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Neurons in the caudal ventrolateral medulla projecting to the paraventricular hypothalamic nucleus receive synaptic inputs from the nucleus of the solitary tract: a light and electron microscopic double-labeling study in the rat
ELSEVIER
Neuroscience Letters 218 (1996) 33-36
N[HOSCIOiC[ IETT[IlS
Neurons in the caudal ventrolateral medulla projecting to the paraventricular hypothalamic nucleus receive synaptic inputs from the nucleus of the solitary tract: a light and electron microscopic double-labeling study in the rat Hitoshi Kawano*,
Sadahiko Masuko
Department of Anatomy, Saga Medical School, Nabeshima 5-1-1, Saga 849, Japan Received 16 May 1996; revised version received 20 September 1996; accepted 21 September 1996
Abstract
We used light and electron microscopic techniques to investigate the possibility that neurons in the nucleus of the solitary tract (NST) might send projection fibers to neurons in the caudal ventrolateral medulla (CVLM) projecting to the paraventricular hypothalamic nucleus (PVN) by the anterograde and retrograde double labeling method in the rat. The retrograde tracer, wheat germ agglutininconjugated horseradish peroxidase-colloidal gold complex, was injected into the PVN, and the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the NST of each rat. Many retrogradely labeled neurons were encountered in the CVLM, including the A1 region. On the other hand, a dense network of anterogradely labeled terminals was found in the CVLM. Electron microscopic examination revealed synaptic contacts between PHA-L-immunoreactive nerve terminals and dendrites of retrogradely labeled neurons in the CVLM. The results indicate that CVLM neurons projecting to the PVN receive axon terminals of NST neurons. Keywords." Caudal ventrolateral medulla; Nucleus of the solitary tract; Paraventricular hypothalamic nucleus; Phaseolus vulgaris leucoagglutinin; Wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex; Electron microscopy; Rat
The activity of vasopressin (VP)-secreting neurons in the paraventricular nucleus of the hypothalamus (PVN) is enhanced by stimulation of the A 1 region in the caudal ventrolateral medulla oblongata (CVLM) [2], and it has been demonstrated morphologically that CVLM neurons send projection fibers to the PVN [1,4,9-12]. Direct projections from the nucleus of the solitary tract (NST) to the A1 region or C V L M have also been shown by the tracttracing methods [11,13]. However, synaptic contacts between axon terminals of NST neurons and CVLM neurons projecting to the PVN have not been demonstrated thus far. Accordingly, in the present study we used light and electron microscopic examinations to determine whether NST neurons might send projection fibers to CVLM neurons sending projection fibers to the PVN. Male Sprague-Dawley rats (body weight 2 2 0 - 2 6 0 g, * Corresponding author. Tel.: +81 952 316511, ext. 2223; fax: +81 952 332518; e-mail: [email protected]
n = 14), anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight), were injected stereotaxically with a small quantity (0.3 #1) of wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex (WGA-HRP-gold) into the left PVN through a glass micropipette with an outer diameter of about 5 0 / z m [5,6]. In ten of these rats, 2.5% Phaseolus vulgaris leucoagglutinin (PHA-L; Vector, CA, USA) in 50 mM Tris buffer (pH 7.6) was also injected iontophoretically (2-5 ~zA positive current, for 2 0 - 3 0 rain) into the ipsilateral medial subnucleus of the NST at the level of the area postrema. Fourteen days after the WGA-HRP-gold injection, the rats were deeply reanesthetized and perfused through the ascending aorta with physiological saline containing heparin sodium (1000 IU/1), followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). The brains were removed, placed in the same fixative without glutaraldehyde for 2 - 2 4 h, and saturated with 20% sucrose
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13115-3
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
34
