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Synaptic contacts between nerve terminals originating from the ventrolateral medullary catecholaminergic area and median preoptic neurons projecting to the paraventricular hypothalamic nucleus
Brain Research 817 Ž1999. 110–116
Research report
Synaptic contacts between nerve terminals originating from the ventrolateral medullary catecholaminergic area and median preoptic neurons projecting to the paraventricular hypothalamic nucleus Hitoshi Kawano ) , Sadahiko Masuko Department of Anatomy, Saga Medical School, Saga 849-8501, Japan Accepted 10 November 1998
Abstract A significant role of catecholaminergic projection to the median preoptic nucleus ŽPOMe. which activates vasopressin-producing cells of the paraventricular hypothalamic nucleus ŽPVN. has been suggested. We investigated the existence of the synaptic contacts between catecholaminergic fibers from the ventrolateral medulla and the POMe neurons projecting to the PVN. Rats received a retrograde tracer in the PVN and subsequently an anterograde tracer into the catecholaminergic area of the ventrolateral medulla at the level of the area postrema. In the POMe, anterogradely labeled nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts onto retrogradely labeled neurons. Additional studies in which a retrograde tracer was injected into the POMe revealed that almost all retrogradely labeled neurons in the ventrolateral medulla at the level of the area postrema were immunoreactive to tyrosine hydroxylase, suggesting that projection to the POMe from the ventrolateral medulla is largely limited to catecholamine neurons. These results provide, for the first time, direct evidence that catecholaminergic inputs from the ventrolateral medulla affect POMe neurons projecting to the PVN by way of direct synaptic contact. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Median preoptic nucleus; Paraventricular hypothalamic nucleus; Ventrolateral medulla; Catecholamine; Anterograde and retrograde tracing; Rat
1. Introduction The median preoptic nucleus ŽPOMe., a lamina terminalis-associated structure, is situated at the anterior wall of the third ventricle and is a member of the anteroventral third ventricle ŽAV3V. region w13x. A rise in mean arterial pressure induced by intravenous injection of angiotensin II ŽA-II. increases the firing frequency of the POMe neurons w7x. Electrical and neurotoxigenic lesions of the ventral AV3V region, including the POMe, block the increase in water intake, pressor and natri- as well as kaliuretic responses induced by systematically or locally applied chemical agents, such as A-II or hypertonic saline w3,5,8,21x. These physiological data indicate that the POMe is critical for the regulation of water and ion balance and cardiovascular control.
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Electrical stimulation of the POMe or injection of hypertonic saline into the POMe increases activity of neuroendocrine cells in the paraventricular hypothalamic nucleus ŽPVN. w12x, while electrical lesions of the POMe block the hypertonic saline-induced increase in plasma vasopressin ŽVP. level w8x. POMe neurons projecting to the PVN are excited under several conditions, which include stimulation of the subfornical organ ŽSFO., injection of A-II into the POMe or the SFO, intravenous injection of hypertonic saline and stimulation of the A1 catecholaminergic area w29,30,32,34x. When A-II antagonist is injected into the POMe or a knife cut is made between the SFO and POMe, several responses, such as A-II-induced drinking as well as A-II-induced or SFO stimulation-induced excitation of PVN neurons, are blocked w17,27,31x. Furthermore, microinjection of local anesthetic lidocaine into the POMe abolishes the excitatory responses with long duration of putative VP-producing PVN cells to electrical stimulation of the SFO w33x. Morphological studies have demonstrated the existence of projections from the SFO to the POMe
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H. Kawano, S. Masukor Brain Research 817 (1999) 110–116
w9,20,22,23x, which contain A-II immunoreactivity w18,19x, and of projections from the POMe to the magnocellular PVN w11,15,16,25,26x. Thus, it is likely that POMe neurons, being sensitive to A-II, receive angiotensinergic afferent fibers originating from the SFO and, in turn, project to the PVN to stimulate VP-producing cells. On the other hand, the POMe receives many catecholaminergic nerve terminals w14,16x. When noradrenaline or adrenaline nerve terminals in the POMe are selectively destroyed by 6-hydroxydopamine, A-II-induced drinking and pressor responses are blocked w1,2,4x. It has also been demonstrated that reduced extracellular fluid volume by subcutaneously applied polyethylene glycol increases noradrenaline release in the POMe region w28,36x. Previous studies have shown that ventrolateral medullary neurons retrogradely labeled from the POMe are immunoreactive to tyrosine hydroxylase ŽTH. and to phenylethanolamine N-methyltransferase ŽPNMT., catecholamine synthesizing enzymes used as a marker for all catecholamines and adrenaline, respectively, suggesting projections to the POMe from both the A1 and the C1 catecholamine groups w24,35x. It has not been clarified as yet, whether nerve terminals of these catecholamine neurons have a direct effect by synaptic contacts onto POMe neurons projecting to the PVN, or an indirect effect via interneurons or by presynaptic interaction with nerve terminals derived from the SFO. The present study was designed to investigate these issues electron microscopically, using a double tract tracing method, in which anterograde and retrograde tracers were injected into the catecholaminergic area of the ventrolateral medulla and the PVN, respectively. The existence of the catecholaminergic projection from the ventrolateral medulla to the POMe was also confirmed by a double labeling experiment for retrograde labeling and TH immunoreactivity in the ventrolateral medullary neurons after injection of a retrograde tracer into the POMe.
