- Email: [email protected]
Difference in topology and numbers of barosensitive catecholaminergic and cholinergic neurons in the medulla between SHR and WKY rats
Journal of the Autonomic Nervous System 70 Ž1998. 200–208
Difference in topology and numbers of barosensitive catecholaminergic and cholinergic neurons in the medulla between SHR and WKY rats Yu Xiong, Junichi Okada, Senichi Tomizawa, Kiyoshige Takayama, Mitsuhiko Miura
)
Department of Physiology 1st DiÕision, Gunma UniÕersity School of Medicine, 3-39-22 Showa-machi, Maebashi-shi 371, Japan Received 21 January 1998; revised 2 March 1998; accepted 2 March 1998
Abstract We hypothesized that there may be a significant difference in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto ŽWKY. and spontaneously hypertensive SHR rats. Using the double-immunoreactive ŽIR. method, the topology and numbers of barosensitive neurons that contain glutamate ŽGlu., glutamic acid decarboxylase ŽGAD., tyrosine hydroxylase ŽTH., phenylethanolamine N-methyltransferase ŽPNMT. and choline acetyltransferase ŽChAT. were compared between the two strains. The control rats were sham-operated only for cannulation of the trachea and femoral arteryrvein. The test rats were injected with the pressor agent phenylephrine to raise blood pressure and stimulate arterial baroreceptors. In both the control and test experiments, the c-FosrGlu-, GAD-, TH- and PNMT-IR neurons were found in the nucleus tractus solitarii ŽNTS. and ventrolateral medulla ŽVLM., while the FosBrChAT-IR neurons were found in the NTS, dorsal motor nucleus of the vagus ŽDMX. and nucleus ambiguus ŽAMB.. In the control experiment, no significant difference in numbers was recognized in any of the double-IR neurons between the two strains. In the test experiment, the numbers of FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. The numbers of c-FosrTH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY. These results suggest that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR, and that a larger number of barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR. q 1998 Elsevier Science B.V. All rights reserved. Keywords: c-Fos; FosB; Nucleus ambiguus; Nucleus tractus solitarii; Tyrosine hydroxylase; Phenylethanolamine N-methyltransferase; Choline acetyltransferase; Ventrolateral medulla
1. Introduction The outline of the baroreceptor reflex pathway in the medulla has been established. The first-order neurons transmit baroreceptor signals to the second-order neurons in the nucleus tractus solitarii ŽNTS. ŽCiriello, 1983; Ciriello et al., 1981; Housley et al., 1991; Miura and Reis, 1969.. The second-order neurons in turn project to the dorsal motor nucleus of the vagus ŽDMX., the ambiguus nucleus ŽAMB. ŽNosaka et al., 1979, 1982. and the ventrolateral medulla ŽVLM. ŽGuyenet, 1990; Miura et al., 1994b; Suzuki et al., 1997.. The baroreceptor-cardiac vagal reflex is transmitted via the NTS–DMXrAMB pathway ŽOkada and Miura, 1997; Ross et al., 1985., while the baroreceptor-sympathetic nerve reflex via the NTS–VLM pathway ) Corresponding author. Tel.: q81 27 2207923; fax: q81 27 2207926; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 5 2 - 6
ŽMatsumoto et al., 1994; Miura et al., 1994a,b; Ross et al., 1985.. Then, we hypothesized that there may be significant differences in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto ŽWKY. and spontaneously hypertensive SHR rats, because the baroreceptor-cardiac vagal reflex is weaker in SHR than in WKY ŽHuang and Leenen, 1994; Luft et al., 1986; Verberne et al., 1988.. Many studies have showed differences among properties of neurons in the NTS, AMB and VLM between WKY and SHR, by using electrical stimulation ŽChan et al., 1990, 1991., chemical stimulation ŽMiura et al., 1991; Smith and Barron, 1990; Yang et al., 1996. and immunohistochemistry ŽMinson et al., 1996; Miura et al., 1994a; Murphy et al., 1994; Takayama and Miura, 1992; Xiong et al., 1997a,b.. Recently, we applied the double-immunoreactive ŽIR. method to normotensive Wistar rats, and succeeded in identifying c-Fosrglutamate ŽGlu.-, glu-
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
tamic acid decarboxylase ŽGAD.-, phenylethanolamine Nmethyltransferase ŽPNMT.- and tyrosine hydroxylase ŽTH.-IR neurons in the NTS and VLM ŽMiura et al., 1996. and FosBrcholine acetyltransferase ŽChAT.-IR neurons in the AMB ŽOkada and Miura, 1997.. To further test the aforementioned hypothesis, we sought evidence of significant differences in the numbers of barosensitive neurons containing Glu, GAD, PNMT, TH or ChAT between WKY and SHR. A preliminary report on the results obtained in the present study was presented in abstract form ŽXiong et al., 1997b.. 2. Materials and methods 2.1. General care Experiments were performed using 10- – 13-week-old male SHR and age-matched male WKY weighing 210–280 g Ž n s 16; Charles River Japan, Yokohama, Japan.. To avoid the restlessness characteristic of rats housed alone, pairs of rats were kept in cages ŽMiura et al., 1996.. To extinguish c-Fos expression that might have been present before isolation, pairs of rats were kept in a dark and soundless room for 12 h before the experiment. Anesthesia was induced with halothane and maintained with fentanyl Žinitially 50 m grkg, i.p., and then 25 m grkg, i.p., every 30 min; Sankyo, Tokyo, Japan. and midazolam Žinitially 5 mgrkg, i.p., and then 2.5 mgrkg, i.p., every 30 min; Yamanouchi, Tokyo, Japan.. Fentanylrmidazolam is the optimal anesthesia for excluding false-positive expression induced by anesthetics themselves ŽTakayama et al., 1994.. EEGs were continuously recorded via a fronto-occipital lead. For standardization of the anesthesia level, the ratio of power in the 8–20 Hz Ž a q b . band to power in the 0–4 Hz Ž d . band was set at 0.5–1.0 ŽDrummond et al., 1991.. The trachea was cannulated with vinyl tubing Ž2 mm i.d.. A femoral artery and vein were cannulated with polyethylene catheter Ž0.58 mm i.d... The relative tidal volume Ž VT . measured by a thermistor ŽPB7-43-S2; Shibaura Electronics, Japan., the respiratory frequency ŽRF. measured by a tachometer ŽAT601G; Nihon Kohden, Japan., the systemic arterial pressure ŽAP. measured by pressure transducer ŽTP602T, Nihon Kohden. and heart rate ŽHR. computed from the pressure pulse by a cardiotachometer ŽRT-5, Nihon Kohden. were displayed on a polygraph ŽRJG-4226, Nihon Kohden.. When necessary, arterial blood was sampled from a femoral artery and drugs were injected from a femoral vein. The head of the animal was placed on a stereotaxic frame Žbite bar set at y3.0 mm.. Rectal temperature was maintained at 378C with an infrared heat lamp. 2.2. Experimental procedure Care was taken to avoid c-Fos expression due to nonspecific stimulation, i.e. light, sound, odor and pain. The
201
control and test experiments were performed using four WKY and four SHR, respectively. The control rats were sham-operated for cannulation of the trachea, femoral artery and vein, and injected with 40–60 nl saline solution to confirm that no cardiovascular response had been induced. The test rats were injected with 40–60 nl of 2.5 mM L-phenylephrine saline solution Ž100 m grkg, i.v.; Sigma, St. Louis, MO, USA. at a speed of one shot per minute ŽSmyth et al., 1969. to induce the pressor and reflex bradycardic responses. Injections were repeated five times at intervals of 3–4 min ŽMiura et al., 1996, 1994b.. Before and after baroreceptor stimulation, 0.1 ml of blood was extracted to measure PaCO 2 , PaO 2 and pH, and a similar volume of compensatory saline solution was injected to prevent a blood pressure fall. Ninety minutes after cessation of the sham operation or administration of phenylephrine, rats were additionally anesthetized with pentobarbital sodium Ž50 mgrkg of body weight, i.p.. and perfused transcardially with heparinized saline, followed by 0.5% glutaraldehyde and 4% paraformaldehyde ŽPFA. in 0.1 M phosphate buffer ŽPB., and with 4% PFA in 0.1 M PB. The perfusate pH was set at 7.4. The brains were removed, postfixed in the same fixative for 2 h and soaked in 10%, 20% and another 20% sucrose PB solution. The medulla oblongata and pons were frozen and cut at 40 m m in frontal series. 2.3. Immunohistochemistry First, five sets of every fifth section were rinsed, then incubated in 0.5% bovine serum albumin ŽBSA. in 0.1 M Tris–HCl buffer for 20 min at 208C. Four sets of sections were incubated with sheep anti-cFos-polyclonal antiserum ŽOA-11-824, Genosys Biotechnologies, UK. at a dilution of 1:1000 for 20 h at 48C. Then, the sections were incubated in 0.1 M Tris–HCl buffer containing biotinylated anti-sheep IgG antiserum ŽVector Labs., USA. for 1 h at 208C. One set of sections was incubated with rabbit anti-FosB polyclonal antiserum Žsc-48, Santa Cruz Biotechnology, USA. at a dilution of 1:1000 for 20 h at 48C. Then, the sections were incubated in 0.1 M Tris–HCl buffer containing biotinylated anti-rabbit IgG antiserum ŽVector Labs.. for 1 h at 208C. Subsequently, all sets of sections were processed with avidin–biotin peroxidase complex ŽABC, Vector Labs.. for 1 h at 208C and treated with diaminobenzidine ŽDAB.nickel solution containing 0.003% H 2 O 2 . Secondly, five sets of sections were rinsed, incubated in 0.5% BSA in 0.1 M Tris–HCl buffer, and incubated for 20 h at 48C with one of the antisera to common neurotransmitters and related enzymes listed below: Ž1. rabbit antiglutamate ŽGlu. polyclonal antiserum Ž1:1000; SFRI, France., Ž2. rabbit anti-glutamic acid decarboxylase ŽGAD. polyclonal antiserum Ž1:500; Chemicon International, USA., Ž3. rabbit anti-tyrosine hydroxylase ŽTH. polyclonal
