Neuroscience Letters, 113 (1990) 1-6
I
Elsevier Scientific Publishers Ireland Ltd. NSL 06836
Simultaneous localization of calcitonin gene-related peptide and neurotensin in rat central amygdaloid nucleus Jari Honkaniemi, M a r k k u Pelto-Huikko, J o r m a Isola and Leena Rechardt Department of Biomedical Sciences, Universityof Tampere, Tampere (Finland) (Received 5 January 1990; Aceepted 16 January 1990)
Key words: Amygdaloid body; Immunocytochemistry; Neuropeptides; Electron microscopy; Innervation; Double staining The distribution of calcitonin gene-related peptide (CGRP) and neurotensin (NT)-like immunoreactivities (LI) was studied in rat central amygdaloid nucleus (ACe) with immunocytoehemieal double staining. A dense network of CGRP- and NT-immunorcactive (IR) nerve fibers and some NT-positive neurons were found in the lateral and lateral capsular subnuclei. Light microscopically CGRP-immunoreactive nerve endings were in close contact to most of the NT-immunoreaetive neurons. Under the electron microscope CGRP-positive terminals formed symmetric axo-somatic synapses with part of the NT-IR neurons. These results indicate that the NT- and CGRP-eontaining neuronal systems are in contact with each other in the ACe. Both peptides have marked effects on the circolatory system when administered intracerebrally. Thus the NT-IR neuronal system receiving synaptic input from CGRP-IR nerve terminals may mediate the cardiovascular effects of these two peptides.
The central amygdaloid nucleus (ACe) is a limbic system structure which regulates endocrine [4] and cardiovascular functions [3, 15]. Morphologically it consists of 2--4 subnuclei [8, 11]. Based on the distribution of certain neuropeptides, the ACe has been divided into 3 subnuclei: lateral capsular (CLC), lateral (CL) and medial (CM) subnucleus [22]. The ACe has several connections with various nuclei in the brain, especially with those in the brainstem involved in central autonomic regulation [6, 12, 13, 14, 17]. The ACe contains several neuropeptides [for review see 16]. One of these, the calcitonin gene-related peptide (CGRP) has profound effects on blood pressure [2, 3, 7]. Recent studies have shown that the ACe is densely innervated by CGRP-immunoreactive nerve fibers [9, 20]. Neurotensin (NT) is an another peptide which is found in the ACe where it is localized in a large number of neurons and nerve fibers [13, 19, 22]. In several brain areas NT is known to function as an excitatory transmitter [16]. Correspondence: J. Honkaniemi, Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
Fig. 1. The distribution o f C G R P in the rat ACe. The C L C holds the densest immunopositive nerve fiber network. The C L contains a moderate n u m b e r o f C G R P - I R nerve fibers, while only sparse immunoposirive fibers can be seen in the CM. Bar represents 130/lm. Fig. 2. The distribution o f NT-LI in a colchicine-treated rat ACe. A b o u t 60 immunopositive neurons can be observed. The localization of N T - I R nerve fibers is similar to the C G R P - I R nerve fibers (compar e Fig. 2 to Fig. 1). Bar represents 130 #m. Fig. 4. A nerve terminal exhibiting C G R P - L I (black arrows) forming a symmetric synapse (white arrows) with a N T - I R neuron. Silver grains demonstrating NT-LI are seen in the cytoplasm. Bar represents 200 rim.
