Antiserum specificity was checked by absorption tests and radioimmunoassay. For absorption tests the sections were divided into two series, one was subjected to the immunoreaction described above. The other was incubated with antiSOM antiserum preabsorbed with synthetic SOM (2/~g/ml at a diluted antiserum) under conditions identical to those of the former series. No immunofluorescent structures were found in the control series. The specificity was also confirmed by radioimmunoassays13,14. The insular cortex was identified according to its cytoarchitecture using the criteria of Krieg 7. Injecting B - H R P into the spinal cord at cervical or lumbal levels revealed numerous tracer-labeled neurons in the insular cortex (Figs. 1A, 2A, C). B-HRPlabeled cell soma were identified by their finely packed green fluorescein granules of FITC, and sometimes processes of these cell soma were also visible.
199 Sections were simultaneously stained with antiSOM antiserum by the immunofluorescent method 1. Accordingly, SOM-positive structures (labeled by Texas red with red fluorescence) were observed in the same field as B-HRP by changing the filter from B to G excitation. The distribution and density of SOM-like immunoreactive (SOMii) structures in the insular cortex was similar to that revealed in previous studiesS,13A4. As shown in Fig. 2A, B (and the higher magnifications in Fig. 2C, D), some SOMIi neurons (red fluorescein) in the insular cortex were labeled simultaneously with retrograde tracing (green fluorescein). These double-labeled cells were found mainly in layers V and VI of insular cortex, and a few were also observed in the endopyriform nucleus. The doublelabeled cells were medium sized (15-20/~m) fusiform or polygonal cells (Fig. 2C, D) and the density was about 20% of B-HRP-labeled cells. These cells
were distributed in the insular cortex to the level of commissura anterior, rostrally, and to the level of hypothalamic paraventricular nucleus, caudally (Fig. 1). The existence of the pathway from the insular cortex to the spinal cord has been described by Shipley using the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) 16. Our preliminary study using HRP failed to confirm the presence of this pathway, while the B-HRP method clearly demonstrated its existence, suggesting that B-HRP has a higher sensitivity than the conventional HRP method but similar to that of the W G A - H R P method. The present study using the BHTI method has demonstrated the SOM-containing system to be one of the major components of this pathway. As this pathway may be related to autonomic functions 16, it is likely that SOM projections from insular cortex to
Fig. 2. Fluorescent photomicrographsshowing double-labeled neurons in layer V of insular cortex after injection of B-HRP into the lumbar spinal cord. A: B-HRP-labeted neurons visualized by avidin-FITCin the insular cortex observed by a B-dichroic mirror filter and IF 520-545 nm interference filter. B: somatostatin-positive cells in the same fields of A, respectively. Observations were carried out by a G-dichroicmirror and 580 nm absorption filter. Double-labeled cells were indicated by arrows. C and D: higher magnification of part A and B as indicated in rectangles in A and B, respectively.
2(I(~ the spinal cord are r e l a t e d to it as well.
sensory system and the third to the m o t o r system.
In addition to this S O M p a t h w a y , the f o l l o w i n g
due to the location of their m a j o r t e r m i n a l fields in
S O M p r o j e c t i o n s h a v e b e e n s h o w n in the spinal cord:
the dorsal h o r n and lamina V I i , respectivelyL t-~luci-
(1) primary sensory a f f e r e n t 3, (2) intrinsic S O M sys-
dation of the exact function of S O M will only be pos-
tem 3 and (3) d e s c e n d i n g p a t h w a y f r o m the central
sible on the basis of f u r t h e r analysis of its origins and
a m y g d a l o i d nucleus 4-e'. T h e first m a y be r e l a t e d to the
pathways.