2. Materials and methods Male Sprague–Dawley rats Ž49 " 1 days old, n s 30., anesthetized by intraperitoneal injection of pentobarbital sodium Ž50 mgrkg b.wt.. before surgical procedures and sacrifice, were used. The rats received a stereotaxical injection of a small quantity Ž0.3 ml. of a retrograde tracer, wheat germ agglutinin–conjugated horseradish peroxidase–colloidal gold complex ŽWGA–HRP–gold. w15x, into the left PVN from the dorsal aspect through a glass micropipette with an outer diameter of about 50 mm w15,16x. Subsequently, the animals received an iontophoretical injection of an anterograde tracer, biotinylated dextran amine ŽBDX; Molecular Probes, OR, USA; 10% in 0.05 M Tris buffer, pH 7.6; 2–5 mA positive current, for 20–30 min. into the ipsilateral ventrolateral medulla at the level of the area postrema. Ten to fourteen days after the
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injections, the rats were perfused through the ascending aorta with physiological saline containing heparin sodium Ž1000 IUrl., followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer ŽpH 7.3.. The brains were removed and fixed by immersion in the same fixative without glutaraldehyde for 2–24 h, and 20 mm thick frontal sections through the entire rostrocaudal extent of the POMe were serially cut on a microslicer ŽD.S.K., Kyoto, Japan.. Retrograde labeling was detected in the free-floating sections through the POMe, using a silver-intensification method described previously w15,16x. The silver intensified POMe sections were then processed for immunohistochemical detection of the anterograde tracer, after being soaked in 0.3% H 2 O 2 in PBS for 10 min to block activities of peroxidase. The sections were incubated first with streptavidin–Texas Red conjugate ŽGibco BRL, Gaithersburg, MD, USA; diluted 1:100., then biotinylated streptoavidin–peroxidase complex ŽVector, Burlingame, CA, USA; diluted 1:1000., and reacted with 0.02% diaminobenzidine tetrahydrochloride ŽDAB; Dotite, Kumamoto, Japan. in Tris buffered saline ŽpH 7.6. containing 0.005% H 2 O 2 for 15–30 min. Finally, the sections were incubated with 0.05% chloroauric acid in 0.1 M phosphate buffer ŽpH 7.3. for 10 min in an ice-cold bath to tone up the retrograde labeling and DAB reaction products, postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer, dehydrated, and embedded in Spurr’s resin ŽNisshin EM, Tokyo, Japan.. Ultrathin sections of the POMe were prepared on a Sorvall MT2-B Ultra Microtome, and observed under a JEOL 100CX electron microscope without metal staining. Only those rats in which both the retrograde and anterograde tracers were successfully injected into the PVN and the ventrolateral medulla, respectively, were used. To verify the injection site of WGA–HRP–gold in the PVN, 40 mm thick frontal sections through the hypothalamus were cut on a freezing microtome, mounted on gelatin-coated glass slides, counterstained with hematoxylin, and observed under a light microscope. To verify the injection site of BDX in the catecholaminergic areas of the ventrolateral medulla, 40 mm thick frontal sections through the medulla were cut on a freezing microtome and every sixth section was processed for double labeling for TH immunoreaction and BDX. Sections were sequentially incubated with mouse monoclonal antibody to TH w10x, and a cocktail containing fluorescein isothiocyanate-labeled swine anti-mouse IgG ŽNordic Immunological Laboratories, Tilburg, The Netherlands; diluted 1:100. and streptoavidin–Texas Red conjugate, and observed under a fluorescent microscope. To verify the anterograde tracing from the catecholaminergic area of the caudal ventrolateral medulla to the POMe, experiments with retrograde tracer injected into the POMe were also performed. Three rats received a stereotaxical injection of WGA–HRP–gold into the POMe
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and were perfused with 4% paraformaldehyde in phosphate buffer ŽpH 7.3. after a 2- to 7-day survival. Medullary sections of 40 mm were obtained and processed for double labeling. Sections were first silver intensified to reveal retrogradely labeled neurons and then immunostained for TH using mouse monoclonal antibody to TH w10x, swine anti-mouse IgG ŽNordic Immunological Laboratories; diluted 1:100. and mouse peroxidase anti-peroxidase complex ŽDAKOPATTS, Glostrup, Denmark; 1:100. followed by visualization with the DAB reaction. To investigate immunoreactivity for noradrenaline and PNMT, two other rats were fixed by perfusion of 5% glutaraldehyde in 0.1 M cacodylate buffer ŽpH 7.4. and 20 mm thick free-floating sections were obtained on a freezing microtome. Sections were immunostained using rabbit polyclonal anti-noradrenaline ŽSFRI Laboratoire, Martignas, France; diluted 1:4000. or anti-PNMT ŽEugene Tech International, Ridgefield Park, NJ, USA; diluted 1:1000., unlabeled swine anti-rabbit IgG ŽDAKOPATTS; diluted 1:40. and rabbit PAP ŽDAKOPATTS; diluted 1:80.. The details for noradrenaline immunohistochemistry have been described elsewhere w14,16x and this procedure was also applied to PNMT immunohistochemistry.