Fig. 5. A N T - I R vesicle (arrows) in the cytoplasm o f a labelled neuron. Silver grains (open arrows) can also be seen diffusely in the cytoplasm. Bar represents 200 n m
The aim of this study was to investigate whether the NT-immunoreactive neurons receive synaptic input from CGRP-positive nerve endings. Immunoelectron microscopy was applied to demonstrate synapses between NT-IR neurons and CGRP-IR nerve terminals. Twelve adult male albino Sprague-Dawley rats aged 3-4 months were used. Six animals were treated with 1% colchicine (10--20 gl intracerebroventricularly) under chloralhydrate anesthesia. The rats were killed 24--48 h after injection. Fixation was carried out using subsequent perfusions with two paraformaldehyde solutions at pH 6.5 and pH 11 [1]. For electron microscopy, 0.2% glutaraldehyde (v/v) was added to the perfusion solutions. After perfusion, the brains were excised and further fixed by immersion at 4°C in the second fixative ( p H I 1.0) for 12 h for light microscopy or for 2--4 h for electron microscopy. Sections (50/tm) were cut with a Vibratome. For light microscopy, double immunostaining was carded out using the peroxidaseantiperoxidase (PAP) method to demonstrate CGRP and the avidin-coupled alkaline phosphatase (AFOS) method for detection of NT. Briefly, the sections were incubated with a rabbit CGRP antiserum, diluted 1:2000 (Peninsula, Belmont, U.S.A.; Amersham, Buckinghamshire, U.K.), goat anti-rabbit (GAR) serum and rabbit PAP. The immunoreaction was visualized with diaminobenzidine (DAB; brown reaction product). Part of the sections were mounted in Aquamount (Gurr). The rest of the sections were then incubated with a rabbit NT antibody (Mersey-Side Laboratories, Merseyside, U.K.), diluted 1:1000, biotinylated GAR and avidin AFOS. The neurotensin immunoreaction was visualized with a solution containing 5 mg Fast blue BB and 1 mg Naphtol AS-MX phosphatase dissolved in 100 #1 of dimethylformamide and 5 ml Tris-saline buffer (blue reaction product). Double labeling for electron microscopy was carried out using the silver intensification of DAB reaction to demonstrate NT and the non-intensified DAB reaction to demonstrate CGRP [10]. After the latter DAB reaction sections were postfixed with 2% osmium tetroxide (30 min), dehydrated, and embedded in Epon under plastic coverslips. Desired areas were photographed in light microscopy and processed further for electron microscopy. The ultrathin sections were examined with a Jeol 1200 EX electron microscope without counterstaining. Controls included normal serum and antiserum preabsorbed with appropriate peptides. No staining was observed in the controls. Both NT- and CGRP-immunoreactive nerve fibers showed a similar distribution in the ACe (Figs. 1 and 2). The lateral capsular subnucleus (CLC) was densely innervated by nerves immunoreactive to both peptides. A moderate number of fibers was present in the lateral part (CL) and only a sparse immunoreactive network of nerve fibers was seen in the medial subnucleus (CM). The CGRP-IR fiber network was more dense in all areas of the ACe than that of NT-immunopositive nerve fibers. Most of the NT-IR neurons were observed in the CL, some in the CLC, and only a few immunoreactive neurons were seen in the CM. In non-treated animals, there were 20-30 NT-containing neurons per section (results not shown). Colchicine treatment clearly increased the number of immunoreactive neurons and about 50-60 NTIR neurons were observed (Fig. 2). The staining intensity of NT-IR neurons was increased whereas the labeling of both NT- and CGRP-immunopositive nerve fibers
was decreased. Colchicine pretreatment did not alter the overall patterns of distribution of the peptides studied. Light microscopically C G R P - I R nerve-endings were in close contact to most of the N T - I R neurons in the CL and CLC (Fig. 3). Some N T - I R neurons were innervated by several CGRP-IR terminals. Electron microscopically NT-immunoreactive neurons had symmetric axo-somatic CGRP-immunoreactive synapses (Fig. 4), Silver-intensified DAB precipitates demonstrating NT-LI were located in the cytoplasmic dense-cored vesicles (diam, 50-500 nm) of the labeled neurons (Fig. 5). CGRPimmunoreaction was however localized diffusely in the nerve endings (Fig. 4). The present results of the distribution of NT- and CGRP-LI are in agreement with previous studies [19, 21]. The origin of C G R P - I R nerve fibers in the ACe is the parabrachial nucleus [PB] as demonstrated by retrograde tracing [18]. The PB is an important mediator of stress-related responses [14]. Both C G R P and N T have several effects on cardiovascular functions. N T causes changes in blood pressure [for review see 16]. C G R P has also intense effects on blood pressure and heart rate [5]. Intravenous injection of C G R P causes vasodilatation [2]. Microinjections of C G R P into the ACe results in an increase in heart rate and blood pressure [3, 15] and an elevation
Fig. 3. CGRP-IR nerve terminals (brown; arrows) in close contact with a NT-IR neuron (blue; star) in the rat ACe. Bar represents 10/an.