1 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 2 Heizman, H. and Richards, F. M., Use of the avidin-biotin complex for specific staining of biological membranes in electronmicroscopy, Proc. nat. Acad. Sci. U.S.A., 71 (1974) 3537-3541. 3 Hunt, S. P., Cytochemistry of the spinal cord. In P. C. Erason (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 53-84. 4 Inagaki, S., Kawai, Y., Matsuzaki, T., Shiosaka, S. and Tohyama, M., Precise terminal fields of the descending somatostatinergic neuron system from the amygdaloid complex of the rat, J. Hirnforsch., 24 (1983) 345-356. 5 Johansson, O., HOkfelt, T. and Elde, R. P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat, Neuroscience, in press. 6 Kawai, Y., Inagaki, S., Shiosaka, S., Senba, E., Hara, Y., Sakanaka, M., Takatsuki, K. and Tohyama, M., Long descending projection from amygdaloid somatostatin-containing cells to the lower brain stem, Brain Research, 239 (1982) 603-607. 7 Krieg, W. J. S., Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas, J. comp. Neurol., 84 (1946) 221-275. 8 Larsson, L.-1., Fahrenkrug, J., Schaffalizky de Muckadell, O., Sundler, F., Hakanson, R. and Rehfeld, J, F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 3197- 3200. 9 Loren, I., Alumets, R., Hakanson, R. and Sundler, F., Distribution of gastrin and CCK-like peptides in rat brain, Histochemistrv, 59 (1979) 249-257. 10 Saper, C. B., Convergence of autonomic and limbic connections in the insular cortex of the rat, J. comp. Neurol., 210 (1982) 163-173. 11 Saper, C. B., Reciprocal parabrachial-cortical connections
in the rat, Brain Research, 242 (1982) 33-40. 12 Shiosaka, S., Shibasaki, S. and Tohyama, M., Bilateral alpha-melanocyte stimulating hormonergic fiber system from zona incerta to cerebral cortex: combined retrograde axonal transport and immunohistochemical study, Brain Research, 309 (1984) 350-353. 13 Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Iida, H., Minagawa, H., Hara, Y. and Tohyama, M., Ontogeny of somatostatincontaining neuron system of the rat: immunohistochemical analysis. II. Forebrain and diencephalon, J. comp. Neurol., 204 (1982) 211-224. 14 Shiosaka, S., Takatsuki, K., Sakanaka, M., lnagaki, S.. Takagi, H., Senba, E., Kawai, Y., Minagawa, H. and Tohyama, M., New somatostatin-containing sites in the diencephalon of the neonatal rat, Neurosci. Lett., 25 (I981) 69-73. 15 Shiosaka, S. and Tohyama, M., Evidence for an a-MSH-ergic hippocampal commissural connection in the rat, revealed by a double-labeling technique, Neurosci. Lett., 49 (1984) 213-216. 16 Shipley, M. T., Insular cortex projection to the nucleus of the solitary tract and brainstem visceromotor regions in the mouse, Brain Res. Bull., 8 (1982) 139-148, 17 Swanson, L. W., Sawchenko, P. E., Rivier, J. and Vale, W. W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an ,mmunohistochemical study, Neuroendocrinology, 36 (1983) 165-186. 18 Titus, J. A., Haugland, R., Sharrow, S. O. and Segal, D. M., Texas red. A hydrophilic, red-emitting fluorephore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies, J. lmmunol. Meth., 50 (1982) 193-204. 19 Van der Kooy, D., Mcginty, J. F., Koda, L, Y., Gerfen, C, R. and Bloom, F. E., Visceral cortex: a direct connection from prefrontal cortex to the solitary nucleus in rat, Neurosci. Lett., 33 (1982) 123-127.
