Neuroscience Letters, 140 (1992) 215-218 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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NSL 08697

Noradrenergic innervation of vasopressin-containing neurons in the rat hypothalamic supraoptic nucleus Seiji Shioda and Yasumitsu Nakai Department of Anatomy, Showa University School of Medicine, Tokyo (Japan) (Received 12 March 1992; Accepted 26 March 1992)

Key words: Synapse; Noradrenaline; Vasopressin; Supraoptic nucleus; Rat; Double-labeling immunocytochemistry; Electron microscopy The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron microscopically by using double-labeling immunocytochemistry combining the pre-embedding peroxidase-anti-peroxidase method with post-embedding immunocolloidal gold staining. Noradrenaline-like immunoreactive axon terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neurons in the SON. This study provides morphological evidence for noradrenergic control of neuronal activity of VP-containing neurons at the SON level.

Several physiological studies indicate that exogenously applied noradrenaline (NA) or cq-agonists activate vasopressin (VP) neurons in the supraoptic nucleus (SON) in vivo [6, 10, 23] and in vitro [10, 17, 24], and promote the release ofVP [1, 2, 11, 12, 14]. Morphological studies using fluorescence histochemistry have revealed a dense catecholaminergic innervation in the SON [4, 9]. Electron microscopic immunocytochemical studies have revealed that dopamine (DA)- or NA-like immunoreactive (LI) axon terminals synapse with magnocellular neurosecretory neurons in the rat SON or PVN [3, 8]. By a technique combining immunocytochemistry using VP antiserum with autoradiography after [3H]NA injection or 5-hydroxydopamine (5-OHDA) uptake, [3H]NA- or 5-OHDA-labeled axon terminals have been found to synapse with VP-LI neurons in the PVN and SON [15]. Recently, double labeling immunocytochemistry indicated that catecholamine synthesizing enzyme, tyrosine hydroxylase (TH)-LI axon terminals synapse with VP-LI neurons in the rat PVN [16]. However, it is not known exactly whether DA or NA neurons make synaptic contacts with VP-containing neurosecretory neurons in the rat hypothalamus. Therefore, we tried to identify noradrenergic innervation of VP-containing neurosecretory neurons in the rat SON by using double labeling immunocytochemistry in which the pre-embedCorrespondence: Y. Nakai, Department of Anatomy, Showa University School of Medicine, Hatanodai 1-5-8, Shinagawa-ku, Tokyo 142, Japan.

ding peroxidase-anti-peroxidase (PAP) method using antiserum to NA itself was combined with post-embedding immunocolloidal gold staining using antiserum to neurophysin II (NP II), the carrier protein of VP. Adult male Wistar rats (170-200 g) were maintained pre- and post-operatively on a 12 h light-dark cycle and given food and water ad libitum. The animals were anesthetized with sodium pentobarbital (20 mg/kg, i.p.) and transcardially perfused with 1% Na2S20~ in 0.1 M cacodylate buffer (pH 6.2), followed by 2.5% glutaraldehyde in 0.1 M cacodylate-l% Na2S205 (pH 7.5). Trans-

Fig. 1. Photomicrograph of a frozen section through the SON after treatment with anti-NA antibody. NA-LI fibers in the SON surround and contact with magnocellular neurons (arrows), more densely in the ventral than in the dorsal part. OC, optic chiasma. Bar = 30/lm.

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Fig. 2. Photomicrographsof ultrathin sections through the SON after treatment with anti-NA antibody. A: NA-LI varicosefiber (*) contactinga cell body containing NP II-LI large neurosecretory granules (arrows). B,C: NA-LI axon terminals (*) making axo-dendritic (B) and axo-somatic (C) synapses (arrowheads) with neurons containing NP II-LI large neurosecretory granules (arrows). G, Golgi apparatus; N, nucleus. Bar - 0.5/am.