202
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
antiserum Ž1:3000; Chemicon International., Ž4. rabbit anti-phenylethanolamine N-methyltransferase ŽPNMT. polyclonal antiserum Ž1:3000; Chemicon International., and Ž5. mouse anti-cholineacetyltransferase ŽChAT. monoclonal antiserum Ž1:500; Chemicon International.. Finally, sections were incubated in 0.1 M Tris–HCl buffer containing: Ž1. – Ž4. biotinylated anti-rabbit IgG antiserum ŽVector Labs..; Ž5. biotinylated anti-mouse IgG antiserum ŽVector Labs.. for 1 h at 208C and in ABC solution for 1 h at 208C and treated with DAB solution containing 0.003% H 2 O 2 . The data sheets provided by the manufacturers indicated the following: Ž1. ELISA showed the anti-Glu antiserum to have cross-reactivity with aspartate Ž0.006%., b-alanine Ž- 0.001%., GABA Ž- 0.001%., glycine Ž0.001%. and taurine Ž- 0.001%.; Ž2. the anti-GAD antiserum was made using recombinant-DNA GAD, and Western blot analysis revealed the anti-GAD antiserum to recognize specifically the larger form of GAD Ž67 kDa.; Ž3. no staining was observed immunocytochemically when the anti-TH and anti-PNMT antisera were preabsorbed with TH and PNMT. Sections were mounted on slides and coverslipped with Permount ŽFisher, USA.. The neurons in the medulla were surveyed light microscopically. Brain histology was checked against the brain atlas of Swanson Ž1992.. Dark blue particles confined to the nucleus were identified as expression products of c-fos and FosB. In the cytoplasma, homogeneously brown particles were identified as reaction products of bioactive substances. The IR neurons of each nucleus in the medulla were counted and summed for each division. The reproducibility of staining was checked by comparing representative stained neurons in sections from different animals, such as GAD-stained Purkinje neurons in the cerebellum. The significance of differences in the numbers of double-IR neurons was evaluated using the Mann– Whitney test, with P - 0.05 being taken as statistically significant.
Žcaudal to the obex., while the 4th–5th divisions belong to the rostral medulla Žrostral to the obex.. According to the criteria described above, the NTS at the level of the 1st division of the medulla was abbreviated as the 1st division of the NTS. The other divisions follow this example.
3. Results 3.1. Effects of pressor stimulation on hemodynamics Effects of administration of phenylephrine Žpressor stimulation. on mean arterial blood pressure ŽMAP. and heart rate ŽHR. were compared before vs. after intravenous administration of 100 m grkg phenylephrine and between WKY and SHR. The pressor stimulation significantly elevated MAP ŽFig. 1A, left., but significantly lowered HR ŽFig. 1A, right.. The increases in MAP were 58 " 8 and
2.4. DiÕision of the medulla In place of the numerical coordinates, we used five divisions of the medulla oblongata according to the topology of the important nuclei located along the rostrocaudal axis ŽMiura et al., 1996.. The 1st division extends from the medullospinal border to the caudal end of the inferior olivary complex Žaverage of four sections per set, 4 = 5 = 40 m m s 0.8 mm.. Ahead of the 1st division, the 2nd division extends to the caudal end of the area postrema Žaverage of six sections, ca. 1.2 mm.. Similarly, the 3rd division extends to the obex Žaverage of four sections, ca. 0.8 mm., the 4th to the rostral end of the inferior olivary complex Žaverage of five sections, ca. 1.0 mm., and the 5th division terminates at the caudal end of the nucleus of the trapezoid body Žaverage of seven sections, ca. 1.4 mm.. The 1st–3rd divisions belong to the caudal medulla
Fig. 1. Effects of intravenous administration of 100 m grkg phenylephrine on hemodynamics in WKY and SHR. ŽA. Significant rise in mean arterial blood pressure Žleft. and significant fall in heart rate Žright. after administration of phenylephrine in both strains. ŽB. No significant difference in MAP changes between the two strains Ž DMAP, left., but difference in heart rate changes is significant Ž DHR, right.. ŽC. and ŽD. Examples of pressor and bradycardic reflex responses elicited by administration of phenylephrine in WKY ŽC. and SHR ŽD.. Abbreviations: a, after; AP, arterial blood pressure; b, before; HR, heart rate; MAP, mean arterial blood pressure; S, SHR; W, WKY. ) P - 0.05; ) ) P - 0.01, estimated by t-test.
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
203
58 " 7 mm Hg in WKY and SHR, respectively ŽFig. 1B, left., not significantly different. On the other hand, the decrease in HR was 90 " 10 bpm in WKY and 44 " 7 bpm in SHR ŽFig. 1B, right., with a statistically significant difference between the two strains Ž P - 0.05.. Fig. 1C, D shows examples of the pressor and reflex bradycardic responses produced by i.v. administration of 100 m grkg phenylephrine. In both strains, the PaCO 2 , PaO 2 and pH of arterial blood were in the range of 40–46 mm Hg, 88–113 mm Hg and 7.32–7.37 prior to stimulation, and 40–46 mm Hg, 84–105 mm Hg and 7.31–7.47 after stimulation, respectively. There were no significant differences in these parameters before vs. after stimulation. Since the PaCO 2 , PaO 2 and arterial blood pH were maintained within normal
Fig. 3. Histograms of averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in five rostrocaudal divisions of the NTS, DMX, AMB and VLM. Open columns, WKY. Oblique-line columns, SHR. ŽA. Control experiment Žmean"S.E.M., ns 4.. ŽB. Test experiment Žmean"S.E.M., ns 4.. 1–5, 1st–5th divisions of the medulla. Dotted line divides the VLM and AMB into their caudal Ž1–3. and rostral Ž4–5. divisions. ) P - 0.05 estimated by Mann–Whitney test.
range, changes in arterial blood gases and pH are unlikely to have influenced c-Fos expression ŽMiura et al., 1996.. 3.2. AÕeraged numbers of double-IR neurons in NTS, AMB and VLM
Fig. 2. Comparison of averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-immunoreactive ŽIR. neurons in the NTS, DMX, AMB and VLM between WKY and SHR. ŽA. Control experiment Žmean"S.E.M., ns 4.. ŽB. Test experiment after administration of phenylephrine Žmean"S.E.M., ns 4.. Open columns, WKY. Obliqueline columns, SHR. ) P - 0.05, estimated by Mann–Whitney test. Abbreviations: ChAT, choline acetyltransferase; GAD, glutamic acid decarboxylase; Glu, glutamate; PNMT, phenylethanolamine N-methyltransferase; TH, tyrosine hydroxylase.
In both control and test experiments Žfour each., all c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons were counted in WKY and SHR Žfour animals each.. They were distributed evenly on both sides of the NTS, DMX, AMB and VLM in the medulla. The NTS, DMX and AMB are distinct nuclei with borders, while the VLM is not a distinct nucleus but part of the reticular formation located in the ventrolateral medulla without borders. Not only in our previous studies ŽMiura et al., 1994b, 1996. but also in the present study, we found that the barosensitive Fos-IR neurons are distributed in the VLM, covering the caudal reticular formation, just dorsal to the nucleus reticularis lateralis at the 1st–3rd divisions of the medulla, and the rostral reticular formation, just ventral to the AMBrretrofacial nucleus in the 4th–5th divisions of the medulla. Numbers of the c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in the NTS, DMX, AMB and VLM were counted, averaged and compared between WKY and SHR. In the control experiment ŽFig. 2A., no signifi-
204
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
Fig. 4. Topology of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons, identified after phenylephrine administration, distributed in the NTS, DMX, AMB and VLM throughout four divisions of the medulla Ž1–4.. ŽA. WKY. ŽB. SHR. One dot corresponds to one neuron for the FosBrChAT-IR neurons, but two neurons for the others.
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
205
Fig. 5. Photomicrographs of double-IR neurons in the ventrolateral medulla of WKY and SHR after administration of phenylephrine. ŽA. – ŽE.: c-FosrGluŽA., GAD- ŽB., TH- ŽC., PNMT- ŽD. and FosBrChAT-IR neuron ŽE.. Left, sites of neurons indicated by arrows. Right, magnified photomicrographs of corresponding neurons. Bar, 500 m m on the left; 20 m m on the right.