in phosphate-buffered saline (PBS). The brains were frozen by immersion in isopentane pre-cooled with acetonedry ice, and then cut serially into 40 /zm thick frontal sections on a freezing microtome. The sections were divided into two series of alternate serial sections. To verify the injection site of WGA-HRP-gold, the sections through the hypothalamus were mounted on gelatin-coated glass slides. Retrograde labeling was observed in free-floating sections through the medulla oblongata by a silver-intensification method [6], which was modified from the method of Lah et al. [7]. The sections were then fixed with 2.5% sodium thiosulfate in 0.02 M HEPES buffer (pH 6.8). The silver intensified sections were soaked in 0.3% H202
in PBS for 10 min to block activities of peroxidase and processed for immunohistochemistry. After preincubation with 2% normal swine serum plus 0.3% Triton X-100 in PBS for 1 h (also used as the diluent of all antisera employed), the sections were sequentially incubated with goat anti-PHA-L (Vector, Lot: 70917; diluted 1:2000) overnight at 4°C, fluorescein isothiocyanate-conjugated swine anti-goat IgG (Dakopatts, Glostrup, Denmark; diluted 1:40) for 1.5 h at room temperature, and goat peroxidase anti-peroxidase complex (PAP; Dakopatts; diluted 1:80) for 2 h at room temperature and then reacted with 0.02% diaminobenzidine-tetrahydrochloride (Dotite, Kumamoto, Japan) in 0.05 M Tris buffered saline (pH 7.6) containing 0.005% H202 for 15-30 min. After the
B Fig. 1. (A) An injection site of WGA-HRP-gold within the PVN. v, Third ventricle. (B) Arrows indicate retrogradely labeled neurons in the CVLM at a level 320 ~m rostral to the obex. Darkfield illumination. (C) An injection site of PHA-L in the medial subnucleus of the NST at a level of the area postrema (200/zm caudal to the obex). The deposit of PHA-L is almost restricted within the medial subnucleus. Fluorescence immunohistochemistry, a, Area postrema; c, central canal; h, hypoglossal nucleus; t, solitary tract; x, dorsal motor nucleus of the vagus. (D) A dense network of PHA-Limmunoreactive fibers in the CVLM at a level about 280/~m rostral to the obex. PAP method. Bars (A-D) 100/zm.
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
incubation with PAP, some sections were photographed under a fluorescence microscope to verify the injection site of PHA-L. For electron microscopic examination, the sections which were subjected to silver-intensification and immunostaining for PHA-L were incubated with 0.05% chloroauric acid in 0.1 M phosphate buffer (pH 7.3) for 10 rain in an ice-cold bath to tone up the retrograde labeling, postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer, dehydrated, and then embedded in Spurr's resin (Nisshin EM, Tokyo, Japan). Ultrathin sections were prepared on a Sorvall MT2-B ultra microtome, and then observed under a JEOL 100C x electron microscope without metal staining. Colocalization of tyrosine hydroxylase (TH) immunoreactivity in the retrogradely labeled neurons was also examined. Some silver-intensified sections for light microscopy were immunostained for TH using mouse monoclonal antibody to TH [3], goat anti-mouse IgG (CooperBiochemical, PA, USA; diluted 1:40) and mouse PAP (Dakopatts; diluted 1:80). After injection of WGA-HRP-gold into the PVN (Fig. 1A), retrogradely labeled neurons were found in many regions of the brain, including the median preoptic nucleus, the subfornical organ, the NST and the ventrolateral medulla oblongata (VLM; Fig. 1B), as reported previously [1,4-6,9-12]. Retrogradely labeled neurons in the VLM were distributed throughout the rostrocaudal levels with an apparent ipsilateral dominance; the majority of these were distributed at levels more caudal than the level 500 ~m rostral to the obex. At these levels, about 60% of the retrogradely labeled neurons showed TH immunoreactivity. Under an electron microscope, retrogradely transported tracer was found in lysosomes in the cell bodies and dendrites of labeled neurons. In the rats injected with PHA-L into the medial subnucleus of the NST at a level of the area postrema (Fig. 1C), a dense network of anterogradely labeled PHA-L-immunoreactive fibers was found in the VLM (Fig. ID), especially at levels 300-400/~m rostral to the injection. The distribution area of PHA-L-immunoreactive fibers overlapped with that of neurons retrogradely labeled with WGAHRP-gold injected into the PVN (Fig. 1B,D). Axon terminals showing PHA-L immunoreactivity contained many small clear