3. Results The POMe contained both noradrenaline- and PNMTimmunoreactive nerve terminals in the entire nucleus ŽFig. 1., especially in the ventral part. The density of noradrenaline-immunoreactive nerve terminals was similar to that of PNMT-immunoreactive nerve terminals in the rostral half of the nucleus, while a few more noradrenaline- than PNMT-immunoreactive nerve terminals were found in the caudal half. In the experiments of retrograde tracing from the PVN, each injection site of the WGA–HRP–gold was mainly confined within the magnocellular and extended somewhat to the parvocellular parts of the PVN ŽFig. 2a.. After these injections, retrogradely labeled neurons were found throughout the POMe, especially in the ventral part of the nucleus at the middle level ŽFig. 2b.. In the anterograde experiments, the injection site of the BDX was located in the ventrolateral medullary catecholaminergic areas, where TH-immunoreactive neurons were clustered ŽFig. 3a,b.. Anterogradely labeled terminals were observed in the POMe and many other brain regions including the medial preoptic area and the PVN. In the
Fig. 1. Serial sections showing immunoreactivity for noradrenaline Ža. and PNMT Žb. in the POMe at the middle level Žabout 60 mm rostral to the crossing of the anterior commissure.. Both immunoreactive nerve varicosities are seen in abundance in the ventral part of the nucleus. More noradrenaline- than PNMT-immunoreactive nerve varicosities are present. Demarcation of the nucleus is indicated by broken lines. v: third ventricle. Bar s 50 mm Ža, b..
H. Kawano, S. Masukor Brain Research 817 (1999) 110–116
Fig. 2. Ža. Injection site of WGA–HRP–gold in the PVN. Deposit of the retrograde tracer is mainly restricted in the magnocellular part and somewhat in the parvocellular part of the nucleus. Demarcation of the nucleus is indicated by a broken line. Žb. Darkfield illumination of the POMe sectioned through the middle level Žabout 180 mm rostral to the crossing of the anterior commissure. of the nucleus showing retrograde labeling after the injection of WGA–HRP–gold into the PVN shown in Ža.. Many bright retrogradely labeled neurons are seen in the nucleus bilaterally. v: third ventricle. Barss100 mm Ža, b..
POMe, anterogradely labeled terminals were found throughout the nucleus, especially in the ventral part of the caudal two thirds of the nucleus ŽFig. 3c.. Electron microscopy revealed that the anterogradely labeled axon terminals in the POMe were round, ovoid and ellipsoid in shape, and contained many small clear round vesicles and some large cored vesicles. These terminals made synaptic contacts onto dendrites and cell bodies in the POMe. Both asymmetric and symmetric synapses were encountered. Among these, synaptic contacts were found between the anterogradely labeled axon terminals and dendrites or cell bodies of the POMe neurons containing retrograde labelings in the lysosomes ŽFig. 4.. Hereupon,
Fig. 3. Ža, b. An identical section of the ventrolateral medulla showing injection site of biotinylated dextran amine Ža. and TH immunoreactivity Žb. under a fluorescence microscope. The level of the injection is the area postrema and corresponds to that shown in Fig. 5b. The location of the anterograde tracer corresponds fairly well to the cluster of TH-immunoreactive neurons, and some neurons in the vicinity of the injection site are labeled for both Žarrowheads.. Žc. Anterogradely labeled afferent nerve varicosities in the POMe after the injection of biotinylated dextran amine into the catecholaminergic area of the ventrolateral medulla shown in Ža.. Dense, bilateral distribution of the labeled varicosities are mainly found in the ventral part of the nucleus. The rostrocaudal level of the section corresponds to that shown in Fig. 1. Barss 50 mm Ža–c..
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both the asymmetric and symmetric synapses as well as both the axo-dendritic and axo-somatic synapses were observed. The retrogradely labeled postsynaptic cell bodies and thick dendrites which make synapses with anterogradely labeled nerve terminals were also in synaptic contact with unlabeled nerve terminals ŽFig. 4.. After an injection of WGA–HRP–gold into the middle level of the rostrocaudal extent of the POMe ŽFig. 5a.,
retrogradely labeled neurons in the medulla oblongata were found in the ventrolateral regions and in the nucleus of the solitary tract. In the ventrolateral medulla, retrogradely labeled neurons were distributed bilaterally in levels between about 1400 mm caudal to the obex and 1900 mm rostral to the obex, occupying parts of catecholamine regions, with a peak number Žabout 10 neurons in a section. at the level of the area postrema. At these levels in which retrogradely labeled neurons were found, a substantial number of neurons were double-labeled for retrograde labeling and TH immunoreactivity. Doublelabeled neurons were more frequently observed at levels caudal to the obex Ž22 neurons in total, representing 12% of 181 TH-immunoreactive neurons and 75% of 29 retrogradely labeled neurons; mean of three rats, counted at intervals of 240 mm for each experiment. than at levels rostral to the obex Ž13 neurons in total, representing 7% of 186 TH-immunoreactive neurons and 52% of 25 retrogradely labeled neurons.. At the level of the area postrema, 16 neurons of 19 retrogradely labeled neurons Ž85%. showed TH immunoreactivity ŽFig. 5b,c..