206
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
cant difference was found between the two. On the other hand, in the test experiment ŽFig. 2B., the averaged numbers of total FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. Inversely, the averaged numbers of total c-FosrTH- and PNMT-IR neurons in the VLM were significantly larger in SHR than in WKY. 3.3. AÕeraged numbers of double-IR neurons in fiÕe diÕisions of the medulla The averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons were calculated in five rostrocaudal divisions of the NTS, DMX, AMB and VLM, and were compared between WKY and SHR. In the control experiment ŽFig. 3A., no significant difference in the averaged numbers between WKY and SHR was recognized. On the other hand, in the test experiment ŽFig. 3B., the averaged numbers of the FosBrChAT-IR neurons in the 3rd and 4th divisions of the AMB were significantly smaller in SHR than in WKY. Inversely, the averaged numbers of the c-FosrTH-IR neurons in the 2nd and 3rd divisions of the VLM were significantly larger in SHR than in WKY. As described above, a significant difference was found in the averaged numbers of the total c-FosrPNMT-IR neurons in the VLM and the total FosBrChAT-IR neurons in the NTS and DMX between the two strains, whereas there was no significant difference in the averaged numbers of these IR neurons in the 1st to 5th divisions. 3.4. Topology of double-IR neurons in WKY and SHR The topology of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in the test experiment was compared against the 1st–4th divisions of the medulla between WKY ŽFig. 4A. and SHR ŽFig. 4B.. The number of double-IR neurons in the 5th division was too low to mark on the map. In the 3rd and 4th divisions of the AMB, the numbers of FosBrChAT-IR neurons were smaller in SHR than in WKY, whereas in the 2nd and 3rd divisions of the VLM, the numbers of c-FosrTH-IR neurons were larger in SHR than in WKY. Fig. 5 shows photomicrographs of the double-IR neurons found in the VLM of WKY and SHR after administration of phenylephrine. Since the nucleoplasm of the double-IR neurons was stained dark and the cytoplasm was homogeneously brown, the double-IR neurons were clearly differentiated from single c-Fos or FosB-labeled neurons and single Glu-, GAD-, TH-, PNMT- and ChAT-IR neurons. The diameter of these double-IR neurons in the VLM was as small as 20 m m in the larger axis, i.e. approximately one-third the diameter of motor neurons in the AMB ŽFig. 5E..
4. Discussion Many studies have shown that in normotensive Wistar rats barosensitive c-Fos-IR neurons are present in the NTS and VLM, and contain Glu, GAD, TH, PNMT and ChAT ŽBadoer et al., 1994; Chan and Sawchenko, 1994; Minson et al., 1996; Miura et al., 1996; Murphy et al., 1994; Okada and Miura, 1997.. In this context, differences in the topology and numbers of these barosensitive neurons between WKY and SHR have been sought. In our previous study ŽXiong et al., 1997a., using the single c-Fos method, we found that the number of barosensitive neurons in the caudal VLM was significantly larger in SHR than in WKY, whereas the number of barosensitive neurons in the rostral VLM was significantly larger in WKY than in SHR. Further, in the present study, stimulation of the arterial baroreceptors revealed differences in the neuronal composition of the baroreceptor reflex pathway in the medulla between WKY and SHR. One important finding was that baroreceptor stimulation made a significant difference in the number of cholinergic neurons. On a larger scale, numbers of the FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY, while on a smaller scale the numbers of FosBrChAT-IR neurons in the 3rd and 4th divisions of the AMB were significantly smaller in SHR than in WKY. Recently, we found that baroreceptor stimulation induced FosB expression in cholinergic neurons in the NTS, DMX and AMB. These cholinergic neurons in the DMX and AMB give off descending cardioinhibitory fibers, as shown by Nosaka et al. Ž1979, 1982., while those in the NTS do not innervate the heart ŽOkada and Miura, 1997.. Many studies demonstrated the presence of cholinergic neurons in the NTS ŽMaley, 1996; Ruggiero et al., 1990; Talman et al., 1994.. Talman and Lewis suggested that cardiovascular regulation is disturbed in SHR, when acetylcholine is injected into the NTS ŽTalman and Lewis, 1991.. However, the physiological role of cholinergic neurons in the NTS remains unknown. In the behavioral study, we confirmed that the bradycardic reflex response to baroreceptor stimulation was significantly weaker in SHR than in WKY. This finding is similar to the results of previous studies by Luft et al. Ž1986. and Verberne et al. Ž1988., that is, the sensitivities of the cardioinhibitory reflexes are more diminished in SHR than in WKY. Therefore, it is possible that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR. Another important finding was that baroreceptor stimulation resulted in a significant difference in the number of catecholaminergic neurons between the two strains. On a larger scale, the numbers of c-FosrTH- and PNMT-IR neurons in the VLM were significantly larger in SHR than in WKY, while on a smaller scale the numbers of cFosrTH-IR neurons in the 2nd and 3rd divisions in the
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
VLM were significantly larger in SHR than in WKY. These results indicate that stimulation of the arterial baroreceptors induced stronger excitation of catecholamninergic neurons in the VLM of SHR. Since the direct effect of phenylephrine on peripheral resistance vessels predominates, the depressor reflex response to phenylephrine was not discernible on the blood pressure record. It is certain, however, that baroreceptor signals were transmitted to neurons of the NTS and VLM in the baroreceptor-sympathetic nerve reflex pathway. Smith and Barron Ž1990. and Yang et al. Ž1996. found that the depressor response to glutamate stimulation of the caudal VLM is significantly greater in SHR than in WKY. Granata et al. Ž1986. reported that the noradrenergic neurons in the caudal VLM tonically inhibit sympathoexcitatory adrenergic neurons in the rostral VLM. Inversely, another group of investigators reported that noradrenergic neurons in the caudal VLM are not involved in the baroreceptor-depressor reflex ŽBlessing and Willoughby, 1987; Day et al., 1983.. Recently, we confirmed that the catecholaminergic projection from the depressor area in the caudal VLM to the pressor area of the rostral VLM is weak, whereas the GABAergic projection is strong ŽSuzuki et al., 1997.. These findings suggest that catecholaminergic neurons in the caudal VLM play a minor role in the inhibition of sympathetic vasomotor neurons in the rostral VLM. Many investigators reported that catecholaminergic neurons in the rostral VLM give off descending fibers to sympathetic vasomotor preganglionic neurons in the intermediolateral nucleus ŽIMLN. of the lower thoracic cord, thereby directly inhibiting these vasomotor neurons ŽJeske and McKenna, 1992; Ruggiero et al., 1994; Tucker et al., 1987.. Although GABAergic neurons in the caudal VLM were verified to project to the IMLN of the lower thoracic cord ŽMatsumoto et al., 1994., no report has demonstrated catecholaminergic neurons projecting from the caudal VLM to the IMLN of the spinal cord. These findings suggest that catecholaminergic neurons in the caudal VLM have no descending projection to the IMLN. On the other hand, ascending projections from the caudal VLM to the paraventricular nucleus of the hypothalamus ŽPVH., a site of vasopressin secretion, merit consideration. Vasopressin mRNA levels are higher in SHR than WKY Žvan Tol et al., 1988., and the plasma level of vasopressin is also higher in SHR than in WKY ŽCrofton et al., 1978.. Since the PVH receives projections from adrenergic and noradrenergic neurons in the caudal VLM ŽTanaka et al., 1985; Tucker et al., 1987., it is possible that these catecholaminergic neurons stimulate vasopressin-secreting neurons in the PVH ŽDay et al., 1984. and antagonize the depressor Žbaroreceptor-sympathetic nerve. reflex. The greater number of c-FosrTH-IR neurons in SHR suggests that the antagonizing effect on the depressor reflex may be more potent in SHR than in WKY. Therefore, we suggest that a larger number of
207
barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR. We conclude that Ž1. without pressor stimulation, there is no significant difference in the numbers of FosBrChAT-IR neurons in the NTS, DMX and AMB between WKY and SHR or in the numbers of c-FosrGlu-, GAD-, TH- and PNMT-IR neurons in the NTS and VLM; Ž2. with pressor stimulation, the numbers of FosBrChATIR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY, which may cause the weaker baroreceptor-cardiac vagal reflex in SHR. Inversely, the numbers of c-FosrTH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY, which may cause involvement of catecholaminergic neurons in the caudal VLM in the stronger baroreceptorvasopressin reflex in SHR.
Acknowledgements This work was supported in part by a Grant-in-Aid for encouragement of visitor scientist from the Sasakawa Health Science Foundation to Y. Xiong.