vesicles and a few large cored vesicles. About 8% of PHA-L-immunoreactive axon terminals in the VLM were in synaptic contact with dendritic profiles. Although both asymmetric and symmetric synapses were encountered, the former were seen more frequently. No PHA-Limmunoreactive axon terminals were observed to make axo-somatic or axo-axonic synapses. Some PHA-L-immunoreactive axon terminals were in synaptic contact with dendritic profiles labeled with WGA-HRP-gold (Fig. 2); most of these synapses were of the asymmetric type showing a thickening of the postsynaptic membrane. The present results indicate that axon terminals of NST neurons made synaptic contacts with VLM neurons pro-
35
Fig. 2. A PHA-L-immunoreactiveaxon terminal (asterisk) in an asym metric synaptic contact with a dendritic profile of a retrogradely labeled neuron. A lysosome which contains retrogradely transported WGAHRP-gold (intensified by silver treatment) is indicated by the arrow. Bar, 0.5 /~m. jecting to the PVN. The previous tract-tracing studies combined with immunohistochemistry indicated that almost all CVLM neurons projecting to the PVN exhibited TH- and dopamine /3-hydroxylase-immunoreactivities [1,11,12]. The majority of the retrogradely labeled VLM neurons observed in the present study also showed TH immunoreactivity. These TH-immunoreactive VLM neurons were considered to be A1 noradrenergic neurons in the CVLM. It has been reported that some populations of NST neurons sending projection fibers to the CVLM show glutamate immunoreactivity [13], and that glutamateimmunoreactive axon terminals usually make asymmetric synapses [8]. In the present study, all synapses between axon terminals of NST neurons and CVLM neurons projecting to the PVN were of the asymmetric type. Thus, it is possible that glutamatergic neurons in the NST exert excitatory influences upon VLM neurons projecting to the PVN. [1] Cunningham,E.T. Jr. and Sawchenko, P.E,, Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypotfialamus, J. Comp. NeuroL, 274 (1988) 60-76. [2] Day, T.A., Ferguson, A.V. and Renaud, L.P., Facilitatory influence
36
[3]
[4]
[5]
[6]
[7]
[8]
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol., 355 (1984) 237-249. Hatanaka, H. and Arimatsu, Y., Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12h ceils with special reference to nerve growth factor-mediated increase of the immunoprecipitable enzymes, Neurosci. Res., 1 (1987) 253-267. Kawano, H. and Masuko, S., Origins of neuropeptide-containing nerve terminals in the hypothalamic paraventricular nucleus, Acta Anat. Nippon. (Abstract), 65 (1990) 231. Kawano, H. and Masuko, S., Synaptic inputs of neuropeptide-Yimmunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohistochemical and retrograde tracing method, Brain Res., 600 (1993) 74-80. Kawano, H. and Masuko, S., Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius, Brain Res., 689 (1995) 136-140. Lah, J.J., Hayes, D.M. and Burry, R.W., A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry, J. Histochem. Cytochem., 38 (1990) 503-508. Maxwell, D.J., Christie, W.M., Brown, A.G., Ottersen, O.P. and
[9]
[10]
[11]
[12]
[13]
Storm-Mathisen, J., Direct observations of synapses between Lglutamate-immunoreactive boutons and identified spinocervical tract neurones in the spinal cord of the cat, J. Comp. Neurol., 326 (1992) 485-500. Moga, M.M. and Saper, C.B., Neuropeptide-immunoreactive neurons projecting to the paraventricular hypothalamic nucleus in the rat, J. Comp. Neurol., 346 (1994) 137-150. Sawchenko, P.E., Arias, C. and Bittencourt, J.C., Inhibin/3, somatostatin, and enkephalin immunoreactivities coexist in caudal medullary neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol., 291 (1990) 269-280. Sawchenko, P.E. and Swanson, L.W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. Sawchenko, P.E., Swanson, L.W., Grzanna, R., Howe, P.R.C., Bloom, S.R. and Polak, J.M., Colocalization of neuropeptide Y immunoreactivity in the brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol., 241 (1985) 138-153. Suzuki, T., Takayama, K. and Miura, M., Distribution of glutamate- and GABA-containing neurons in the nucleus tractus solitarii projecting to the depressor area of the caudal ventrolateral medulla, Neurosci. Res. Suppl. (Abstract), 19 (1994) $113.