4. Discussion
Fig. 4. Electron micrographs of the POMe showing asymmetric Ža. and symmetric Žb. synaptic contacts of an afferent axon terminal anterogradely labeled from the ventrolateral medulla onto a dendrite retrogradely labeled from the PVN. Note that an unlabeled axon terminal Žasterisk. is also in synaptic contact with the identical dendrite in Ža.. Retrograde labelings on the lysosomes are indicated by arrows. Barss 0.5 mm.
The present study, using an anterograde tracer, demonstrated, for the first time, that neurons in the catecholaminergic areas of the ventrolateral medulla at the level of the area postrema project to the POMe. Immunostaining for TH at the present injection sites of the anterograde tracer confirmed that the injections were made into the ventrolateral medullary catecholamine area. The retrograde experiments with the tracer injected into the POMe and subsequent immunohistochemistry for TH also revealed that almost all retrogradely labeled neurons exhibited TH immunoreactivity in the ventrolateral medulla at the level of the area postrema, the exact region where the anterograde tracer was applied in the present study. Thus, it is likely that almost all anterogradely labeled nerve terminals observed in the POMe are derived from the catecholamine neurons of the ventrolateral medulla. Using WGA–HRP– gold as a retrograde tracer, we clarified that about 10% of TH-immunoreactive neurons in the ventrolateral medulla projected to the middle level of the POMe. Since this tracer has minimal diffusion, the injection site was quite restricted to a small part of the POMe. Accordingly, the actual proportion of TH-immunoreactive projections from the ventrolateral medulla to the POMe must be higher. The existence of the catecholaminergic projections from the ventrolateral medulla to the POMe is generally in agreement with the previous reports w24,35x. These studies have used retrograde labeling and immunoreactivities for TH and PNMT to demonstrate that both noradrenaline and adrenaline neurons project to the POMe from the entire rostrocaudal ventrolateral medullary regions. Tucker et al.
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Fig. 5. Ža. Injection site of WGA–HRP–gold in the POMe at the middle level Žalmost the same, but slightly rostral to that shown in Fig. 2b.. Deposit of the retrograde tracer is nearly confined to the nucleus. Demarcation of the nucleus is indicated by a broken line. Žb. Medullary section showing retrogradely labeled neurons in the nucleus of the solitary tract Žarrowheads. and in the ventrolateral medulla Žrectangle. at a level of the area postrema Žap. after injection of WGA–HRP–gold into the POMe shown in Ža.. Žc. High power microgram of the rectangle in the section adjacent to Žb. after silver intensification and TH immunohistochemistry of the ventrolateral medullary region. Note that retrogradely labeled neurons containing granules of silver intensified gold are immunoreactive to TH Žarrowheads.. v: third ventricle. Bars s 100 mm Ža. and 50 mm Žb, c..
w35x have reported that many more adrenergic than noradrenergic neurons project to the POMe from the level of the area postrema, the level corresponding to the transition area from the A1 to the C1 area. However, the present study in which the injection of the anterograde tracer was made at this transition area showed a denser distribution of anterogradely labeled afferent terminals in the caudal twothirds of the POMe, where more noradrenaline- than PNMT-immunoreactive nerve terminals were found. Thus, it is conceivable that, although both the A1 and C1 catecholamine neurons project to the POMe, noradrenergic nerve terminals arborize more predominantly than adrenergic nerve terminals and are distributed mainly in the caudal two-thirds of the nucleus. The present study demonstrated synaptic contacts between POMe neurons retrogradely labeled from the PVN and axon terminals anterogradely labeled from the ventrolateral medulla. The injection site of the retrograde tracer in the PVN was mostly located in the magnocellular parts. Thus, the retrogradely labeled POMe neurons appear to project to VP-producing PVN cells. Our previous tract tracing study combined with immunohistochemistry has revealed that POMe neurons retrogradely labeled from the PVN receive synaptic inputs of axon terminals immunoreactive to TH, NA and neuropeptide Y w16x. Neuropeptide Y-immunoreactive projection from the ventrolateral medullary catecholamine group to the POMe has been found w6x. The morphological features and the types of the synapse of the present anterogradely labeled axon terminals were fairly consistent with those observed in the previous study w16x. The present study confirmed, there-
fore, that POMe neurons projecting to the region of VPproducing PVN cells receive noradrenergic, adrenergic and neuropeptide Y-immunoreactive synaptic inputs from the ventrolateral medullary A1 and C1 catecholamine cell groups. In the present study, anterogradely labeled axon terminals showed axo-dendritic and axo-somatic, but not axoaxonic synaptic contacts with POMe neurons projecting to the PVN. In addition to these anterogradely labeled axon terminals, a few unlabeled axon terminals were found to make synaptic contacts onto the same cell bodies and thick dendrites containing retrograde labelings, suggesting multiple synaptic inputs onto POMe neurons projecting to the PVN. The most likely candidate of unlabeled axon terminals is angiotensinergic terminals derived from the SFO w18,19x. We propose, therefore, that the reinforcing interaction between SFO-derived angiotensinergic and catecholaminergic inputs w1x affects the POMe by way of which two projection fibers Žone is a catecholaminergic fiber from the ventrolateral medulla, and the other from the SFO. converge onto single POMe neurons rather than via interneurons or by presynaptic interactions between the two-fiber systems before acting on POMe neurons.