in p l a s m a n o r e p i n e p h r i n e c o n c e n t r a t i o n [3]. Since the C G R P b i n d i n g sites a r e l o c a t e d in the C L a n d C L C [9], the effects o f C G R P a r e p r o b a b l y m e d i a t e d t h r o u g h the neur o n s in these subnuclei, which give origin to the efferent p r o j e c t i o n s f r o m the A C e to the d o r s a l v a g a l c o m p l e x ( D V C ) a n d the PB [6, 13]. These b r a i n s t e m nuclei c o n t a i n N T - I R nerve fibers [7, for review see 16] a n d the function in a u t o n o m i c r e g u l a t i o n o f the c a r d i o v a s c u l a r system [12, 14, 17]. A b o u t 11% o f N T - I R n e u r o n s [6] in the A C e p r o j e c t to the D V C a n d a b o u t 50% [13] to the PB. T h e origin o f N T - I R projection to the PB a n d the D V C has been localized p r i n c i p a l l y to the C L [6, 13]. H e r e we d e m o n s t r a t e d C G R P - I R nerve fibers m a i n l y in the s a m e area. T h u s it is suggested t h a t the c a r d i o v a s c u l a r effects o f the C G R P in the A C e are m e d i a t e d p a r t l y b y the N T - I R n e u r o n s p r o j e c t i n g to the PB a n d D V C . 1 Berod, A., Hartman, B.K. and Pujol, J.F., Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase, J. Histochem. Cytochem., 29 (1981) 844-850. 2 Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R. and Mclntyre, I., Calcitonin gene-related peptide is a potent vasodilatator, Nature (Lond.), 313 (1985) 54-56. 3 Brown, M.R. and Gray, T.S., Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines, Reg. Pep., 21 (1988) 95-106. 4 Ellendorf, F. and Parvizi, N., Neuroendocrinology of amygdaloid control of gonadotropin secretion. In Y. Ben-Ari (Ed.), The Amygdaloid Complex, Elsevier/North Holland, Amsterdam, 1981, pp. 239-250. 5 Fisher, L.A., Kikkawa, D.O., Rivier, J.E., Amara, S.G., Evanst, R.M., Rosenfeld, M.G., Vale, W.W. and Brown, M.R., Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide, Nature (Lond.), 305 (1980) 534-536. 6 Gray, T.S. and Magnuson, D.J., Neuropeptide neuronal efferents from the bed nucleus of stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat, J. Comp. Neurol., 262 (1987) 365-374. 7 Higgins, G.A., Hoffman, G.E., Wray, S. and Schwaber, J.S., Distribution of neurotensin-immunoreactivity within baroreceptive portions of the nucleus of the tractus solitarius and dorsal vagal nucleus of the rat, J. Comp. Neurol., 226 (1984) 155-164. 8 Krettek, J.E. and Price, J.L., A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid axonal connections, J. Comp. Neurol., 178 (1978) 255-280. 9 Kruger, L., Mantyh, P.W., Sternini, C., Brecha, N.C. and Mantyh, C.R., Calcitonin gene-related peptide (CGRP) in the rat central nervous system: patterns of immunoreactivity and receptor binding sites, Brain. Res., 463 (1988) 223-244. 10 Liposits, Zs., Sherman, D., Phelix, C. and Paull, W.K., A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus in the rat, Histochemistry, 85 (1986) 95-106. l 1 McDonald, A.J., Cytoarchitecture of the central amygdaloid nucleus of the rat, J. Comp. Neurol., 208 (1982) 401-418. 12 Miura, M. and Reis, D.J., The role of the solitary and the paramedian reticular nuclei in mediating cardiovascular reflex responses from carotid baro- and chemoreceptors, J. Physiol. (Lond.), 223 (1972) 525-548. 13 Moga, M.M. and Gray, T.S., Evidence for corticotropin-releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus, J. Comp. Neurol., 241 (1985) 275-284. 14 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain. Res., 232 (1982) 57-75.