Neuroscience Letters 218 (1996) 33-36
N[HOSCIOiC[ IETT[IlS
Neurons in the caudal ventrolateral medulla projecting to the paraventricular hypothalamic nucleus receive synaptic inputs from the nucleus of the solitary tract: a light and electron microscopic double-labeling study in the rat Hitoshi Kawano*,
Sadahiko Masuko
Department of Anatomy, Saga Medical School, Nabeshima 5-1-1, Saga 849, Japan Received 16 May 1996; revised version received 20 September 1996; accepted 21 September 1996
Abstract
We used light and electron microscopic techniques to investigate the possibility that neurons in the nucleus of the solitary tract (NST) might send projection fibers to neurons in the caudal ventrolateral medulla (CVLM) projecting to the paraventricular hypothalamic nucleus (PVN) by the anterograde and retrograde double labeling method in the rat. The retrograde tracer, wheat germ agglutininconjugated horseradish peroxidase-colloidal gold complex, was injected into the PVN, and the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the NST of each rat. Many retrogradely labeled neurons were encountered in the CVLM, including the A1 region. On the other hand, a dense network of anterogradely labeled terminals was found in the CVLM. Electron microscopic examination revealed synaptic contacts between PHA-L-immunoreactive nerve terminals and dendrites of retrogradely labeled neurons in the CVLM. The results indicate that CVLM neurons projecting to the PVN receive axon terminals of NST neurons. Keywords." Caudal ventrolateral medulla; Nucleus of the solitary tract; Paraventricular hypothalamic nucleus; Phaseolus vulgaris leucoagglutinin; Wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex; Electron microscopy; Rat
The activity of vasopressin (VP)-secreting neurons in the paraventricular nucleus of the hypothalamus (PVN) is enhanced by stimulation of the A 1 region in the caudal ventrolateral medulla oblongata (CVLM) [2], and it has been demonstrated morphologically that CVLM neurons send projection fibers to the PVN [1,4,9-12]. Direct projections from the nucleus of the solitary tract (NST) to the A1 region or C V L M have also been shown by the tracttracing methods [11,13]. However, synaptic contacts between axon terminals of NST neurons and CVLM neurons projecting to the PVN have not been demonstrated thus far. Accordingly, in the present study we used light and electron microscopic examinations to determine whether NST neurons might send projection fibers to CVLM neurons sending projection fibers to the PVN. Male Sprague-Dawley rats (body weight 2 2 0 - 2 6 0 g, * Corresponding author. Tel.: +81 952 316511, ext. 2223; fax: +81 952 332518; e-mail: [email protected]
n = 14), anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight), were injected stereotaxically with a small quantity (0.3 #1) of wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex (WGA-HRP-gold) into the left PVN through a glass micropipette with an outer diameter of about 5 0 / z m [5,6]. In ten of these rats, 2.5% Phaseolus vulgaris leucoagglutinin (PHA-L; Vector, CA, USA) in 50 mM Tris buffer (pH 7.6) was also injected iontophoretically (2-5 ~zA positive current, for 2 0 - 3 0 rain) into the ipsilateral medial subnucleus of the NST at the level of the area postrema. Fourteen days after the WGA-HRP-gold injection, the rats were deeply reanesthetized and perfused through the ascending aorta with physiological saline containing heparin sodium (1000 IU/1), followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). The brains were removed, placed in the same fixative without glutaraldehyde for 2 - 2 4 h, and saturated with 20% sucrose
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13115-3
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
34
in phosphate-buffered saline (PBS). The brains were frozen by immersion in isopentane pre-cooled with acetonedry ice, and then cut serially into 40 /zm thick frontal sections on a freezing microtome. The sections were divided into two series of alternate serial sections. To verify the injection site of WGA-HRP-gold, the sections through the hypothalamus were mounted on gelatin-coated glass slides. Retrograde labeling was observed in free-floating sections through the medulla oblongata by a silver-intensification method [6], which was modified from the method of Lah et al. [7]. The sections were then fixed with 2.5% sodium thiosulfate in 0.02 M HEPES buffer (pH 6.8). The silver intensified sections were soaked in 0.3% H202