Acknowledgements The authors wish to thank Miss Y. Honda for her technical assistance.
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Research report
Synaptic contacts between nerve terminals originating from the ventrolateral medullary catecholaminergic area and median preoptic neurons projecting to the paraventricular hypothalamic nucleus Hitoshi Kawano ) , Sadahiko Masuko Department of Anatomy, Saga Medical School, Saga 849-8501, Japan Accepted 10 November 1998
Abstract A significant role of catecholaminergic projection to the median preoptic nucleus ŽPOMe. which activates vasopressin-producing cells of the paraventricular hypothalamic nucleus ŽPVN. has been suggested. We investigated the existence of the synaptic contacts between catecholaminergic fibers from the ventrolateral medulla and the POMe neurons projecting to the PVN. Rats received a retrograde tracer in the PVN and subsequently an anterograde tracer into the catecholaminergic area of the ventrolateral medulla at the level of the area postrema. In the POMe, anterogradely labeled nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts onto retrogradely labeled neurons. Additional studies in which a retrograde tracer was injected into the POMe revealed that almost all retrogradely labeled neurons in the ventrolateral medulla at the level of the area postrema were immunoreactive to tyrosine hydroxylase, suggesting that projection to the POMe from the ventrolateral medulla is largely limited to catecholamine neurons. These results provide, for the first time, direct evidence that catecholaminergic inputs from the ventrolateral medulla affect POMe neurons projecting to the PVN by way of direct synaptic contact. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Median preoptic nucleus; Paraventricular hypothalamic nucleus; Ventrolateral medulla; Catecholamine; Anterograde and retrograde tracing; Rat
1. Introduction The median preoptic nucleus ŽPOMe., a lamina terminalis-associated structure, is situated at the anterior wall of the third ventricle and is a member of the anteroventral third ventricle ŽAV3V. region w13x. A rise in mean arterial pressure induced by intravenous injection of angiotensin II ŽA-II. increases the firing frequency of the POMe neurons w7x. Electrical and neurotoxigenic lesions of the ventral AV3V region, including the POMe, block the increase in water intake, pressor and natri- as well as kaliuretic responses induced by systematically or locally applied chemical agents, such as A-II or hypertonic saline w3,5,8,21x. These physiological data indicate that the POMe is critical for the regulation of water and ion balance and cardiovascular control.
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Electrical stimulation of the POMe or injection of hypertonic saline into the POMe increases activity of neuroendocrine cells in the paraventricular hypothalamic nucleus ŽPVN. w12x, while electrical lesions of the POMe block the hypertonic saline-induced increase in plasma vasopressin ŽVP. level w8x. POMe neurons projecting to the PVN are excited under several conditions, which include stimulation of the subfornical organ ŽSFO., injection of A-II into the POMe or the SFO, intravenous injection of hypertonic saline and stimulation of the A1 catecholaminergic area w29,30,32,34x. When A-II antagonist is injected into the POMe or a knife cut is made between the SFO and POMe, several responses, such as A-II-induced drinking as well as A-II-induced or SFO stimulation-induced excitation of PVN neurons, are blocked w17,27,31x. Furthermore, microinjection of local anesthetic lidocaine into the POMe abolishes the excitatory responses with long duration of putative VP-producing PVN cells to electrical stimulation of the SFO w33x. Morphological studies have demonstrated the existence of projections from the SFO to the POMe
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 3 3 - 5
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w9,20,22,23x, which contain A-II immunoreactivity w18,19x, and of projections from the POMe to the magnocellular PVN w11,15,16,25,26x. Thus, it is likely that POMe neurons, being sensitive to A-II, receive angiotensinergic afferent fibers originating from the SFO and, in turn, project to the PVN to stimulate VP-producing cells. On the other hand, the POMe receives many catecholaminergic nerve terminals w14,16x. When noradrenaline or adrenaline nerve terminals in the POMe are selectively destroyed by 6-hydroxydopamine, A-II-induced drinking and pressor responses are blocked w1,2,4x. It has also been demonstrated that reduced extracellular fluid volume by subcutaneously applied polyethylene glycol increases noradrenaline release in the POMe region w28,36x. Previous studies have shown that ventrolateral medullary neurons retrogradely labeled from the POMe are immunoreactive to tyrosine hydroxylase ŽTH. and to phenylethanolamine N-methyltransferase ŽPNMT., catecholamine synthesizing enzymes used as a marker for all catecholamines and adrenaline, respectively, suggesting projections to the POMe from both the A1 and the C1 catecholamine groups w24,35x. It has not been clarified as yet, whether nerve terminals of these catecholamine neurons have a direct effect by synaptic contacts onto POMe neurons projecting to the PVN, or an indirect effect via interneurons or by presynaptic interaction with nerve terminals derived from the SFO. The present study was designed to investigate these issues electron microscopically, using a double tract tracing method, in which anterograde and retrograde tracers were injected into the catecholaminergic area of the ventrolateral medulla and the PVN, respectively. The existence of the catecholaminergic projection from the ventrolateral medulla to the POMe was also confirmed by a double labeling experiment for retrograde labeling and TH immunoreactivity in the ventrolateral medullary neurons after injection of a retrograde tracer into the POMe.