197
BRE 20596
Somatostatinergic neurons in the insular cortex project to the spinal cord: combined retrograde axonal transport and immunohistochemical study SHOICHI SHIMADA. SADAO SHIOSAKA. KENJI TAKAMI, MARIKO YAMANO and MASAYA TOHYAMA Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan)
(Accepted September 4th, 1984) Key words: somatostatin - - combination - - retrograde axonal transport - - biotin-HRP - immunohistochemistry - - insular cortex - - spinal cord
A double-labeling method combining immunohistochemistry and a retrograde tracer technique using biotin-horseradish peroxidase (B-HRP) was employed to identify a descending somatostatinergic fiber system from the insular cortex to the spinal cord. Injection of B-HRP into the spinal cord at cervical or lumbar levels resulted in the labeling of a number of neurons in the insular cortex. Simultaneous immunostaining revealed the existence of double-labeled neurons in the insular cortex. The result provides direct evidence for the presence of a descending somatostatinergic pathway from the insular cortex to lumbar levels of the spinal cord. Retrograde tracing methods have revealed many afferent fiber pathways to the insular cortex from visceral relay centers and limbic areas such as the solitary nucleus, parabrachial nuclei, and hypothalamus 10,11. Efferents from the insular cortex to the caudate nucleus, thalamus, parabrachial nucleus, solitary tract nucleus, and spinal cord have been found using the anterograde tracing technique16.19. As a result of these studies, it is speculated that the insular cortex is one of the important integrative centers of the autonomic nervous system converging visceral and limbic inputs 1°,16,19. On the other hand, the insular cortex is known to contain various peptidergic neurons, such as somatostatin (SOM) 5.13,14,vasoactive intestinal polypeptide (VIp)8, cholecystokinin-8 (CCK-8) 9 and corticotropin releasing factor (CRF) 17. For elucidating the function of these peptides, it is necessary to reveal their neural circuits. However, such studies have been hampered by experimental techniques lacking the necessary sensitivity and specificity for evaluating peptidergic fiber connections. The present study has attempted to reveal somatostatinergic neuron projections from cortex to spinal cord, using the newly developed sensitive combination method of biotin
conjugated horseradish peroxidase (B-HRP) as the tracer and immunohistochemistry using Texas red as the fluorochrome ( B H T I method)12,15. Details of the procedures have been described elsewhere 12,15. Briefly, 10 adult Wistar rats (50-120 g) were anesthetized with sodium pentobarbital (Nembutal, 30 mg/kg, i.p.). Then, 1 kti of 5% of B - H R P solubilized in 0.05 M Tris-HC1 buffer, pH 7.5 (Vector Lab. Inc., CA, U.S.A.) was injected into the upper cervical ( C 3 - C 5 ) or lumbar cord ( L 3 - L 6 ) of the rats using a 10-~tl Hamilton's microsyringe. Twenty-four to 48 h later, animals were anesthetized as above and perfused intracardially with 4.0% paraformaldehyde-0.1 M sodium phosphate buffer (pH 7.4). Brains were removed immediately and immersed in the same fixative for 12 h followed by immersion for 24 h in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Next, the brain was sectioned at 20 ktm in a cryostat and rinsed in 0.02 M sodium phosphate-buffered saline (PBS), pH 7.4. The sections were first incubated for 24 h with SOM antiserum diluted with PBS (1:1000) and avidin-fluorescein isothiocyanate (FITC): (1:250, E Y Laboratory, U . S . A . ) PBS solution at 15 °C. Next, they were washed 3 times for 10
Correspondence: S. Shimada, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57, Nakanoshima, Kitaku, Osaka (530), Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V.
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B Fig. 1. Distribution of neurons labeled retrogradely by B-HRP after injection into lumbar spinal cord (open circles), somatostatin-positive cells (open triangles), and double-labeled cells (black dots) (A and B) and (C) a precise illustration of cortical region of level (A). Abbreviations: AH, anterior hypothalamic nucleus; CA, anterior commissura; CC; corpus catlosum; CL, claustrum; CO, optic chiasma; E, endopyriform nucleus; IC, insular cortex; PC, pyriform cortex; RS, rhinal sulcus; I, II, III, IV, V and VI, layers of insular cortex.