References Badoer, E., McKinley, M.J., Oldfield, B.J., McAllen, R.M., 1994. Localization of barosensitive neurons in the caudal ventrolateral medulla which project to the rostral ventrolateral medulla. Brain Res. 657, 258–268. Blessing, W.W., Willoughby, J.O., 1987. Depressor neurons in rabbit caudal medulla do not transmit the baroreceptor-vasomotor reflex. Am. J. Physiol. 253, H777–786. Chan, P.K.W., Sawchenko, P.E., 1994. Spatially and temporally differentiated patterns of fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J. Comp. Neurol. 348, 433–460. Chan, R.K.W., Chan, T.S., Wong, M., 1990. Cardiovascular responses to electrical stimulation of the ventrolateral medulla of the spontaneously hypertensive rat. Brain Res. 522, 99–106. Chan, R.K.W., Chan, T.S., Wong, M., 1991. Electrophysiological properties of neurons in the rostral ventrolateral medulla of normotensive Wistar Kyoto and spontaneously hypertensive rats. Brain Res. 549, 118–127. Ciriello, J., 1983. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci. Lett. 36, 37–42. Ciriello, J., Hrycyshyn, A.W., Calaresu, F.R., 1981. Glossopharyngeal and vagal afferent projections to the brain stem of the cat: a horseradish peroxidase study. J. Auton. Nerv. Syst. 4, 63–79. Crofton, J.T., Share, L., Shade, R.E., Allen, C., Tarnowski, D., 1978. Vasopressin in the rat with spontaneous hypertension. Am. J. Physiol. 235, H361–H366. Day, T.A., Ro, A., Renaud, L.P., 1983. Depressor area within caudal ventrolateral medulla of the rat does not correspond to the A1 catecholamine cell group. Brain Res. 279, 299–302. Day, T.A., Ferguson, A.V., Renaud, L.P., 1984. Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells. J. Physiol. ŽLondon. 355, 237–249.
208
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
Drummond, J.C., Brann, C.A., Perkins, D.E., Wolfe, D.E., 1991. A comparison of median frequency, spectral edge frequency, a frequency band power ratio, total power, and dominance shift the determination of depth of anesthesia. Acta Anaesthesiol. Scand. 35, 693–699. Granata, A.R., Numao, Y., Kumada, M., Reis, D.J., 1986. A1 noradrenergic neurons tonically inhibit sympatho-excitatory neurons of C1 area in rat brainstem. Brain Res. 377, 127–146. Guyenet, P.G., 1990. Role of the ventral medulla oblongata in blood pressure regulation. In: Loewy, A.D., Spyer, K.M. ŽEds.., Central Regulation of Autonomic Functions. Oxford Univ. Press, Oxford, pp. 145–167. Housley, G.D., Martin-Body, R.L., Dawson, N.J., Sinclair, J.D., 1991. Brainstem projections of the glossopharyngeal nerve and its carotid sinus branch in the rat. Neuroscience 22, 237–250. Huang, B.S., Leenen, F.H., 1994. Dietary Na and baroreflex modulation of blood pressure and RSNA in normotensive vs. spontaneously hypertensive rats. Am. J. Physiol. 266, H496–H502. Jeske, I., McKenna, K.E., 1992. Quantitative analysis of bulbospinal projections from the rostral ventrolateral medulla: contribution of C1-adrenergic and noradrenergic neurons. J. Comp. Neurol. 324, 1–13. Luft, F.C., Demmert, G., Rohmeiss, P., Unger, T., 1986. Baroreceptor reflex effect on sympathetic nerve activity in stroke-prone spontaneously hypertensive rats. J. Auton. Nerv. Syst. 17, 199–209. Maley, B.E., 1996. Immunohistochemical localization of neuropeptides and neurotransmitters in the nucleus solitarius. Chem. Senses 21, 367–376. Matsumoto, M., Takayama, K., Miura, M., 1994. Distribution of glutamate and GABA-immunoreactive neurons projecting vasomotor center of the intermediolateral nucleus of the lower thoracic cord of Wistar rats: a double-labeling study. Neurosci. Lett. 174, 165–168. Minson, J., Arnolda, L., Llewellyn-Smith, I., Pilowsky, P., Chalmers, J., 1996. Altered c-fos in rostral medulla and spinal cord of spontaneously hypertensive rats. Hypertension 27, 433–441. Miura, M., Reis, D.J., 1969. Termination and secondary projections of carotid sinus nerve in the cat brain stem. Am. J. Physiol. 217, 142–153. Miura, M., Takayama, K., Okada, J., 1991. Difference in sensitivity in cardiovascular and respiratory control neurons in the subretrofacial nucleus to glutamate receptor subtype agonists in SHR, WKY and cats. J. Auton. Nerv. Syst. 36, 1–12. Miura, M., Takayama, K., Okada, J., 1994a. Distribution of glutamateand GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats: a double-labeling study. Brain Res. 638, 139– 150. Miura, M., Takayama, K., Okada, J., 1994b. Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation. J. Auton. Nerv. Syst. 50, 31–43. Miura, M., Okada, J., Takayama, K., Jingu, H., 1996. Barosensitive and chemosensitive neurons in the rat medulla: a double labeling study with c-Fosrglutamate, GAD, PNMT and calbindin. J. Auton. Nerv. Syst. 61, 17–25. Murphy, A.Z., Ennis, M., Shipley, M.T., Behbehani, M.M., 1994. Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: a double-labeling study for fos and catecholamines. J. Comp. Neurol. 349, 36–50. Nosaka, S., Yamamoto, T., Yasunaga, K., 1979. Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem. J. Comp. Neurol. 186, 79–92. Nosaka, S., Yasunaga, K., Tamai, S., 1982. Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges. Am. J. Physiol. 243, R92–98. Okada, J., Miura, M., 1997. Barosensitive cardioinhibitory neurons in the
medulla: comparison of FosBrChAT positive neurons with CT-HRP labelled neurons. J. Auton. Nerv. Syst. 64, 85–90. Ross, C.A., Ruggiero, D.A., Reis, D.J., 1985. Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol. 242, 511–534. Ruggiero, D.A., Giuliano, R., Anwar, M., Stornetta, R., Reis, D.J., 1990. Anatomical substrates of cholinergic-autonomic regulation in the rat. J. Comp. Neurol. 292, 1–53. Ruggiero, D.A., Cravo, S.L., Golanov, E., Gomez, R., Anwar, M., Reis, D.J., 1994. Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis. Brain Res. 663, 107–120. Smith, J.K., Barron, K.W., 1990. Cardiovascular effects of L-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats. Brain Res. 506, 1–8. Smyth, M.A., Sleight, P., Pickering, G.W., 1969. Reflex regulation of arterial pressure during sleep in man: a quantitative method of assessing baroreflex sensitivity. Circ. Res. 24, 109–121. Suzuki, T., Takayama, K., Miura, M., 1997. Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats. Neurosci. Res. 27, 9–19. Swanson, L.W., 1992. Brain Maps: Structure of the Rat Brain. Elsevier, Amsterdam, p. 240. Takayama, K., Miura, M., 1992. Difference in distribution of glutamateimmunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: a double-labeling study. Brain Res. 570, 259–266. Takayama, K., Suzuki, T., Miura, M., 1994. The comparison of effects of various anesthetics on expression of fos protein in the rat brain. Neurosci. Lett. 176, 59–62. Talman, W.T., Lewis, S.J., 1991. Altered cardiovascular responses to glutamate and acetylcholine microinjection into the nucleus tractus solitarii of the SHR. Clin. Exp. Hypertens. A 13, 661–668. Talman, W.T., Wellendorf, L., Martinez, D., Ellison, S., Li, X., Cassel, M., Ohta, H., 1994. Glycine elicits release of acetylcholine from the nucleus tractus solitarii in rat. Brain Res. 650, 253–259. Tanaka, J.H., Kaba, H., Sato, H., Seto, K., 1985. Inputs from the A1 noradrenergic region to the hypothalamus paraventricular neurons in the rat. Brain Res. 335, 368–371. Tucker, D.C., Saper, C.B., Ruggiero, D.A., Reis, D.J., 1987. Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord. J. Comp. Neurol. 259, 591–603. van Tol, H.H., van den Buuse, M., de Jong, W., Burbach, J.P., 1988. Vasopressin and oxytocin gene expression in the supraoptic and paraventricular nucleus of the spontaneously hypertensive rat ŽSHR. during development of hypertension. Brain Res. 464, 303–311. Verberne, A.J.M., Young, N.A., Louis, W.J., 1988. Impairment of inhibitory cardiopulmonary vagal reflexes in spontaneously hypertensive rats. J. Auton. Nerv. Syst. 23, 63–68. Xiong, Y., Takayama, K., Miura, M., 1997a. Differences in the density of barosensitive neurons in the medulla of spontaneously hypertensive and Wistar Kyoto rats. Clin. Exp. Pharm. Physiol. 24, 398–402. Xiong, Y., Takayama, K., Miura, M., 1997b. Difference in distribution of barosensitive neurons in the medulla between SHR and WKY, estimated by regional density of FOSrglutamate, GAD, TH or PNMT and FosBrChAT-immunoreactive neurons. Proceedings of XXXIII International Congress of Physiological Sciences IUPS. St. Petersburg, Russia, PO58.34. Yang, Z., Chan, Y.S., Wong, T.M., 1996. Effects of kainic acid administered to the caudal ventrolateral medulla on arterial blood pressure in the spontaneously hypertensive and normotensive Wistar Kyoto rats. Neurosci. Lett. 202, 145–148.