15 Nguyen, K.Q., Sills, M.A. and Jacobwitz, D.M., Cardiovascular effects produced by microinjection of calcitonin gene-related peptide into the rat central amygdaloid nucleus, Peptides, 7 (1986) 337 ~33q. 16 Nieuwenhuys, R., Chemoarchitecture of the brain, Springer, Berlin, 1985, 246 pp. 17 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Afferent projections to cardiovascular portions of the nucleus of the tractus solitarius of the rat, Brain Res., 223 (1981) 402-408. 18 Schwaber, J.S., Sternini, C., Brecha, N.C., Rogers, W.T. and Card, J.P., Neurons containing calcitonin gene-related peptide in the parabrachial nucleus project to the central nucleus of the amygdala, J. Comp. Neurol., 270 (1988) 41 f~426. 19 Shimada, S., lnagaki, S., Kubota, Y., Ogawa, N., Shibasaki, T. and Takagi, H., Coexistence of peptides (corticotropin releasing factor/neurotensin and substance P/somatostatin) in the bed nucleus of the stria terminalis and central amygdaloid nucleus of the rat, Neuroscience, 30 (1989) 377 -383. 20 Skofitsch, G. and Jacobwitz, D.M., Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system, Peptides, 6 (1985) 721-745. 21 Tay, S.S., Williams, T.H. and Jew, J.Y., Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach, Peptides, I0 (1989) 113 [20. 22 Wray, S. and Hoffman, G.E. Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat, Peptides, 4 (1983) 525-541.
I
Elsevier Scientific Publishers Ireland Ltd. NSL 06836
Simultaneous localization of calcitonin gene-related peptide and neurotensin in rat central amygdaloid nucleus Jari Honkaniemi, M a r k k u Pelto-Huikko, J o r m a Isola and Leena Rechardt Department of Biomedical Sciences, Universityof Tampere, Tampere (Finland) (Received 5 January 1990; Aceepted 16 January 1990)
Key words: Amygdaloid body; Immunocytochemistry; Neuropeptides; Electron microscopy; Innervation; Double staining The distribution of calcitonin gene-related peptide (CGRP) and neurotensin (NT)-like immunoreactivities (LI) was studied in rat central amygdaloid nucleus (ACe) with immunocytoehemieal double staining. A dense network of CGRP- and NT-immunorcactive (IR) nerve fibers and some NT-positive neurons were found in the lateral and lateral capsular subnuclei. Light microscopically CGRP-immunoreactive nerve endings were in close contact to most of the NT-immunoreaetive neurons. Under the electron microscope CGRP-positive terminals formed symmetric axo-somatic synapses with part of the NT-IR neurons. These results indicate that the NT- and CGRP-eontaining neuronal systems are in contact with each other in the ACe. Both peptides have marked effects on the circolatory system when administered intracerebrally. Thus the NT-IR neuronal system receiving synaptic input from CGRP-IR nerve terminals may mediate the cardiovascular effects of these two peptides.