min each with PBS at 4 °C and incubated with Texas red conjugated donkey anti-rabbit IgG (Amersham, U.K.) at a dilution of 1:100 with PBS for 12 h at 15 °C. The sections were then washed 3 times for 10 min with PBS at 4 °C and mounted in a glycerin-PBS (1:1) mixture. For observation of B - H R P reacted with avidin-FITC, a Nikon EF microscope equipped with a B dichroic mirror filter and IF520-545 interference filter (Nikon Co, Japan) was used. For antigen reacted with Texas red conjugated anti-rabbit IgG the same microscope with G dichroic mirror filter and 580 nm absorption filter was used. Texas red has a strong red fluorescence of 615 nm length emission upon 596 nm length excitation, while FITC has a 517 nm length emission upon 492 nm length excitation. Thus, these two substances were easily discriminated without overlap of fluorescein emission 18. The antiserum to SOM was obtained after immunization of a rabbit with a synthetic SOMj~-bovine se-
rum albumin conjugate. Antiserum specificity was checked by absorption tests and radioimmunoassay. For absorption tests the sections were divided into two series, one was subjected to the immunoreaction described above. The other was incubated with antiSOM antiserum preabsorbed with synthetic SOM (2/~g/ml at a diluted antiserum) under conditions identical to those of the former series. No immunofluorescent structures were found in the control series. The specificity was also confirmed by radioimmunoassays13,14. The insular cortex was identified according to its cytoarchitecture using the criteria of Krieg 7. Injecting B - H R P into the spinal cord at cervical or lumbal levels revealed numerous tracer-labeled neurons in the insular cortex (Figs. 1A, 2A, C). B-HRPlabeled cell soma were identified by their finely packed green fluorescein granules of FITC, and sometimes processes of these cell soma were also visible.
199 Sections were simultaneously stained with antiSOM antiserum by the immunofluorescent method 1. Accordingly, SOM-positive structures (labeled by Texas red with red fluorescence) were observed in the same field as B-HRP by changing the filter from B to G excitation. The distribution and density of SOM-like immunoreactive (SOMii) structures in the insular cortex was similar to that revealed in previous studiesS,13A4. As shown in Fig. 2A, B (and the higher magnifications in Fig. 2C, D), some SOMIi neurons (red fluorescein) in the insular cortex were labeled simultaneously with retrograde tracing (green fluorescein). These double-labeled cells were found mainly in layers V and VI of insular cortex, and a few were also observed in the endopyriform nucleus. The doublelabeled cells were medium sized (15-20/~m) fusiform or polygonal cells (Fig. 2C, D) and the density was about 20% of B-HRP-labeled cells. These cells
were distributed in the insular cortex to the level of commissura anterior, rostrally, and to the level of hypothalamic paraventricular nucleus, caudally (Fig. 1). The existence of the pathway from the insular cortex to the spinal cord has been described by Shipley using the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) 16. Our preliminary study using HRP failed to confirm the presence of this pathway, while the B-HRP method clearly demonstrated its existence, suggesting that B-HRP has a higher sensitivity than the conventional HRP method but similar to that of the W G A - H R P method. The present study using the BHTI method has demonstrated the SOM-containing system to be one of the major components of this pathway. As this pathway may be related to autonomic functions 16, it is likely that SOM projections from insular cortex to
Fig. 2. Fluorescent photomicrographsshowing double-labeled neurons in layer V of insular cortex after injection of B-HRP into the lumbar spinal cord. A: B-HRP-labeted neurons visualized by avidin-FITCin the insular cortex observed by a B-dichroic mirror filter and IF 520-545 nm interference filter. B: somatostatin-positive cells in the same fields of A, respectively. Observations were carried out by a G-dichroicmirror and 580 nm absorption filter. Double-labeled cells were indicated by arrows. C and D: higher magnification of part A and B as indicated in rectangles in A and B, respectively.
2(I(~ the spinal cord are r e l a t e d to it as well.
sensory system and the third to the m o t o r system.
In addition to this S O M p a t h w a y , the f o l l o w i n g
due to the location of their m a j o r t e r m i n a l fields in
S O M p r o j e c t i o n s h a v e b e e n s h o w n in the spinal cord:
the dorsal h o r n and lamina V I i , respectivelyL t-~luci-
(1) primary sensory a f f e r e n t 3, (2) intrinsic S O M sys-
dation of the exact function of S O M will only be pos-
tem 3 and (3) d e s c e n d i n g p a t h w a y f r o m the central
sible on the basis of f u r t h e r analysis of its origins and
a m y g d a l o i d nucleus 4-e'. T h e first m a y be r e l a t e d to the
pathways.