Difference in topology and numbers of barosensitive catecholaminergic and cholinergic neurons in the medulla between SHR and WKY rats Yu Xiong, Junichi Okada, Senichi Tomizawa, Kiyoshige Takayama, Mitsuhiko Miura
)
Department of Physiology 1st DiÕision, Gunma UniÕersity School of Medicine, 3-39-22 Showa-machi, Maebashi-shi 371, Japan Received 21 January 1998; revised 2 March 1998; accepted 2 March 1998
Abstract We hypothesized that there may be a significant difference in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto ŽWKY. and spontaneously hypertensive SHR rats. Using the double-immunoreactive ŽIR. method, the topology and numbers of barosensitive neurons that contain glutamate ŽGlu., glutamic acid decarboxylase ŽGAD., tyrosine hydroxylase ŽTH., phenylethanolamine N-methyltransferase ŽPNMT. and choline acetyltransferase ŽChAT. were compared between the two strains. The control rats were sham-operated only for cannulation of the trachea and femoral arteryrvein. The test rats were injected with the pressor agent phenylephrine to raise blood pressure and stimulate arterial baroreceptors. In both the control and test experiments, the c-FosrGlu-, GAD-, TH- and PNMT-IR neurons were found in the nucleus tractus solitarii ŽNTS. and ventrolateral medulla ŽVLM., while the FosBrChAT-IR neurons were found in the NTS, dorsal motor nucleus of the vagus ŽDMX. and nucleus ambiguus ŽAMB.. In the control experiment, no significant difference in numbers was recognized in any of the double-IR neurons between the two strains. In the test experiment, the numbers of FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. The numbers of c-FosrTH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY. These results suggest that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR, and that a larger number of barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR. q 1998 Elsevier Science B.V. All rights reserved. Keywords: c-Fos; FosB; Nucleus ambiguus; Nucleus tractus solitarii; Tyrosine hydroxylase; Phenylethanolamine N-methyltransferase; Choline acetyltransferase; Ventrolateral medulla
1. Introduction The outline of the baroreceptor reflex pathway in the medulla has been established. The first-order neurons transmit baroreceptor signals to the second-order neurons in the nucleus tractus solitarii ŽNTS. ŽCiriello, 1983; Ciriello et al., 1981; Housley et al., 1991; Miura and Reis, 1969.. The second-order neurons in turn project to the dorsal motor nucleus of the vagus ŽDMX., the ambiguus nucleus ŽAMB. ŽNosaka et al., 1979, 1982. and the ventrolateral medulla ŽVLM. ŽGuyenet, 1990; Miura et al., 1994b; Suzuki et al., 1997.. The baroreceptor-cardiac vagal reflex is transmitted via the NTS–DMXrAMB pathway ŽOkada and Miura, 1997; Ross et al., 1985., while the baroreceptor-sympathetic nerve reflex via the NTS–VLM pathway ) Corresponding author. Tel.: q81 27 2207923; fax: q81 27 2207926; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 5 2 - 6
ŽMatsumoto et al., 1994; Miura et al., 1994a,b; Ross et al., 1985.. Then, we hypothesized that there may be significant differences in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto ŽWKY. and spontaneously hypertensive SHR rats, because the baroreceptor-cardiac vagal reflex is weaker in SHR than in WKY ŽHuang and Leenen, 1994; Luft et al., 1986; Verberne et al., 1988.. Many studies have showed differences among properties of neurons in the NTS, AMB and VLM between WKY and SHR, by using electrical stimulation ŽChan et al., 1990, 1991., chemical stimulation ŽMiura et al., 1991; Smith and Barron, 1990; Yang et al., 1996. and immunohistochemistry ŽMinson et al., 1996; Miura et al., 1994a; Murphy et al., 1994; Takayama and Miura, 1992; Xiong et al., 1997a,b.. Recently, we applied the double-immunoreactive ŽIR. method to normotensive Wistar rats, and succeeded in identifying c-Fosrglutamate ŽGlu.-, glu-
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
tamic acid decarboxylase ŽGAD.-, phenylethanolamine Nmethyltransferase ŽPNMT.- and tyrosine hydroxylase ŽTH.-IR neurons in the NTS and VLM ŽMiura et al., 1996. and FosBrcholine acetyltransferase ŽChAT.-IR neurons in the AMB ŽOkada and Miura, 1997.. To further test the aforementioned hypothesis, we sought evidence of significant differences in the numbers of barosensitive neurons containing Glu, GAD, PNMT, TH or ChAT between WKY and SHR. A preliminary report on the results obtained in the present study was presented in abstract form ŽXiong et al., 1997b.. 2. Materials and methods 2.1. General care Experiments were performed using 10- – 13-week-old male SHR and age-matched male WKY weighing 210–280 g Ž n s 16; Charles River Japan, Yokohama, Japan.. To avoid the restlessness characteristic of rats housed alone, pairs of rats were kept in cages ŽMiura et al., 1996.. To extinguish c-Fos expression that might have been present before isolation, pairs of rats were kept in a dark and soundless room for 12 h before the experiment. Anesthesia was induced with halothane and maintained with fentanyl Žinitially 50 m grkg, i.p., and then 25 m grkg, i.p., every 30 min; Sankyo, Tokyo, Japan. and midazolam Žinitially 5 mgrkg, i.p., and then 2.5 mgrkg, i.p., every 30 min; Yamanouchi, Tokyo, Japan.. Fentanylrmidazolam is the optimal anesthesia for excluding false-positive expression induced by anesthetics themselves ŽTakayama et al., 1994.. EEGs were continuously recorded via a fronto-occipital lead. For standardization of the anesthesia level, the ratio of power in the 8–20 Hz Ž a q b . band to power in the 0–4 Hz Ž d . band was set at 0.5–1.0 ŽDrummond et al., 1991.. The trachea was cannulated with vinyl tubing Ž2 mm i.d.. A femoral artery and vein were cannulated with polyethylene catheter Ž0.58 mm i.d... The relative tidal volume Ž VT . measured by a thermistor ŽPB7-43-S2; Shibaura Electronics, Japan., the respiratory frequency ŽRF. measured by a tachometer ŽAT601G; Nihon Kohden, Japan., the systemic arterial pressure ŽAP. measured by pressure transducer ŽTP602T, Nihon Kohden. and heart rate ŽHR. computed from the pressure pulse by a cardiotachometer ŽRT-5, Nihon Kohden. were displayed on a polygraph ŽRJG-4226, Nihon Kohden.. When necessary, arterial blood was sampled from a femoral artery and drugs were injected from a femoral vein. The head of the animal was placed on a stereotaxic frame Žbite bar set at y3.0 mm.. Rectal temperature was maintained at 378C with an infrared heat lamp. 2.2. Experimental procedure Care was taken to avoid c-Fos expression due to nonspecific stimulation, i.e. light, sound, odor and pain. The
201
control and test experiments were performed using four WKY and four SHR, respectively. The control rats were sham-operated for cannulation of the trachea, femoral artery and vein, and injected with 40–60 nl saline solution to confirm that no cardiovascular response had been induced. The test rats were injected with 40–60 nl of 2.5 mM L-phenylephrine saline solution Ž100 m grkg, i.v.; Sigma, St. Louis, MO, USA. at a speed of one shot per minute ŽSmyth et al., 1969. to induce the pressor and reflex bradycardic responses. Injections were repeated five times at intervals of 3–4 min ŽMiura et al., 1996, 1994b.. Before and after baroreceptor stimulation, 0.1 ml of blood was extracted to measure PaCO 2 , PaO 2 and pH, and a similar volume of compensatory saline solution was injected to prevent a blood pressure fall. Ninety minutes after cessation of the sham operation or administration of phenylephrine, rats were additionally anesthetized with pentobarbital sodium Ž50 mgrkg of body weight, i.p.. and perfused transcardially with heparinized saline, followed by 0.5% glutaraldehyde and 4% paraformaldehyde ŽPFA. in 0.1 M phosphate buffer ŽPB., and with 4% PFA in 0.1 M PB. The perfusate pH was set at 7.4. The brains were removed, postfixed in the same fixative for 2 h and soaked in 10%, 20% and another 20% sucrose PB solution. The medulla oblongata and pons were frozen and cut at 40 m m in frontal series. 2.3. Immunohistochemistry First, five sets of every fifth section were rinsed, then incubated in 0.5% bovine serum albumin ŽBSA. in 0.1 M Tris–HCl buffer for 20 min at 208C. Four sets of sections were incubated with sheep anti-cFos-polyclonal antiserum ŽOA-11-824, Genosys Biotechnologies, UK. at a dilution of 1:1000 for 20 h at 48C. Then, the sections were incubated in 0.1 M Tris–HCl buffer containing biotinylated anti-sheep IgG antiserum ŽVector Labs., USA. for 1 h at 208C. One set of sections was incubated with rabbit anti-FosB polyclonal antiserum Žsc-48, Santa Cruz Biotechnology, USA. at a dilution of 1:1000 for 20 h at 48C. Then, the sections were incubated in 0.1 M Tris–HCl buffer containing biotinylated anti-rabbit IgG antiserum ŽVector Labs.. for 1 h at 208C. Subsequently, all sets of sections were processed with avidin–biotin peroxidase complex ŽABC, Vector Labs.. for 1 h at 208C and treated with diaminobenzidine ŽDAB.nickel solution containing 0.003% H 2 O 2 . Secondly, five sets of sections were rinsed, incubated in 0.5% BSA in 0.1 M Tris–HCl buffer, and incubated for 20 h at 48C with one of the antisera to common neurotransmitters and related enzymes listed below: Ž1. rabbit antiglutamate ŽGlu. polyclonal antiserum Ž1:1000; SFRI, France., Ž2. rabbit anti-glutamic acid decarboxylase ŽGAD. polyclonal antiserum Ž1:500; Chemicon International, USA., Ž3. rabbit anti-tyrosine hydroxylase ŽTH. polyclonal
202
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
antiserum Ž1:3000; Chemicon International., Ž4. rabbit anti-phenylethanolamine N-methyltransferase ŽPNMT. polyclonal antiserum Ž1:3000; Chemicon International., and Ž5. mouse anti-cholineacetyltransferase ŽChAT. monoclonal antiserum Ž1:500; Chemicon International.. Finally, sections were incubated in 0.1 M Tris–HCl buffer containing: Ž1. – Ž4. biotinylated anti-rabbit IgG antiserum ŽVector Labs..; Ž5. biotinylated anti-mouse IgG antiserum ŽVector Labs.. for 1 h at 208C and in ABC solution for 1 h at 208C and treated with DAB solution containing 0.003% H 2 O 2 . The data sheets provided by the manufacturers indicated the following: Ž1. ELISA showed the anti-Glu antiserum to have cross-reactivity with aspartate Ž0.006%., b-alanine Ž- 0.001%., GABA Ž- 0.001%., glycine Ž0.001%. and taurine Ž- 0.001%.; Ž2. the anti-GAD antiserum was made using recombinant-DNA GAD, and Western blot analysis revealed the anti-GAD antiserum to recognize specifically the larger form of GAD Ž67 kDa.; Ž3. no staining was observed immunocytochemically when the anti-TH and anti-PNMT antisera were preabsorbed with TH and PNMT. Sections were mounted on slides and coverslipped with Permount ŽFisher, USA.. The neurons in the medulla were surveyed light microscopically. Brain histology was checked against the brain atlas of Swanson Ž1992.. Dark blue particles confined to the nucleus were identified as expression products of c-fos and FosB. In the cytoplasma, homogeneously brown particles were identified as reaction products of bioactive substances. The IR neurons of each nucleus in the medulla were counted and summed for each division. The reproducibility of staining was checked by comparing representative stained neurons in sections from different animals, such as GAD-stained Purkinje neurons in the cerebellum. The significance of differences in the numbers of double-IR neurons was evaluated using the Mann– Whitney test, with P - 0.05 being taken as statistically significant.