in PBS for 10 min to block activities of peroxidase and processed for immunohistochemistry. After preincubation with 2% normal swine serum plus 0.3% Triton X-100 in PBS for 1 h (also used as the diluent of all antisera employed), the sections were sequentially incubated with goat anti-PHA-L (Vector, Lot: 70917; diluted 1:2000) overnight at 4°C, fluorescein isothiocyanate-conjugated swine anti-goat IgG (Dakopatts, Glostrup, Denmark; diluted 1:40) for 1.5 h at room temperature, and goat peroxidase anti-peroxidase complex (PAP; Dakopatts; diluted 1:80) for 2 h at room temperature and then reacted with 0.02% diaminobenzidine-tetrahydrochloride (Dotite, Kumamoto, Japan) in 0.05 M Tris buffered saline (pH 7.6) containing 0.005% H202 for 15-30 min. After the
B Fig. 1. (A) An injection site of WGA-HRP-gold within the PVN. v, Third ventricle. (B) Arrows indicate retrogradely labeled neurons in the CVLM at a level 320 ~m rostral to the obex. Darkfield illumination. (C) An injection site of PHA-L in the medial subnucleus of the NST at a level of the area postrema (200/zm caudal to the obex). The deposit of PHA-L is almost restricted within the medial subnucleus. Fluorescence immunohistochemistry, a, Area postrema; c, central canal; h, hypoglossal nucleus; t, solitary tract; x, dorsal motor nucleus of the vagus. (D) A dense network of PHA-Limmunoreactive fibers in the CVLM at a level about 280/~m rostral to the obex. PAP method. Bars (A-D) 100/zm.
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
incubation with PAP, some sections were photographed under a fluorescence microscope to verify the injection site of PHA-L. For electron microscopic examination, the sections which were subjected to silver-intensification and immunostaining for PHA-L were incubated with 0.05% chloroauric acid in 0.1 M phosphate buffer (pH 7.3) for 10 rain in an ice-cold bath to tone up the retrograde labeling, postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer, dehydrated, and then embedded in Spurr's resin (Nisshin EM, Tokyo, Japan). Ultrathin sections were prepared on a Sorvall MT2-B ultra microtome, and then observed under a JEOL 100C x electron microscope without metal staining. Colocalization of tyrosine hydroxylase (TH) immunoreactivity in the retrogradely labeled neurons was also examined. Some silver-intensified sections for light microscopy were immunostained for TH using mouse monoclonal antibody to TH [3], goat anti-mouse IgG (CooperBiochemical, PA, USA; diluted 1:40) and mouse PAP (Dakopatts; diluted 1:80). After injection of WGA-HRP-gold into the PVN (Fig. 1A), retrogradely labeled neurons were found in many regions of the brain, including the median preoptic nucleus, the subfornical organ, the NST and the ventrolateral medulla oblongata (VLM; Fig. 1B), as reported previously [1,4-6,9-12]. Retrogradely labeled neurons in the VLM were distributed throughout the rostrocaudal levels with an apparent ipsilateral dominance; the majority of these were distributed at levels more caudal than the level 500 ~m rostral to the obex. At these levels, about 60% of the retrogradely labeled neurons showed TH immunoreactivity. Under an electron microscope, retrogradely transported tracer was found in lysosomes in the cell bodies and dendrites of labeled neurons. In the rats injected with PHA-L into the medial subnucleus of the NST at a level of the area postrema (Fig. 1C), a dense network of anterogradely labeled PHA-L-immunoreactive fibers was found in the VLM (Fig. ID), especially at levels 300-400/~m rostral to the injection. The distribution area of PHA-L-immunoreactive fibers overlapped with that of neurons retrogradely labeled with WGAHRP-gold injected into the PVN (Fig. 1B,D). Axon terminals showing PHA-L immunoreactivity contained many small clear vesicles and a few large cored vesicles. About 8% of PHA-L-immunoreactive axon terminals in the VLM were in synaptic contact with dendritic profiles. Although both asymmetric and symmetric synapses were encountered, the former were seen more frequently. No PHA-Limmunoreactive axon terminals were observed to make axo-somatic or axo-axonic synapses. Some PHA-L-immunoreactive axon terminals were in synaptic contact with dendritic profiles labeled with WGA-HRP-gold (Fig. 2); most of these synapses were of the asymmetric type showing a thickening of the postsynaptic membrane. The present results indicate that axon terminals of NST neurons made synaptic contacts with VLM neurons pro-