The central amygdaloid nucleus (ACe) is a limbic system structure which regulates endocrine [4] and cardiovascular functions [3, 15]. Morphologically it consists of 2--4 subnuclei [8, 11]. Based on the distribution of certain neuropeptides, the ACe has been divided into 3 subnuclei: lateral capsular (CLC), lateral (CL) and medial (CM) subnucleus [22]. The ACe has several connections with various nuclei in the brain, especially with those in the brainstem involved in central autonomic regulation [6, 12, 13, 14, 17]. The ACe contains several neuropeptides [for review see 16]. One of these, the calcitonin gene-related peptide (CGRP) has profound effects on blood pressure [2, 3, 7]. Recent studies have shown that the ACe is densely innervated by CGRP-immunoreactive nerve fibers [9, 20]. Neurotensin (NT) is an another peptide which is found in the ACe where it is localized in a large number of neurons and nerve fibers [13, 19, 22]. In several brain areas NT is known to function as an excitatory transmitter [16]. Correspondence: J. Honkaniemi, Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
Fig. 1. The distribution o f C G R P in the rat ACe. The C L C holds the densest immunopositive nerve fiber network. The C L contains a moderate n u m b e r o f C G R P - I R nerve fibers, while only sparse immunoposirive fibers can be seen in the CM. Bar represents 130/lm. Fig. 2. The distribution o f NT-LI in a colchicine-treated rat ACe. A b o u t 60 immunopositive neurons can be observed. The localization of N T - I R nerve fibers is similar to the C G R P - I R nerve fibers (compar e Fig. 2 to Fig. 1). Bar represents 130 #m. Fig. 4. A nerve terminal exhibiting C G R P - L I (black arrows) forming a symmetric synapse (white arrows) with a N T - I R neuron. Silver grains demonstrating NT-LI are seen in the cytoplasm. Bar represents 200 rim.
Fig. 5. A N T - I R vesicle (arrows) in the cytoplasm o f a labelled neuron. Silver grains (open arrows) can also be seen diffusely in the cytoplasm. Bar represents 200 n m
The aim of this study was to investigate whether the NT-immunoreactive neurons receive synaptic input from CGRP-positive nerve endings. Immunoelectron microscopy was applied to demonstrate synapses between NT-IR neurons and CGRP-IR nerve terminals. Twelve adult male albino Sprague-Dawley rats aged 3-4 months were used. Six animals were treated with 1% colchicine (10--20 gl intracerebroventricularly) under chloralhydrate anesthesia. The rats were killed 24--48 h after injection. Fixation was carried out using subsequent perfusions with two paraformaldehyde solutions at pH 6.5 and pH 11 [1]. For electron microscopy, 0.2% glutaraldehyde (v/v) was added to the perfusion solutions. After perfusion, the brains were excised and further fixed by immersion at 4°C in the second fixative ( p H I 1.0) for 12 h for light microscopy or for 2--4 h for electron microscopy. Sections (50/tm) were cut with a Vibratome. For light microscopy, double immunostaining was carded out using the peroxidaseantiperoxidase (PAP) method to demonstrate CGRP and the avidin-coupled alkaline phosphatase (AFOS) method for detection of NT. Briefly, the sections were incubated with a rabbit CGRP antiserum, diluted 1:2000 (Peninsula, Belmont, U.S.A.; Amersham, Buckinghamshire, U.K.), goat anti-rabbit (GAR) serum and rabbit PAP. The immunoreaction was visualized with diaminobenzidine (DAB; brown reaction product). Part of the sections were mounted in Aquamount (Gurr). The rest of the sections were then incubated with a rabbit NT antibody (Mersey-Side Laboratories, Merseyside, U.K.), diluted 1:1000, biotinylated GAR and avidin AFOS. The neurotensin immunoreaction was visualized with a solution containing 5 mg Fast blue BB and 1 mg Naphtol AS-MX phosphatase dissolved in 100 #1 of dimethylformamide and 5 ml Tris-saline buffer (blue reaction product). Double labeling for electron microscopy was carried out using the silver intensification of DAB reaction to demonstrate NT and the