Žcaudal to the obex., while the 4th–5th divisions belong to the rostral medulla Žrostral to the obex.. According to the criteria described above, the NTS at the level of the 1st division of the medulla was abbreviated as the 1st division of the NTS. The other divisions follow this example.
3. Results 3.1. Effects of pressor stimulation on hemodynamics Effects of administration of phenylephrine Žpressor stimulation. on mean arterial blood pressure ŽMAP. and heart rate ŽHR. were compared before vs. after intravenous administration of 100 m grkg phenylephrine and between WKY and SHR. The pressor stimulation significantly elevated MAP ŽFig. 1A, left., but significantly lowered HR ŽFig. 1A, right.. The increases in MAP were 58 " 8 and
2.4. DiÕision of the medulla In place of the numerical coordinates, we used five divisions of the medulla oblongata according to the topology of the important nuclei located along the rostrocaudal axis ŽMiura et al., 1996.. The 1st division extends from the medullospinal border to the caudal end of the inferior olivary complex Žaverage of four sections per set, 4 = 5 = 40 m m s 0.8 mm.. Ahead of the 1st division, the 2nd division extends to the caudal end of the area postrema Žaverage of six sections, ca. 1.2 mm.. Similarly, the 3rd division extends to the obex Žaverage of four sections, ca. 0.8 mm., the 4th to the rostral end of the inferior olivary complex Žaverage of five sections, ca. 1.0 mm., and the 5th division terminates at the caudal end of the nucleus of the trapezoid body Žaverage of seven sections, ca. 1.4 mm.. The 1st–3rd divisions belong to the caudal medulla
Fig. 1. Effects of intravenous administration of 100 m grkg phenylephrine on hemodynamics in WKY and SHR. ŽA. Significant rise in mean arterial blood pressure Žleft. and significant fall in heart rate Žright. after administration of phenylephrine in both strains. ŽB. No significant difference in MAP changes between the two strains Ž DMAP, left., but difference in heart rate changes is significant Ž DHR, right.. ŽC. and ŽD. Examples of pressor and bradycardic reflex responses elicited by administration of phenylephrine in WKY ŽC. and SHR ŽD.. Abbreviations: a, after; AP, arterial blood pressure; b, before; HR, heart rate; MAP, mean arterial blood pressure; S, SHR; W, WKY. ) P - 0.05; ) ) P - 0.01, estimated by t-test.
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
203
58 " 7 mm Hg in WKY and SHR, respectively ŽFig. 1B, left., not significantly different. On the other hand, the decrease in HR was 90 " 10 bpm in WKY and 44 " 7 bpm in SHR ŽFig. 1B, right., with a statistically significant difference between the two strains Ž P - 0.05.. Fig. 1C, D shows examples of the pressor and reflex bradycardic responses produced by i.v. administration of 100 m grkg phenylephrine. In both strains, the PaCO 2 , PaO 2 and pH of arterial blood were in the range of 40–46 mm Hg, 88–113 mm Hg and 7.32–7.37 prior to stimulation, and 40–46 mm Hg, 84–105 mm Hg and 7.31–7.47 after stimulation, respectively. There were no significant differences in these parameters before vs. after stimulation. Since the PaCO 2 , PaO 2 and arterial blood pH were maintained within normal
Fig. 3. Histograms of averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in five rostrocaudal divisions of the NTS, DMX, AMB and VLM. Open columns, WKY. Oblique-line columns, SHR. ŽA. Control experiment Žmean"S.E.M., ns 4.. ŽB. Test experiment Žmean"S.E.M., ns 4.. 1–5, 1st–5th divisions of the medulla. Dotted line divides the VLM and AMB into their caudal Ž1–3. and rostral Ž4–5. divisions. ) P - 0.05 estimated by Mann–Whitney test.
range, changes in arterial blood gases and pH are unlikely to have influenced c-Fos expression ŽMiura et al., 1996.. 3.2. AÕeraged numbers of double-IR neurons in NTS, AMB and VLM
Fig. 2. Comparison of averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-immunoreactive ŽIR. neurons in the NTS, DMX, AMB and VLM between WKY and SHR. ŽA. Control experiment Žmean"S.E.M., ns 4.. ŽB. Test experiment after administration of phenylephrine Žmean"S.E.M., ns 4.. Open columns, WKY. Obliqueline columns, SHR. ) P - 0.05, estimated by Mann–Whitney test. Abbreviations: ChAT, choline acetyltransferase; GAD, glutamic acid decarboxylase; Glu, glutamate; PNMT, phenylethanolamine N-methyltransferase; TH, tyrosine hydroxylase.
In both control and test experiments Žfour each., all c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons were counted in WKY and SHR Žfour animals each.. They were distributed evenly on both sides of the NTS, DMX, AMB and VLM in the medulla. The NTS, DMX and AMB are distinct nuclei with borders, while the VLM is not a distinct nucleus but part of the reticular formation located in the ventrolateral medulla without borders. Not only in our previous studies ŽMiura et al., 1994b, 1996. but also in the present study, we found that the barosensitive Fos-IR neurons are distributed in the VLM, covering the caudal reticular formation, just dorsal to the nucleus reticularis lateralis at the 1st–3rd divisions of the medulla, and the rostral reticular formation, just ventral to the AMBrretrofacial nucleus in the 4th–5th divisions of the medulla. Numbers of the c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in the NTS, DMX, AMB and VLM were counted, averaged and compared between WKY and SHR. In the control experiment ŽFig. 2A., no signifi-
204
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
Fig. 4. Topology of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons, identified after phenylephrine administration, distributed in the NTS, DMX, AMB and VLM throughout four divisions of the medulla Ž1–4.. ŽA. WKY. ŽB. SHR. One dot corresponds to one neuron for the FosBrChAT-IR neurons, but two neurons for the others.
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
205
Fig. 5. Photomicrographs of double-IR neurons in the ventrolateral medulla of WKY and SHR after administration of phenylephrine. ŽA. – ŽE.: c-FosrGluŽA., GAD- ŽB., TH- ŽC., PNMT- ŽD. and FosBrChAT-IR neuron ŽE.. Left, sites of neurons indicated by arrows. Right, magnified photomicrographs of corresponding neurons. Bar, 500 m m on the left; 20 m m on the right.