versal brain blocks (about 3 mm thick) including the hypothalamus were isolated by hand and subsequently immersed in the same fixative for 2 h at 4°C. Sections were cut at 40 p m on a Vibratome (Oxford) in 0.05 M TrisHCI-I% Na2S205 (pH 7.5). The immunocytochemical procedures were as follows: (1) incubation in N A antise-

rum diluted at 1:2000-1:4000 in Tris buffer for 24 h at 4°C, (2) washing and subsequent incubation in 0.05 M Tris buffer (pH 7.5), (3) incubation in goat anti-rabbit IgG diluted at 1:150 for 60 min at room temperature, (4) washing, (5) PAP complex diluted at 1:100 for 60 min at room temperature, (6) washing, (7) reaction with 3,3'-

217 diaminobenzidine (Dojin, 1.0 mg/ml) and 0.005% H202 (Wako) in 0.05 M Tris buffer (pH 7.6) for 10-15 min, (8) treatment with 1% OsO4 in 0.1 M cacodylate buffer for 1 h, (9) washing, (10) dehydration in graded ethanol, and (11) flat embedding in an Epon-Araldite mixture. U1trathin sections mounted on nickel grids were treated with sodium metaperiodate, and processed by post-embedding immunocolloidal gold staining [19]. The sections were incubated with rabbit antiserum to N P II (diluted at 1:2000) for 24 h at 4°C, and then treated with gold (10 nm diameter)-labeled goat anti-rabbit IgG (Janssen) for 1 h at room temperature. They were contrasted with uranyl acetate and lead citrate and then examined with a J E O L JEM-1200 EXII electron microscope. The antisera to N A and N P II were purchased from Biodesign International (Lot 429) and I N C S T A R Science (Lot 8 512024), respectively. The specificity of the N A antiserum was tested by using antiserum preabsorbed with N A and DA, and examined in sections prepared for light and electron microscopy. The minimal concentration of N A required to completely block staining was 1.0 flg/ml. D A (10/tg/ml) did not alter the staining with the N A antiserum. Numerous varicose N A - L I fibers appeared to be present in equal density throughout the SON from the rostral to the caudal part. These fibers were also concentrated in the ventral part of the SON, but some N A - L I fibers could be found in the dorsal part (Fig. 1). Some N A - L I fibers appeared to surround and contact directly with magnocellular neurons in the SON (Fig. 1). N o N A - L I cell bodies were observed in the SON, in the PVN, or in other hypothalamic areas. Electron microscopy of the SON revealed many NALI fibers contacting (Fig. 2A) or making synapses with cell processes (Fig. 2B) and cell bodies (Fig. 2C) that contained N P II-LI large dense neurosecretory granules (150-200 nm diameter). These contacts appeared generally as symmetrical synapses. In the SON, the N A - L I presynaptic terminals contained some dense granular vesicles (70-90 nm diameter) and many small clear vesicles (30-50 nm diameter). Reaction products were seen on the external membranes of mitochondria, the inner surfaces of plasma membranes, outer surfaces of small clear vesicles, and in granular vesicles. The present study is the first to use double labeling immunocytochemistry to show that N A - L I axon terminals make synaptic contacts with N P II-LI cell bodies and their processes in the SON. Catecholaminergic afferent fibers to VP-containing neurosecretory neurons in the rat hypothalamus are known to arise primarily from A I noradrenergic neurons in the caudal ventrolateral medulla [5, 13, 18]. Less dense catecholaminergic afferent fibers in the SON, which arise from A2 neurons in the

caudal nucleus tractus solitarii, were observed by light microscopic anterograde and retrograde transport methods [5, 7, 22]. We have shown, by anterograde tracing plus immunocytochemistry, that VP-containing neurosecretory neurons in the PVN receive monosynaptic input from medullary A1/C1 cells which probably represent a catecholaminergic neuron system [20]. In conclusion, our results provide morphological evidence that VP-containing neurons in the SON receive synaptic input from NA-containing neurons. We thank Dr. A. Simpson for help with the manuscript. This study was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture of Japan.

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Noradrenergic innervation of vasopressin-containing neurons in the rat hypothalamic supraoptic nucleus.

The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron micr...
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