Synaptic contacts between CGRP-immunoreactive terminals and enkephalin-immunoreactive neurons in the central amygdaloid nucleus of the rat

Neuroscience Letters, 134 (1992) 243-246 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 03.50

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Synaptic contacts between CGRP-immunoreactive terminals and enkephalin-immunoreactive neurons in the central amygdaloid nucleus of the rat S. Shimada 1, S. Inagaki 2, N. Narita 2 and H. Takagi 2 ~Second Department of Anatomy, Osaka University Medical School, Suita (Japan.) and 2FirstDepartment of Anatomy, Osaka City University Medical School Osaka (Japan) (Received 27 September 1991; Accepted 4 October 1991)

Key words: Calcitonin gene-related peptide; Enkephalin; Central amygdaloid nucleus; Synaptic interaction An immunoelectron microscopic method combined with immunofluorescence double staining was carried out to examine the relationship between calcitonin gene-related peptide (CGRP)-like immunoreactive (LI) axon terminals and enkephalin (ENK)-LI neurons in the central amygdaloid nucleus (Ce) of the rat. The latter method showed that many ENK-LI cell bodies are densely surrounded by CGRP-LI axons in the lateral subdivision of the Ce (CeL). After taking fluorescence micrographs, the immunoperoxidase technique was used to examine the CGRP-LI axonal profiles under an electron microscope. CGRP-LI terminals were frequently found to form axo-somatic synaptic contacts with ENK-LI neurons in the CeL.

The central amygdaloid nucleus (Ce) is extensively innervated by brainstem regions relaying visceral inputs from the peripheral organs [12, 15]. Especially the parabrachial nucleus (PB)-Ce pathway is considered to be involved in cardiovascular [3, 7, 10, 11], respiratory [4, 16], gustatory [12, 22] and gastrointestinal activities [5]. Experimental immunocytochemical studies have demonstrated that calcitonin-gene related peptide (CGRP) is abundantly contained in the PB-Ce pathway [17, 19], and CGRP nerve terminals in the Ce make numerous synaptic contacts, the most striking feature of which is heavy synaptic inputs to the perikarya in the form of large long CGRP boutons [18]. Therefore, studies on the property of postsynaptic targets to the CGRP afferents may provide a morphological basis for understanding of the functional roles of the Ce. Thus, in order to demonstrate the synaptic relationship between CGRP terminals and enkephalin (ENK)-containing cells which are one of the biochemically defined neurons frequently seen in the Ce, we performed an immunoelectron microscopic study combined with the immunofluorescence double staining method modified from the technique developed by Morishima et al. [8].

Correspondence: H. Takagi, First Department of Anatomy, Osaka City University Medical School, 1-4-54, Asahimachi, Abenoku, Osaka, 545 Japan.

A total of 7 Wistar male rats weighing 100-150 g were used. Four of them received colchicine injections (4/~1, 4 mg/4 ml) into the lateral ventricle 48 h before sacrifice. All the animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The animals were perfused through the ascending aorta, first with 50 ml of saline, then with 200 ml of fixative (0.05% glutaraldehyde, 4% paraformaldehyde, 0.2~ picric acid and 0.1 M phosphate buffer) [21]. The brain was removed from the skull, cut into small blocks and put into the same fresh fixative for 2 h at 4°C. Subsequently, the samples were cut into 30-40 am thick sections using a Vibratome. The sections were first incubated with a mixture of polyclonal rabbit CGRP antiserum (diluted 1:2000, obtained from Dr. I. Maclntyre [9]) and monoclonal mouse Leu°ENK antiserum (diluted 1:500, Sera Lab.) in 0.25 M phosphatebuffered saline (PBS) for 48 h at 4°C. After washing in PBS 3 times, the sections were incubated with a mixture of the secondary antibody: biotinylated donkey anti-rabbit IgG (1:500, Amersham) and fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antiserum (1:1000, Cappel) for 24 h at 4°C. We tested the immunohistochemical specificities of the primary and secondary antisera as shown previously [23]. There was no crossreactivity between the secondary antisera, or between the primary antisera and non-specific secondary antisera. After washing in PBS, the sections were incubated with Texas Red streptavidin for 2 h at room temperature

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(RT). Subsequently, the sections were mounted on glass slides and analyzed under a fluorescence microscope (Nikon) equipped with a B dichroic mirror system (excitation wave-length at 495 nm, LP 515 stop filter) for observing FITC labeling and with a G dichroic mirror system (excitation wave-length at 545 nm, LP 580 stop filter) for Texas Red labeling. After taking fluorescence micrographs, the sections were removed from the glass slides and further incubated with biotinylated peroxidase (Vector) for 2 h at RT. In order to visualize the CGRP-

like immunoreactivity at the electron microscopic level, the sections were placed in 0.05% 3,3'-diaminobenzidine tetra-HC1 (DAB) (Dotite) and 0.01% hydrogen peroxide dissolved in 0.05 M Tris-HC1 buffer for 5-10 min at RT. After washing, the sections were postfixed in 1% OsO4 in 0.I M phosphate buffer and dehydrated. The sections were stained with 1% uranyl acetate at the 70% alcohol dehydration stage. All the sections were flat-embedded in epoxy resin on siliconized glass slides. Under a light microscope, we searched the same area in the sections