206
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
cant difference was found between the two. On the other hand, in the test experiment ŽFig. 2B., the averaged numbers of total FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. Inversely, the averaged numbers of total c-FosrTH- and PNMT-IR neurons in the VLM were significantly larger in SHR than in WKY. 3.3. AÕeraged numbers of double-IR neurons in fiÕe diÕisions of the medulla The averaged numbers of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons were calculated in five rostrocaudal divisions of the NTS, DMX, AMB and VLM, and were compared between WKY and SHR. In the control experiment ŽFig. 3A., no significant difference in the averaged numbers between WKY and SHR was recognized. On the other hand, in the test experiment ŽFig. 3B., the averaged numbers of the FosBrChAT-IR neurons in the 3rd and 4th divisions of the AMB were significantly smaller in SHR than in WKY. Inversely, the averaged numbers of the c-FosrTH-IR neurons in the 2nd and 3rd divisions of the VLM were significantly larger in SHR than in WKY. As described above, a significant difference was found in the averaged numbers of the total c-FosrPNMT-IR neurons in the VLM and the total FosBrChAT-IR neurons in the NTS and DMX between the two strains, whereas there was no significant difference in the averaged numbers of these IR neurons in the 1st to 5th divisions. 3.4. Topology of double-IR neurons in WKY and SHR The topology of c-FosrGlu-, GAD-, TH-, PNMT- and FosBrChAT-IR neurons in the test experiment was compared against the 1st–4th divisions of the medulla between WKY ŽFig. 4A. and SHR ŽFig. 4B.. The number of double-IR neurons in the 5th division was too low to mark on the map. In the 3rd and 4th divisions of the AMB, the numbers of FosBrChAT-IR neurons were smaller in SHR than in WKY, whereas in the 2nd and 3rd divisions of the VLM, the numbers of c-FosrTH-IR neurons were larger in SHR than in WKY. Fig. 5 shows photomicrographs of the double-IR neurons found in the VLM of WKY and SHR after administration of phenylephrine. Since the nucleoplasm of the double-IR neurons was stained dark and the cytoplasm was homogeneously brown, the double-IR neurons were clearly differentiated from single c-Fos or FosB-labeled neurons and single Glu-, GAD-, TH-, PNMT- and ChAT-IR neurons. The diameter of these double-IR neurons in the VLM was as small as 20 m m in the larger axis, i.e. approximately one-third the diameter of motor neurons in the AMB ŽFig. 5E..
4. Discussion Many studies have shown that in normotensive Wistar rats barosensitive c-Fos-IR neurons are present in the NTS and VLM, and contain Glu, GAD, TH, PNMT and ChAT ŽBadoer et al., 1994; Chan and Sawchenko, 1994; Minson et al., 1996; Miura et al., 1996; Murphy et al., 1994; Okada and Miura, 1997.. In this context, differences in the topology and numbers of these barosensitive neurons between WKY and SHR have been sought. In our previous study ŽXiong et al., 1997a., using the single c-Fos method, we found that the number of barosensitive neurons in the caudal VLM was significantly larger in SHR than in WKY, whereas the number of barosensitive neurons in the rostral VLM was significantly larger in WKY than in SHR. Further, in the present study, stimulation of the arterial baroreceptors revealed differences in the neuronal composition of the baroreceptor reflex pathway in the medulla between WKY and SHR. One important finding was that baroreceptor stimulation made a significant difference in the number of cholinergic neurons. On a larger scale, numbers of the FosBrChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY, while on a smaller scale the numbers of FosBrChAT-IR neurons in the 3rd and 4th divisions of the AMB were significantly smaller in SHR than in WKY. Recently, we found that baroreceptor stimulation induced FosB expression in cholinergic neurons in the NTS, DMX and AMB. These cholinergic neurons in the DMX and AMB give off descending cardioinhibitory fibers, as shown by Nosaka et al. Ž1979, 1982., while those in the NTS do not innervate the heart ŽOkada and Miura, 1997.. Many studies demonstrated the presence of cholinergic neurons in the NTS ŽMaley, 1996; Ruggiero et al., 1990; Talman et al., 1994.. Talman and Lewis suggested that cardiovascular regulation is disturbed in SHR, when acetylcholine is injected into the NTS ŽTalman and Lewis, 1991.. However, the physiological role of cholinergic neurons in the NTS remains unknown. In the behavioral study, we confirmed that the bradycardic reflex response to baroreceptor stimulation was significantly weaker in SHR than in WKY. This finding is similar to the results of previous studies by Luft et al. Ž1986. and Verberne et al. Ž1988., that is, the sensitivities of the cardioinhibitory reflexes are more diminished in SHR than in WKY. Therefore, it is possible that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR. Another important finding was that baroreceptor stimulation resulted in a significant difference in the number of catecholaminergic neurons between the two strains. On a larger scale, the numbers of c-FosrTH- and PNMT-IR neurons in the VLM were significantly larger in SHR than in WKY, while on a smaller scale the numbers of cFosrTH-IR neurons in the 2nd and 3rd divisions in the
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
VLM were significantly larger in SHR than in WKY. These results indicate that stimulation of the arterial baroreceptors induced stronger excitation of catecholamninergic neurons in the VLM of SHR. Since the direct effect of phenylephrine on peripheral resistance vessels predominates, the depressor reflex response to phenylephrine was not discernible on the blood pressure record. It is certain, however, that baroreceptor signals were transmitted to neurons of the NTS and VLM in the baroreceptor-sympathetic nerve reflex pathway. Smith and Barron Ž1990. and Yang et al. Ž1996. found that the depressor response to glutamate stimulation of the caudal VLM is significantly greater in SHR than in WKY. Granata et al. Ž1986. reported that the noradrenergic neurons in the caudal VLM tonically inhibit sympathoexcitatory adrenergic neurons in the rostral VLM. Inversely, another group of investigators reported that noradrenergic neurons in the caudal VLM are not involved in the baroreceptor-depressor reflex ŽBlessing and Willoughby, 1987; Day et al., 1983.. Recently, we confirmed that the catecholaminergic projection from the depressor area in the caudal VLM to the pressor area of the rostral VLM is weak, whereas the GABAergic projection is strong ŽSuzuki et al., 1997.. These findings suggest that catecholaminergic neurons in the caudal VLM play a minor role in the inhibition of sympathetic vasomotor neurons in the rostral VLM. Many investigators reported that catecholaminergic neurons in the rostral VLM give off descending fibers to sympathetic vasomotor preganglionic neurons in the intermediolateral nucleus ŽIMLN. of the lower thoracic cord, thereby directly inhibiting these vasomotor neurons ŽJeske and McKenna, 1992; Ruggiero et al., 1994; Tucker et al., 1987.. Although GABAergic neurons in the caudal VLM were verified to project to the IMLN of the lower thoracic cord ŽMatsumoto et al., 1994., no report has demonstrated catecholaminergic neurons projecting from the caudal VLM to the IMLN of the spinal cord. These findings suggest that catecholaminergic neurons in the caudal VLM have no descending projection to the IMLN. On the other hand, ascending projections from the caudal VLM to the paraventricular nucleus of the hypothalamus ŽPVH., a site of vasopressin secretion, merit consideration. Vasopressin mRNA levels are higher in SHR than WKY Žvan Tol et al., 1988., and the plasma level of vasopressin is also higher in SHR than in WKY ŽCrofton et al., 1978.. Since the PVH receives projections from adrenergic and noradrenergic neurons in the caudal VLM ŽTanaka et al., 1985; Tucker et al., 1987., it is possible that these catecholaminergic neurons stimulate vasopressin-secreting neurons in the PVH ŽDay et al., 1984. and antagonize the depressor Žbaroreceptor-sympathetic nerve. reflex. The greater number of c-FosrTH-IR neurons in SHR suggests that the antagonizing effect on the depressor reflex may be more potent in SHR than in WKY. Therefore, we suggest that a larger number of
207
barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR. We conclude that Ž1. without pressor stimulation, there is no significant difference in the numbers of FosBrChAT-IR neurons in the NTS, DMX and AMB between WKY and SHR or in the numbers of c-FosrGlu-, GAD-, TH- and PNMT-IR neurons in the NTS and VLM; Ž2. with pressor stimulation, the numbers of FosBrChATIR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY, which may cause the weaker baroreceptor-cardiac vagal reflex in SHR. Inversely, the numbers of c-FosrTH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY, which may cause involvement of catecholaminergic neurons in the caudal VLM in the stronger baroreceptorvasopressin reflex in SHR.
Acknowledgements This work was supported in part by a Grant-in-Aid for encouragement of visitor scientist from the Sasakawa Health Science Foundation to Y. Xiong.