2. Materials and methods Male Sprague–Dawley rats Ž49 " 1 days old, n s 30., anesthetized by intraperitoneal injection of pentobarbital sodium Ž50 mgrkg b.wt.. before surgical procedures and sacrifice, were used. The rats received a stereotaxical injection of a small quantity Ž0.3 ml. of a retrograde tracer, wheat germ agglutinin–conjugated horseradish peroxidase–colloidal gold complex ŽWGA–HRP–gold. w15x, into the left PVN from the dorsal aspect through a glass micropipette with an outer diameter of about 50 mm w15,16x. Subsequently, the animals received an iontophoretical injection of an anterograde tracer, biotinylated dextran amine ŽBDX; Molecular Probes, OR, USA; 10% in 0.05 M Tris buffer, pH 7.6; 2–5 mA positive current, for 20–30 min. into the ipsilateral ventrolateral medulla at the level of the area postrema. Ten to fourteen days after the
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injections, the rats were perfused through the ascending aorta with physiological saline containing heparin sodium Ž1000 IUrl., followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer ŽpH 7.3.. The brains were removed and fixed by immersion in the same fixative without glutaraldehyde for 2–24 h, and 20 mm thick frontal sections through the entire rostrocaudal extent of the POMe were serially cut on a microslicer ŽD.S.K., Kyoto, Japan.. Retrograde labeling was detected in the free-floating sections through the POMe, using a silver-intensification method described previously w15,16x. The silver intensified POMe sections were then processed for immunohistochemical detection of the anterograde tracer, after being soaked in 0.3% H 2 O 2 in PBS for 10 min to block activities of peroxidase. The sections were incubated first with streptavidin–Texas Red conjugate ŽGibco BRL, Gaithersburg, MD, USA; diluted 1:100., then biotinylated streptoavidin–peroxidase complex ŽVector, Burlingame, CA, USA; diluted 1:1000., and reacted with 0.02% diaminobenzidine tetrahydrochloride ŽDAB; Dotite, Kumamoto, Japan. in Tris buffered saline ŽpH 7.6. containing 0.005% H 2 O 2 for 15–30 min. Finally, the sections were incubated with 0.05% chloroauric acid in 0.1 M phosphate buffer ŽpH 7.3. for 10 min in an ice-cold bath to tone up the retrograde labeling and DAB reaction products, postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M phosphate buffer, dehydrated, and embedded in Spurr’s resin ŽNisshin EM, Tokyo, Japan.. Ultrathin sections of the POMe were prepared on a Sorvall MT2-B Ultra Microtome, and observed under a JEOL 100CX electron microscope without metal staining. Only those rats in which both the retrograde and anterograde tracers were successfully injected into the PVN and the ventrolateral medulla, respectively, were used. To verify the injection site of WGA–HRP–gold in the PVN, 40 mm thick frontal sections through the hypothalamus were cut on a freezing microtome, mounted on gelatin-coated glass slides, counterstained with hematoxylin, and observed under a light microscope. To verify the injection site of BDX in the catecholaminergic areas of the ventrolateral medulla, 40 mm thick frontal sections through the medulla were cut on a freezing microtome and every sixth section was processed for double labeling for TH immunoreaction and BDX. Sections were sequentially incubated with mouse monoclonal antibody to TH w10x, and a cocktail containing fluorescein isothiocyanate-labeled swine anti-mouse IgG ŽNordic Immunological Laboratories, Tilburg, The Netherlands; diluted 1:100. and streptoavidin–Texas Red conjugate, and observed under a fluorescent microscope. To verify the anterograde tracing from the catecholaminergic area of the caudal ventrolateral medulla to the POMe, experiments with retrograde tracer injected into the POMe were also performed. Three rats received a stereotaxical injection of WGA–HRP–gold into the POMe
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and were perfused with 4% paraformaldehyde in phosphate buffer ŽpH 7.3. after a 2- to 7-day survival. Medullary sections of 40 mm were obtained and processed for double labeling. Sections were first silver intensified to reveal retrogradely labeled neurons and then immunostained for TH using mouse monoclonal antibody to TH w10x, swine anti-mouse IgG ŽNordic Immunological Laboratories; diluted 1:100. and mouse peroxidase anti-peroxidase complex ŽDAKOPATTS, Glostrup, Denmark; 1:100. followed by visualization with the DAB reaction. To investigate immunoreactivity for noradrenaline and PNMT, two other rats were fixed by perfusion of 5% glutaraldehyde in 0.1 M cacodylate buffer ŽpH 7.4. and 20 mm thick free-floating sections were obtained on a freezing microtome. Sections were immunostained using rabbit polyclonal anti-noradrenaline ŽSFRI Laboratoire, Martignas, France; diluted 1:4000. or anti-PNMT ŽEugene Tech International, Ridgefield Park, NJ, USA; diluted 1:1000., unlabeled swine anti-rabbit IgG ŽDAKOPATTS; diluted 1:40. and rabbit PAP ŽDAKOPATTS; diluted 1:80.. The details for noradrenaline immunohistochemistry have been described elsewhere w14,16x and this procedure was also applied to PNMT immunohistochemistry.