1 Coons, A. H., Fluorescent antibody methods. In J. F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 2 Heizman, H. and Richards, F. M., Use of the avidin-biotin complex for specific staining of biological membranes in electronmicroscopy, Proc. nat. Acad. Sci. U.S.A., 71 (1974) 3537-3541. 3 Hunt, S. P., Cytochemistry of the spinal cord. In P. C. Erason (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 53-84. 4 Inagaki, S., Kawai, Y., Matsuzaki, T., Shiosaka, S. and Tohyama, M., Precise terminal fields of the descending somatostatinergic neuron system from the amygdaloid complex of the rat, J. Hirnforsch., 24 (1983) 345-356. 5 Johansson, O., HOkfelt, T. and Elde, R. P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat, Neuroscience, in press. 6 Kawai, Y., Inagaki, S., Shiosaka, S., Senba, E., Hara, Y., Sakanaka, M., Takatsuki, K. and Tohyama, M., Long descending projection from amygdaloid somatostatin-containing cells to the lower brain stem, Brain Research, 239 (1982) 603-607. 7 Krieg, W. J. S., Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas, J. comp. Neurol., 84 (1946) 221-275. 8 Larsson, L.-1., Fahrenkrug, J., Schaffalizky de Muckadell, O., Sundler, F., Hakanson, R. and Rehfeld, J, F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 3197- 3200. 9 Loren, I., Alumets, R., Hakanson, R. and Sundler, F., Distribution of gastrin and CCK-like peptides in rat brain, Histochemistrv, 59 (1979) 249-257. 10 Saper, C. B., Convergence of autonomic and limbic connections in the insular cortex of the rat, J. comp. Neurol., 210 (1982) 163-173. 11 Saper, C. B., Reciprocal parabrachial-cortical connections
in the rat, Brain Research, 242 (1982) 33-40. 12 Shiosaka, S., Shibasaki, S. and Tohyama, M., Bilateral alpha-melanocyte stimulating hormonergic fiber system from zona incerta to cerebral cortex: combined retrograde axonal transport and immunohistochemical study, Brain Research, 309 (1984) 350-353. 13 Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Iida, H., Minagawa, H., Hara, Y. and Tohyama, M., Ontogeny of somatostatincontaining neuron system of the rat: immunohistochemical analysis. II. Forebrain and diencephalon, J. comp. Neurol., 204 (1982) 211-224. 14 Shiosaka, S., Takatsuki, K., Sakanaka, M., lnagaki, S.. Takagi, H., Senba, E., Kawai, Y., Minagawa, H. and Tohyama, M., New somatostatin-containing sites in the diencephalon of the neonatal rat, Neurosci. Lett., 25 (I981) 69-73. 15 Shiosaka, S. and Tohyama, M., Evidence for an a-MSH-ergic hippocampal commissural connection in the rat, revealed by a double-labeling technique, Neurosci. Lett., 49 (1984) 213-216. 16 Shipley, M. T., Insular cortex projection to the nucleus of the solitary tract and brainstem visceromotor regions in the mouse, Brain Res. Bull., 8 (1982) 139-148, 17 Swanson, L. W., Sawchenko, P. E., Rivier, J. and Vale, W. W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an ,mmunohistochemical study, Neuroendocrinology, 36 (1983) 165-186. 18 Titus, J. A., Haugland, R., Sharrow, S. O. and Segal, D. M., Texas red. A hydrophilic, red-emitting fluorephore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies, J. lmmunol. Meth., 50 (1982) 193-204. 19 Van der Kooy, D., Mcginty, J. F., Koda, L, Y., Gerfen, C, R. and Bloom, F. E., Visceral cortex: a direct connection from prefrontal cortex to the solitary nucleus in rat, Neurosci. Lett., 33 (1982) 123-127.