i i

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Fig. 1. A,B: immunofluorescence micrographs of the same section of the CeL after double staining with antisera to ENK (A) and CGRP (B). C: bright-field micrograph of the same section as A and B, showing CGRP-LI fibers restained by modifying the avidin-biotin-peroxidase method [61. NI and N4 cells are ENK-LI (A), whereas N2 and N3 cells are immunonegative for ENK. NI and N2 cells are surrounded by CGRP-LI fibers and the other two cells are not (B,C). D: low-power electron micrograph showing cells indicated as NL N4 in C. A capillary (asterisk in C) is also seen. E,F: high-power electron micrographs showing synaptic contacts (small arrows) between Nt cell and CGRP-LI terminal (asterisk, arrow in D) (E), and between N2 cell and CGRP-LI terminal (F). D and F are serial sections. Consequently, CGRP-LI terminals make axo-somatic synapses with both ENK positive and negative neurons in the CeL. Scales (A C) 10 #m; (D) 10 pro; (E,F) 0.5 itm.

245 that had been photographed under a fluorescence microscope. Then the selected area was cut from the section and stuck on a polymerized cylindrical block with glue. Serial ultrathin sections were cut, poststained with lead citrate and examined under an electron microscope. The nomenclature used here was based upon that found in the atlas of Paxinos and Watson [14]. In the case of colchicine-untreated animals, only a small number of E N K - L I cells were seen in the lateral subdivision of the Ce (CeL), and no E N K - L I cells in the lateral capsular and medial subdivisions of the Ce (CeLC and CeM), whereas in the case of colchicine-treated animals, a large number of E N K - L I cells could be visualized in the CeL and a small number of E N K - L I cells in the CeLC and CeM. On the other hand, a very high concentration of C G R P - L I fibers was distributed in the CeL and CeLC, and only a low concentration of them was seen in the CeM in colchicine-untreated animals. Colchicine treatment slightly reduced C G R P - L I fibers in the CeL and CeLC. In both treated and untreated animals, a significant number of C G R P - L I fibers characteristically surrounded the cell somata in the CeL and CeLC as reported previously [18]. The immunofluorescence double staining method revealed that about 70% of the E N K - L I cells were surrounded by C G R P - L I fibers in the CeL (Fig. I A,B). C G R P - L I fibers also surrounded E N K negative cells (Fig. 1A,B). After selecting areas where E N K - L I cells were surrounded by C G R P - L I fibers in the CeL, the C G R P - L I fibers were restained with DAB reaction (Fig. 1C), and examined under an electron microscope (Fig. 1D F). The immunoelectron microscopic study revealed that C G R P - L I terminals formed axosomatic synaptic contacts with the cells which had displayed ENK-like immunoreactivity by the immunofluorescence double staining (Fig. 1E). The characteristic of the C G R P - L I terminals was the presence of large long boutons, each of which surrounded a cell soma and made symmetrical synaptic contacts. The C G R P - L I terminals also made synaptic contacts with the cell bodies which had not shown ENK-like immunoreactivity (Fig. IF). The present results have elucidated the fact that E N K LI cells are postsynaptic targets of C G R P - L I afferents in the Ce. Oertel et al. [13] have demonstrated that opioid and glutamic acid decarboxylase ( G A D ) coexist within single neurons in the Ce. Therefore, C G R P - L I axon terminals may also have synaptic interaction with GABA-ergic cells. G r a y et al. [2] have described that VIP-LI neurons in the Ce receive synaptic inputs from large VIP-negative terminals in the Ce, which are morphologically very similar to C G R P - L I terminals. Thus, C G R P - L I axons may also form synapses with VIP-LI cells in the Ce. A correlation study between Golgi

impregnation and immunohistochemistry suggests that E N K - L I cells belong to the medium-sized neuron type with densely spinous dendrites and VIP-LI cells belong to the sparsely spinous neuron type [1]. Therefore, E N K immunonegative cells in synaptic contacts with C G R P LI terminals in the present study may partly be VIPergic. On the other hand, Y a m a n o et al. have demonstrated that some of the C G R P - L I neurons in the PB which project to the Ce also display neurotensin- or substance Plike immunoreactivity [20, 23]. This raises the possibility that C G R P - L I axons which synapse with E N K - L I cells in the Ce may also contain neurotensin- or substance Plike immunoreactivity. Microinjection of C G R P into the Ce resulted in increases in heart rate and blood pressure I11]. E N K neurons in the Ce may contribute to amygdaloid car~liovascular control via synaptic input from C G R P afferents that originate in the PB known as the autonomic brainstem region. We are very grateful to Dr. I. MacIntyre (Department of Chemical Pathology, University of London, Royal Postgraduate Medical School, H a m m e r s m i t h Hospital, U.K.) for generously supplying us with the antiserum to CGRP. I Cassel,M.D. and Gray, T.S., Morphology ofpeptide-immunoreactive neurons in the rat central nucleus of the amygdala, J. Comp. Neurol., 281 (1989) 320-333. 2 Gray, T.S., Cassell, M.D., Nilaver, G., Zimmerman, E.A. and Williams, T.H., The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala, Neuroscience, l I (1984) 399~,08. 3 Hamilton, R.B., Ellenberger, H., Liskowsky, D. and Schneiderman, N., Parabrachial area as mediator of bradycardia in rabbit, J. Autonom. Nerv. Syst., 4 (1981) 261-281. 4 Harper, R.M., Frysinger, R.C., Trelease, R.B. and Marks, J.D., State-dependent alteration of respiratory cycle timing by stimulation of the central nucleus of the amygdala, Brain Res., 306 (1984) 1 8.

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