3. Results The POMe contained both noradrenaline- and PNMTimmunoreactive nerve terminals in the entire nucleus ŽFig. 1., especially in the ventral part. The density of noradrenaline-immunoreactive nerve terminals was similar to that of PNMT-immunoreactive nerve terminals in the rostral half of the nucleus, while a few more noradrenaline- than PNMT-immunoreactive nerve terminals were found in the caudal half. In the experiments of retrograde tracing from the PVN, each injection site of the WGA–HRP–gold was mainly confined within the magnocellular and extended somewhat to the parvocellular parts of the PVN ŽFig. 2a.. After these injections, retrogradely labeled neurons were found throughout the POMe, especially in the ventral part of the nucleus at the middle level ŽFig. 2b.. In the anterograde experiments, the injection site of the BDX was located in the ventrolateral medullary catecholaminergic areas, where TH-immunoreactive neurons were clustered ŽFig. 3a,b.. Anterogradely labeled terminals were observed in the POMe and many other brain regions including the medial preoptic area and the PVN. In the
Fig. 1. Serial sections showing immunoreactivity for noradrenaline Ža. and PNMT Žb. in the POMe at the middle level Žabout 60 mm rostral to the crossing of the anterior commissure.. Both immunoreactive nerve varicosities are seen in abundance in the ventral part of the nucleus. More noradrenaline- than PNMT-immunoreactive nerve varicosities are present. Demarcation of the nucleus is indicated by broken lines. v: third ventricle. Bar s 50 mm Ža, b..
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Fig. 2. Ža. Injection site of WGA–HRP–gold in the PVN. Deposit of the retrograde tracer is mainly restricted in the magnocellular part and somewhat in the parvocellular part of the nucleus. Demarcation of the nucleus is indicated by a broken line. Žb. Darkfield illumination of the POMe sectioned through the middle level Žabout 180 mm rostral to the crossing of the anterior commissure. of the nucleus showing retrograde labeling after the injection of WGA–HRP–gold into the PVN shown in Ža.. Many bright retrogradely labeled neurons are seen in the nucleus bilaterally. v: third ventricle. Barss100 mm Ža, b..
POMe, anterogradely labeled terminals were found throughout the nucleus, especially in the ventral part of the caudal two thirds of the nucleus ŽFig. 3c.. Electron microscopy revealed that the anterogradely labeled axon terminals in the POMe were round, ovoid and ellipsoid in shape, and contained many small clear round vesicles and some large cored vesicles. These terminals made synaptic contacts onto dendrites and cell bodies in the POMe. Both asymmetric and symmetric synapses were encountered. Among these, synaptic contacts were found between the anterogradely labeled axon terminals and dendrites or cell bodies of the POMe neurons containing retrograde labelings in the lysosomes ŽFig. 4.. Hereupon,
Fig. 3. Ža, b. An identical section of the ventrolateral medulla showing injection site of biotinylated dextran amine Ža. and TH immunoreactivity Žb. under a fluorescence microscope. The level of the injection is the area postrema and corresponds to that shown in Fig. 5b. The location of the anterograde tracer corresponds fairly well to the cluster of TH-immunoreactive neurons, and some neurons in the vicinity of the injection site are labeled for both Žarrowheads.. Žc. Anterogradely labeled afferent nerve varicosities in the POMe after the injection of biotinylated dextran amine into the catecholaminergic area of the ventrolateral medulla shown in Ža.. Dense, bilateral distribution of the labeled varicosities are mainly found in the ventral part of the nucleus. The rostrocaudal level of the section corresponds to that shown in Fig. 1. Barss 50 mm Ža–c..
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both the asymmetric and symmetric synapses as well as both the axo-dendritic and axo-somatic synapses were observed. The retrogradely labeled postsynaptic cell bodies and thick dendrites which make synapses with anterogradely labeled nerve terminals were also in synaptic contact with unlabeled nerve terminals ŽFig. 4.. After an injection of WGA–HRP–gold into the middle level of the rostrocaudal extent of the POMe ŽFig. 5a.,
retrogradely labeled neurons in the medulla oblongata were found in the ventrolateral regions and in the nucleus of the solitary tract. In the ventrolateral medulla, retrogradely labeled neurons were distributed bilaterally in levels between about 1400 mm caudal to the obex and 1900 mm rostral to the obex, occupying parts of catecholamine regions, with a peak number Žabout 10 neurons in a section. at the level of the area postrema. At these levels in which retrogradely labeled neurons were found, a substantial number of neurons were double-labeled for retrograde labeling and TH immunoreactivity. Doublelabeled neurons were more frequently observed at levels caudal to the obex Ž22 neurons in total, representing 12% of 181 TH-immunoreactive neurons and 75% of 29 retrogradely labeled neurons; mean of three rats, counted at intervals of 240 mm for each experiment. than at levels rostral to the obex Ž13 neurons in total, representing 7% of 186 TH-immunoreactive neurons and 52% of 25 retrogradely labeled neurons.. At the level of the area postrema, 16 neurons of 19 retrogradely labeled neurons Ž85%. showed TH immunoreactivity ŽFig. 5b,c..