35
Fig. 2. A PHA-L-immunoreactiveaxon terminal (asterisk) in an asym metric synaptic contact with a dendritic profile of a retrogradely labeled neuron. A lysosome which contains retrogradely transported WGAHRP-gold (intensified by silver treatment) is indicated by the arrow. Bar, 0.5 /~m. jecting to the PVN. The previous tract-tracing studies combined with immunohistochemistry indicated that almost all CVLM neurons projecting to the PVN exhibited TH- and dopamine /3-hydroxylase-immunoreactivities [1,11,12]. The majority of the retrogradely labeled VLM neurons observed in the present study also showed TH immunoreactivity. These TH-immunoreactive VLM neurons were considered to be A1 noradrenergic neurons in the CVLM. It has been reported that some populations of NST neurons sending projection fibers to the CVLM show glutamate immunoreactivity [13], and that glutamateimmunoreactive axon terminals usually make asymmetric synapses [8]. In the present study, all synapses between axon terminals of NST neurons and CVLM neurons projecting to the PVN were of the asymmetric type. Thus, it is possible that glutamatergic neurons in the NST exert excitatory influences upon VLM neurons projecting to the PVN. [1] Cunningham,E.T. Jr. and Sawchenko, P.E,, Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypotfialamus, J. Comp. NeuroL, 274 (1988) 60-76. [2] Day, T.A., Ferguson, A.V. and Renaud, L.P., Facilitatory influence
36
[3]
[4]
[5]
[6]
[7]
[8]
H. Kawano, S Masuko / Neuroscience Letters 218 (1996) 33-36
of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol., 355 (1984) 237-249. Hatanaka, H. and Arimatsu, Y., Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12h ceils with special reference to nerve growth factor-mediated increase of the immunoprecipitable enzymes, Neurosci. Res., 1 (1987) 253-267. Kawano, H. and Masuko, S., Origins of neuropeptide-containing nerve terminals in the hypothalamic paraventricular nucleus, Acta Anat. Nippon. (Abstract), 65 (1990) 231. Kawano, H. and Masuko, S., Synaptic inputs of neuropeptide-Yimmunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohistochemical and retrograde tracing method, Brain Res., 600 (1993) 74-80. Kawano, H. and Masuko, S., Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius, Brain Res., 689 (1995) 136-140. Lah, J.J., Hayes, D.M. and Burry, R.W., A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry, J. Histochem. Cytochem., 38 (1990) 503-508. Maxwell, D.J., Christie, W.M., Brown, A.G., Ottersen, O.P. and
[9]
[10]
[11]
[12]
[13]
Storm-Mathisen, J., Direct observations of synapses between Lglutamate-immunoreactive boutons and identified spinocervical tract neurones in the spinal cord of the cat, J. Comp. Neurol., 326 (1992) 485-500. Moga, M.M. and Saper, C.B., Neuropeptide-immunoreactive neurons projecting to the paraventricular hypothalamic nucleus in the rat, J. Comp. Neurol., 346 (1994) 137-150. Sawchenko, P.E., Arias, C. and Bittencourt, J.C., Inhibin/3, somatostatin, and enkephalin immunoreactivities coexist in caudal medullary neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol., 291 (1990) 269-280. Sawchenko, P.E. and Swanson, L.W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. Sawchenko, P.E., Swanson, L.W., Grzanna, R., Howe, P.R.C., Bloom, S.R. and Polak, J.M., Colocalization of neuropeptide Y immunoreactivity in the brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus, J. Comp. Neurol., 241 (1985) 138-153. Suzuki, T., Takayama, K. and Miura, M., Distribution of glutamate- and GABA-containing neurons in the nucleus tractus solitarii projecting to the depressor area of the caudal ventrolateral medulla, Neurosci. Res. Suppl. (Abstract), 19 (1994) $113.