References Badoer, E., McKinley, M.J., Oldfield, B.J., McAllen, R.M., 1994. Localization of barosensitive neurons in the caudal ventrolateral medulla which project to the rostral ventrolateral medulla. Brain Res. 657, 258–268. Blessing, W.W., Willoughby, J.O., 1987. Depressor neurons in rabbit caudal medulla do not transmit the baroreceptor-vasomotor reflex. Am. J. Physiol. 253, H777–786. Chan, P.K.W., Sawchenko, P.E., 1994. Spatially and temporally differentiated patterns of fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J. Comp. Neurol. 348, 433–460. Chan, R.K.W., Chan, T.S., Wong, M., 1990. Cardiovascular responses to electrical stimulation of the ventrolateral medulla of the spontaneously hypertensive rat. Brain Res. 522, 99–106. Chan, R.K.W., Chan, T.S., Wong, M., 1991. Electrophysiological properties of neurons in the rostral ventrolateral medulla of normotensive Wistar Kyoto and spontaneously hypertensive rats. Brain Res. 549, 118–127. Ciriello, J., 1983. Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci. Lett. 36, 37–42. Ciriello, J., Hrycyshyn, A.W., Calaresu, F.R., 1981. Glossopharyngeal and vagal afferent projections to the brain stem of the cat: a horseradish peroxidase study. J. Auton. Nerv. Syst. 4, 63–79. Crofton, J.T., Share, L., Shade, R.E., Allen, C., Tarnowski, D., 1978. Vasopressin in the rat with spontaneous hypertension. Am. J. Physiol. 235, H361–H366. Day, T.A., Ro, A., Renaud, L.P., 1983. Depressor area within caudal ventrolateral medulla of the rat does not correspond to the A1 catecholamine cell group. Brain Res. 279, 299–302. Day, T.A., Ferguson, A.V., Renaud, L.P., 1984. Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells. J. Physiol. ŽLondon. 355, 237–249.
208
Y. Xiong et al.r Journal of the Autonomic NerÕous System 70 (1998) 200–208
Drummond, J.C., Brann, C.A., Perkins, D.E., Wolfe, D.E., 1991. A comparison of median frequency, spectral edge frequency, a frequency band power ratio, total power, and dominance shift the determination of depth of anesthesia. Acta Anaesthesiol. Scand. 35, 693–699. Granata, A.R., Numao, Y., Kumada, M., Reis, D.J., 1986. A1 noradrenergic neurons tonically inhibit sympatho-excitatory neurons of C1 area in rat brainstem. Brain Res. 377, 127–146. Guyenet, P.G., 1990. Role of the ventral medulla oblongata in blood pressure regulation. In: Loewy, A.D., Spyer, K.M. ŽEds.., Central Regulation of Autonomic Functions. Oxford Univ. Press, Oxford, pp. 145–167. Housley, G.D., Martin-Body, R.L., Dawson, N.J., Sinclair, J.D., 1991. Brainstem projections of the glossopharyngeal nerve and its carotid sinus branch in the rat. Neuroscience 22, 237–250. Huang, B.S., Leenen, F.H., 1994. Dietary Na and baroreflex modulation of blood pressure and RSNA in normotensive vs. spontaneously hypertensive rats. Am. J. Physiol. 266, H496–H502. Jeske, I., McKenna, K.E., 1992. Quantitative analysis of bulbospinal projections from the rostral ventrolateral medulla: contribution of C1-adrenergic and noradrenergic neurons. J. Comp. Neurol. 324, 1–13. Luft, F.C., Demmert, G., Rohmeiss, P., Unger, T., 1986. Baroreceptor reflex effect on sympathetic nerve activity in stroke-prone spontaneously hypertensive rats. J. Auton. Nerv. Syst. 17, 199–209. Maley, B.E., 1996. Immunohistochemical localization of neuropeptides and neurotransmitters in the nucleus solitarius. Chem. Senses 21, 367–376. Matsumoto, M., Takayama, K., Miura, M., 1994. Distribution of glutamate and GABA-immunoreactive neurons projecting vasomotor center of the intermediolateral nucleus of the lower thoracic cord of Wistar rats: a double-labeling study. Neurosci. Lett. 174, 165–168. Minson, J., Arnolda, L., Llewellyn-Smith, I., Pilowsky, P., Chalmers, J., 1996. Altered c-fos in rostral medulla and spinal cord of spontaneously hypertensive rats. Hypertension 27, 433–441. Miura, M., Reis, D.J., 1969. Termination and secondary projections of carotid sinus nerve in the cat brain stem. Am. J. Physiol. 217, 142–153. Miura, M., Takayama, K., Okada, J., 1991. Difference in sensitivity in cardiovascular and respiratory control neurons in the subretrofacial nucleus to glutamate receptor subtype agonists in SHR, WKY and cats. J. Auton. Nerv. Syst. 36, 1–12. Miura, M., Takayama, K., Okada, J., 1994a. Distribution of glutamateand GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats: a double-labeling study. Brain Res. 638, 139– 150. Miura, M., Takayama, K., Okada, J., 1994b. Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation. J. Auton. Nerv. Syst. 50, 31–43. Miura, M., Okada, J., Takayama, K., Jingu, H., 1996. Barosensitive and chemosensitive neurons in the rat medulla: a double labeling study with c-Fosrglutamate, GAD, PNMT and calbindin. J. Auton. Nerv. Syst. 61, 17–25. Murphy, A.Z., Ennis, M., Shipley, M.T., Behbehani, M.M., 1994. Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: a double-labeling study for fos and catecholamines. J. Comp. Neurol. 349, 36–50. Nosaka, S., Yamamoto, T., Yasunaga, K., 1979. Localization of vagal cardioinhibitory preganglionic neurons within rat brain stem. J. Comp. Neurol. 186, 79–92. Nosaka, S., Yasunaga, K., Tamai, S., 1982. Vagal cardiac preganglionic neurons: distribution, cell types, and reflex discharges. Am. J. Physiol. 243, R92–98. Okada, J., Miura, M., 1997. Barosensitive cardioinhibitory neurons in the
medulla: comparison of FosBrChAT positive neurons with CT-HRP labelled neurons. J. Auton. Nerv. Syst. 64, 85–90. Ross, C.A., Ruggiero, D.A., Reis, D.J., 1985. Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol. 242, 511–534. Ruggiero, D.A., Giuliano, R., Anwar, M., Stornetta, R., Reis, D.J., 1990. Anatomical substrates of cholinergic-autonomic regulation in the rat. J. Comp. Neurol. 292, 1–53. Ruggiero, D.A., Cravo, S.L., Golanov, E., Gomez, R., Anwar, M., Reis, D.J., 1994. Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis. Brain Res. 663, 107–120. Smith, J.K., Barron, K.W., 1990. Cardiovascular effects of L-glutamate and tetrodotoxin microinjected into the rostral and caudal ventrolateral medulla in normotensive and spontaneously hypertensive rats. Brain Res. 506, 1–8. Smyth, M.A., Sleight, P., Pickering, G.W., 1969. Reflex regulation of arterial pressure during sleep in man: a quantitative method of assessing baroreflex sensitivity. Circ. Res. 24, 109–121. Suzuki, T., Takayama, K., Miura, M., 1997. Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats. Neurosci. Res. 27, 9–19. Swanson, L.W., 1992. Brain Maps: Structure of the Rat Brain. Elsevier, Amsterdam, p. 240. Takayama, K., Miura, M., 1992. Difference in distribution of glutamateimmunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: a double-labeling study. Brain Res. 570, 259–266. Takayama, K., Suzuki, T., Miura, M., 1994. The comparison of effects of various anesthetics on expression of fos protein in the rat brain. Neurosci. Lett. 176, 59–62. Talman, W.T., Lewis, S.J., 1991. Altered cardiovascular responses to glutamate and acetylcholine microinjection into the nucleus tractus solitarii of the SHR. Clin. Exp. Hypertens. A 13, 661–668. Talman, W.T., Wellendorf, L., Martinez, D., Ellison, S., Li, X., Cassel, M., Ohta, H., 1994. Glycine elicits release of acetylcholine from the nucleus tractus solitarii in rat. Brain Res. 650, 253–259. Tanaka, J.H., Kaba, H., Sato, H., Seto, K., 1985. Inputs from the A1 noradrenergic region to the hypothalamus paraventricular neurons in the rat. Brain Res. 335, 368–371. Tucker, D.C., Saper, C.B., Ruggiero, D.A., Reis, D.J., 1987. Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord. J. Comp. Neurol. 259, 591–603. van Tol, H.H., van den Buuse, M., de Jong, W., Burbach, J.P., 1988. Vasopressin and oxytocin gene expression in the supraoptic and paraventricular nucleus of the spontaneously hypertensive rat ŽSHR. during development of hypertension. Brain Res. 464, 303–311. Verberne, A.J.M., Young, N.A., Louis, W.J., 1988. Impairment of inhibitory cardiopulmonary vagal reflexes in spontaneously hypertensive rats. J. Auton. Nerv. Syst. 23, 63–68. Xiong, Y., Takayama, K., Miura, M., 1997a. Differences in the density of barosensitive neurons in the medulla of spontaneously hypertensive and Wistar Kyoto rats. Clin. Exp. Pharm. Physiol. 24, 398–402. Xiong, Y., Takayama, K., Miura, M., 1997b. Difference in distribution of barosensitive neurons in the medulla between SHR and WKY, estimated by regional density of FOSrglutamate, GAD, TH or PNMT and FosBrChAT-immunoreactive neurons. Proceedings of XXXIII International Congress of Physiological Sciences IUPS. St. Petersburg, Russia, PO58.34. Yang, Z., Chan, Y.S., Wong, T.M., 1996. Effects of kainic acid administered to the caudal ventrolateral medulla on arterial blood pressure in the spontaneously hypertensive and normotensive Wistar Kyoto rats. Neurosci. Lett. 202, 145–148.