non-intensified DAB reaction to demonstrate CGRP [10]. After the latter DAB reaction sections were postfixed with 2% osmium tetroxide (30 min), dehydrated, and embedded in Epon under plastic coverslips. Desired areas were photographed in light microscopy and processed further for electron microscopy. The ultrathin sections were examined with a Jeol 1200 EX electron microscope without counterstaining. Controls included normal serum and antiserum preabsorbed with appropriate peptides. No staining was observed in the controls. Both NT- and CGRP-immunoreactive nerve fibers showed a similar distribution in the ACe (Figs. 1 and 2). The lateral capsular subnucleus (CLC) was densely innervated by nerves immunoreactive to both peptides. A moderate number of fibers was present in the lateral part (CL) and only a sparse immunoreactive network of nerve fibers was seen in the medial subnucleus (CM). The CGRP-IR fiber network was more dense in all areas of the ACe than that of NT-immunopositive nerve fibers. Most of the NT-IR neurons were observed in the CL, some in the CLC, and only a few immunoreactive neurons were seen in the CM. In non-treated animals, there were 20-30 NT-containing neurons per section (results not shown). Colchicine treatment clearly increased the number of immunoreactive neurons and about 50-60 NTIR neurons were observed (Fig. 2). The staining intensity of NT-IR neurons was increased whereas the labeling of both NT- and CGRP-immunopositive nerve fibers
was decreased. Colchicine pretreatment did not alter the overall patterns of distribution of the peptides studied. Light microscopically C G R P - I R nerve-endings were in close contact to most of the N T - I R neurons in the CL and CLC (Fig. 3). Some N T - I R neurons were innervated by several CGRP-IR terminals. Electron microscopically NT-immunoreactive neurons had symmetric axo-somatic CGRP-immunoreactive synapses (Fig. 4), Silver-intensified DAB precipitates demonstrating NT-LI were located in the cytoplasmic dense-cored vesicles (diam, 50-500 nm) of the labeled neurons (Fig. 5). CGRPimmunoreaction was however localized diffusely in the nerve endings (Fig. 4). The present results of the distribution of NT- and CGRP-LI are in agreement with previous studies [19, 21]. The origin of C G R P - I R nerve fibers in the ACe is the parabrachial nucleus [PB] as demonstrated by retrograde tracing [18]. The PB is an important mediator of stress-related responses [14]. Both C G R P and N T have several effects on cardiovascular functions. N T causes changes in blood pressure [for review see 16]. C G R P has also intense effects on blood pressure and heart rate [5]. Intravenous injection of C G R P causes vasodilatation [2]. Microinjections of C G R P into the ACe results in an increase in heart rate and blood pressure [3, 15] and an elevation
Fig. 3. CGRP-IR nerve terminals (brown; arrows) in close contact with a NT-IR neuron (blue; star) in the rat ACe. Bar represents 10/an.
in p l a s m a n o r e p i n e p h r i n e c o n c e n t r a t i o n [3]. Since the C G R P b i n d i n g sites a r e l o c a t e d in the C L a n d C L C [9], the effects o f C G R P a r e p r o b a b l y m e d i a t e d t h r o u g h the neur o n s in these subnuclei, which give origin to the efferent p r o j e c t i o n s f r o m the A C e to the d o r s a l v a g a l c o m p l e x ( D V C ) a n d the PB [6, 13]. These b r a i n s t e m nuclei c o n t a i n N T - I R nerve fibers [7, for review see 16] a n d the function in a u t o n o m i c r e g u l a t i o n o f the c a r d i o v a s c u l a r system [12, 14, 17]. A b o u t 11% o f N T - I R n e u r o n s [6] in the A C e p r o j e c t to the D V C a n d a b o u t 50% [13] to the PB. T h e origin o f N T - I R projection to the PB a n d the D V C has been localized p r i n c i p a l l y to the C L [6, 13]. H e r e we d e m o n s t r a t e d C G R P - I R nerve fibers m a i n l y in the s a m e area. T h u s it is suggested t h a t the c a r d i o v a s c u l a r effects o f the C G R P in the A C e are m e d i a t e d p a r t l y b y the N T - I R n e u r o n s p r o j e c t i n g to the PB a n d D V C . 