4. Discussion
Fig. 4. Electron micrographs of the POMe showing asymmetric Ža. and symmetric Žb. synaptic contacts of an afferent axon terminal anterogradely labeled from the ventrolateral medulla onto a dendrite retrogradely labeled from the PVN. Note that an unlabeled axon terminal Žasterisk. is also in synaptic contact with the identical dendrite in Ža.. Retrograde labelings on the lysosomes are indicated by arrows. Barss 0.5 mm.
The present study, using an anterograde tracer, demonstrated, for the first time, that neurons in the catecholaminergic areas of the ventrolateral medulla at the level of the area postrema project to the POMe. Immunostaining for TH at the present injection sites of the anterograde tracer confirmed that the injections were made into the ventrolateral medullary catecholamine area. The retrograde experiments with the tracer injected into the POMe and subsequent immunohistochemistry for TH also revealed that almost all retrogradely labeled neurons exhibited TH immunoreactivity in the ventrolateral medulla at the level of the area postrema, the exact region where the anterograde tracer was applied in the present study. Thus, it is likely that almost all anterogradely labeled nerve terminals observed in the POMe are derived from the catecholamine neurons of the ventrolateral medulla. Using WGA–HRP– gold as a retrograde tracer, we clarified that about 10% of TH-immunoreactive neurons in the ventrolateral medulla projected to the middle level of the POMe. Since this tracer has minimal diffusion, the injection site was quite restricted to a small part of the POMe. Accordingly, the actual proportion of TH-immunoreactive projections from the ventrolateral medulla to the POMe must be higher. The existence of the catecholaminergic projections from the ventrolateral medulla to the POMe is generally in agreement with the previous reports w24,35x. These studies have used retrograde labeling and immunoreactivities for TH and PNMT to demonstrate that both noradrenaline and adrenaline neurons project to the POMe from the entire rostrocaudal ventrolateral medullary regions. Tucker et al.
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Fig. 5. Ža. Injection site of WGA–HRP–gold in the POMe at the middle level Žalmost the same, but slightly rostral to that shown in Fig. 2b.. Deposit of the retrograde tracer is nearly confined to the nucleus. Demarcation of the nucleus is indicated by a broken line. Žb. Medullary section showing retrogradely labeled neurons in the nucleus of the solitary tract Žarrowheads. and in the ventrolateral medulla Žrectangle. at a level of the area postrema Žap. after injection of WGA–HRP–gold into the POMe shown in Ža.. Žc. High power microgram of the rectangle in the section adjacent to Žb. after silver intensification and TH immunohistochemistry of the ventrolateral medullary region. Note that retrogradely labeled neurons containing granules of silver intensified gold are immunoreactive to TH Žarrowheads.. v: third ventricle. Bars s 100 mm Ža. and 50 mm Žb, c..
w35x have reported that many more adrenergic than noradrenergic neurons project to the POMe from the level of the area postrema, the level corresponding to the transition area from the A1 to the C1 area. However, the present study in which the injection of the anterograde tracer was made at this transition area showed a denser distribution of anterogradely labeled afferent terminals in the caudal twothirds of the POMe, where more noradrenaline- than PNMT-immunoreactive nerve terminals were found. Thus, it is conceivable that, although both the A1 and C1 catecholamine neurons project to the POMe, noradrenergic nerve terminals arborize more predominantly than adrenergic nerve terminals and are distributed mainly in the caudal two-thirds of the nucleus. The present study demonstrated synaptic contacts between POMe neurons retrogradely labeled from the PVN and axon terminals anterogradely labeled from the ventrolateral medulla. The injection site of the retrograde tracer in the PVN was mostly located in the magnocellular parts. Thus, the retrogradely labeled POMe neurons appear to project to VP-producing PVN cells. Our previous tract tracing study combined with immunohistochemistry has revealed that POMe neurons retrogradely labeled from the PVN receive synaptic inputs of axon terminals immunoreactive to TH, NA and neuropeptide Y w16x. Neuropeptide Y-immunoreactive projection from the ventrolateral medullary catecholamine group to the POMe has been found w6x. The morphological features and the types of the synapse of the present anterogradely labeled axon terminals were fairly consistent with those observed in the previous study w16x. The present study confirmed, there-
fore, that POMe neurons projecting to the region of VPproducing PVN cells receive noradrenergic, adrenergic and neuropeptide Y-immunoreactive synaptic inputs from the ventrolateral medullary A1 and C1 catecholamine cell groups. In the present study, anterogradely labeled axon terminals showed axo-dendritic and axo-somatic, but not axoaxonic synaptic contacts with POMe neurons projecting to the PVN. In addition to these anterogradely labeled axon terminals, a few unlabeled axon terminals were found to make synaptic contacts onto the same cell bodies and thick dendrites containing retrograde labelings, suggesting multiple synaptic inputs onto POMe neurons projecting to the PVN. The most likely candidate of unlabeled axon terminals is angiotensinergic terminals derived from the SFO w18,19x. We propose, therefore, that the reinforcing interaction between SFO-derived angiotensinergic and catecholaminergic inputs w1x affects the POMe by way of which two projection fibers Žone is a catecholaminergic fiber from the ventrolateral medulla, and the other from the SFO. converge onto single POMe neurons rather than via interneurons or by presynaptic interactions between the two-fiber systems before acting on POMe neurons.
Acknowledgements The authors wish to thank Miss Y. Honda for her technical assistance.
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