1 Berod, A., Hartman, B.K. and Pujol, J.F., Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase, J. Histochem. Cytochem., 29 (1981) 844-850. 2 Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R. and Mclntyre, I., Calcitonin gene-related peptide is a potent vasodilatator, Nature (Lond.), 313 (1985) 54-56. 3 Brown, M.R. and Gray, T.S., Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines, Reg. Pep., 21 (1988) 95-106. 4 Ellendorf, F. and Parvizi, N., Neuroendocrinology of amygdaloid control of gonadotropin secretion. In Y. Ben-Ari (Ed.), The Amygdaloid Complex, Elsevier/North Holland, Amsterdam, 1981, pp. 239-250. 5 Fisher, L.A., Kikkawa, D.O., Rivier, J.E., Amara, S.G., Evanst, R.M., Rosenfeld, M.G., Vale, W.W. and Brown, M.R., Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide, Nature (Lond.), 305 (1980) 534-536. 6 Gray, T.S. and Magnuson, D.J., Neuropeptide neuronal efferents from the bed nucleus of stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat, J. Comp. Neurol., 262 (1987) 365-374. 7 Higgins, G.A., Hoffman, G.E., Wray, S. and Schwaber, J.S., Distribution of neurotensin-immunoreactivity within baroreceptive portions of the nucleus of the tractus solitarius and dorsal vagal nucleus of the rat, J. Comp. Neurol., 226 (1984) 155-164. 8 Krettek, J.E. and Price, J.L., A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid axonal connections, J. Comp. Neurol., 178 (1978) 255-280. 9 Kruger, L., Mantyh, P.W., Sternini, C., Brecha, N.C. and Mantyh, C.R., Calcitonin gene-related peptide (CGRP) in the rat central nervous system: patterns of immunoreactivity and receptor binding sites, Brain. Res., 463 (1988) 223-244. 10 Liposits, Zs., Sherman, D., Phelix, C. and Paull, W.K., A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus in the rat, Histochemistry, 85 (1986) 95-106. l 1 McDonald, A.J., Cytoarchitecture of the central amygdaloid nucleus of the rat, J. Comp. Neurol., 208 (1982) 401-418. 12 Miura, M. and Reis, D.J., The role of the solitary and the paramedian reticular nuclei in mediating cardiovascular reflex responses from carotid baro- and chemoreceptors, J. Physiol. (Lond.), 223 (1972) 525-548. 13 Moga, M.M. and Gray, T.S., Evidence for corticotropin-releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus, J. Comp. Neurol., 241 (1985) 275-284. 14 Mraovitch, S., Kumada, M. and Reis, D.J., Role of the nucleus parabrachialis in cardiovascular regulation in cat, Brain. Res., 232 (1982) 57-75.
15 Nguyen, K.Q., Sills, M.A. and Jacobwitz, D.M., Cardiovascular effects produced by microinjection of calcitonin gene-related peptide into the rat central amygdaloid nucleus, Peptides, 7 (1986) 337 ~33q. 16 Nieuwenhuys, R., Chemoarchitecture of the brain, Springer, Berlin, 1985, 246 pp. 17 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Afferent projections to cardiovascular portions of the nucleus of the tractus solitarius of the rat, Brain Res., 223 (1981) 402-408. 18 Schwaber, J.S., Sternini, C., Brecha, N.C., Rogers, W.T. and Card, J.P., Neurons containing calcitonin gene-related peptide in the parabrachial nucleus project to the central nucleus of the amygdala, J. Comp. Neurol., 270 (1988) 41 f~426. 19 Shimada, S., lnagaki, S., Kubota, Y., Ogawa, N., Shibasaki, T. and Takagi, H., Coexistence of peptides (corticotropin releasing factor/neurotensin and substance P/somatostatin) in the bed nucleus of the stria terminalis and central amygdaloid nucleus of the rat, Neuroscience, 30 (1989) 377 -383. 20 Skofitsch, G. and Jacobwitz, D.M., Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system, Peptides, 6 (1985) 721-745. 21 Tay, S.S., Williams, T.H. and Jew, J.Y., Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach, Peptides, I0 (1989) 113 [20. 22 Wray, S. and Hoffman, G.E. Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat, Peptides, 4 (1983) 525-541.
