Brain Research, 570 (1992) 259-266 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

259

BRES 17382

Difference in distribution of glutamate-immunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: a double-labeling study Kiyoshige Takayama and Mitsuhiko Miura Department of Physiology 1st Division, Gunma University School of Medicine, Maebashi (Japan) (Accepted 10 September 1991)

Key words: Glutamate; Subretrofacial nucleus; Immunohistochemistry; Rostral ventrolateral medulla; Spontaneously hypertensive rat; Hypertension; Lateral parabrachial nucleus; Nucleus tractus solitarii

Glutamate immunoreactivity was found in 19% and 21% of the neurons of the central autonomic nuclei projecting into the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), respectively, using a double-labeling technique in combination with glutamate immunocytochemistry. Double-labeled neurons were distributed in 22 nuclei or subnuclei in the limbic system, hypothalamus, midbrain, pons and medulla. The average number of glutamate-immunoreactive neurons per thousand in SHR was significantly higher in the ipsilateral lateral parabrachial nucleus (P < 0.05) and Koelliker-Fuse nucleus (P < 0.01) than in WKY, while it was significantly lower in the ipsilateral medial subnucleus (P < 0.05) and the commissure subnucleus (P < 0.05) of the nucleus tractus solitarii in SHR than in WKY. The results indicate that: (1) glutamate-immunoreactive neurons (possibly glutamatergic) in many central autonomic nuclei project into the sympathetic vasomotor control neurons in the SRF; (2) the large population of glutamate-immunoreactive neurons in the lateral parabrachial nucleus and the Koelhker-Fuse nucleus of SHR is likely to increase excitatory inputs to the SRF vasomotor control neurons, while the smaller population of glutamate-immunoreactive neurons in the medial and commissure subnuclei of the nucleus tractus solitarii is likely to decrease excitatory inputs to the GABAergic neurons intrinsic to the SRF. INTRODUCTION

an immunocytochemical marker, and at the same time

Vasomotor tone is controlled by the tonic and phasic activity of sympathetic p r e m o t o r n e u r o n s in the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla (RVLM) 2'5'16'27'28'29, however, the source of the

identify their projection into the SRF by means of the retrograde transport of horseradish peroxidase (HRP). We also sought to determine whether the projection of glutamate-immunoreactive n e u r o n s differs in S H R and WKY. A preliminary report of this study has been presented in the form of an abstract 36.

drive which maintains the activity of the sympathetic premotor n e u r o n s in the SRF of the R V L M and the transmitter involved in the synaptic process remains unknown. We recently found evidence that the vasomotor control SRF n e u r o n s of the spontaneously hypertensive rat (SHR) are more sensitive to glutamate receptor subtype agonists than those of the W i s t a r - K y o t o rat (WKY) 2°. This suggests that glutamate may be a candidate for the transmitter which drives the activity of the SRF vasomotor control neurons. Although m a n y anatomical studies have demonstrated the presence of afferent projections from supramedullary and medullary structures 14'31'33'37 to the R V L M , no systematic survey has b e e n performed to determine whether glutamatergic neurons project to the SRF. In this study, we attempted to identify glutamateimmunoreactive n e u r o n s using antiserum to glutamate as

MATERIALS AND METHODS

General care Experiments were performed on 16-20-week-old male SHR (n = 3) and age-matched male WKY (n = 3) weighing 320-360 g. The rats were anesthetized with urethane (initially 600 mg/kg, i.p.; thereafter 60 mg/kg, i.v., every hour and a-chloralose (initially 60 mg/kg, i.p.; thereafter 6 mg/kg, i.v., every hour). The trachea was cannulated with vinyl tubing (2 mm in diameter) to sample airway gas (Beckman, LB2 and OMll) and measure tidal volume (Nihon Kohden, AQ-601G and AR-601G). The femoral vein was cannuluted to inject anesthetic drugs. The femoral artery was cannulated to measure arterial blood pressure and sample blood, paCO2 and PaO2 were maintained at 40 + 5 mm Hg and 90 + 10 mm Fig, respectively. Heart rate was counted using a cardiotachometer triggered by the R wave of ECG. Arterial blood pressure, mean arterial blood pressure, heart rate, CO2 and 0 2 concentration in the airway gases, and tidal volume were displayed on a polygraph. Rectal temperature was maintained at 37°C with a heating lamp. The heads of the animals were placed in a stereotaxic frame inclined

Correspondence: M. Miura, Department of Physiology 1st Division, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashishi 371, Japan. Fax: (81) (272) 33 7600.

260 10°. Part of the cerebellum and the floor of the fourth ventricle were exposed and covered with 0.9% NaC1 solution containing 1.0% agar to prevent evaporative cooling of the brain surface.

Electrode A triple-barreled glass microelectrode, developed in our own laboratory 19, was used (Fig. 1). The tip of the electrode was beveled obliquely to 20-25/~m along the minor axis and 80-95/~m along the major axis. The first barrel was filled with a solution consisting of a mixture of 2% WGA-HRP, 0.5% Fast green and either 1 /~M quisqualate (Sigma), in the case of SHR, or 50/~M quisqualate, in the case of WKY, dissolved in HEPES buffer 24, maintained at 300 mOsm/kg H20 and pH 7.4. Fast green dye was used to determine the level of the meniscus of the mixed solution in the electrode. The volume ejected was determined by measuring the downward displacement of the meniscus using a low-power microscope. Since the inner diameter of the barrel was 0.5 mm, downward displacement of the meniscus by 0.1 mm corresponded to ejection of 20 nl of solution. Quisqualate was used to confirm initiation of the pressor response to quisqualate to which the SRF vasomotor control neurons are very sensitive2°. The first barrel was connected to a microinjector (Narishige, U.S.A., IM 200), and 10-30 nl of mixed solution was delivered by applying pulsed pressure (50 ms, 50 kpa) triggered by an externally applied 10 Hz electrical signal. The second barrel was filled with 3 M NaCI solution and used for either the recording of multi-unit potentials or electrical stimulation of the brain. The third barrel was filled with 3 M NaCI solution and used as a reserve for the second barrel (not shown in Fig. 1). The tip resistance of all three barrels ranged from 1-2 MQ.

and double-labeled neurons were counted in each nucleus both ipsilaterally and contralateraUy to the side of injection. These labeling counts were averaged in 3 SHR and 3 WKY. The significance of differences in the labeling counts obtained in SHR and WKY was evaluated using Student's t-test. RESULTS

Genealogical differences between SHR and W K Y A d i f f e r e n c e in t h e h e m o d y n a m i c s of 1 6 - 2 0 - w e e k - o l d S H R and a g e - m a t c h e d W K Y was c o n f i r m e d . Systolic, diastolic, m e a n b l o o d p r e s s u r e s and h e a r t rate in the 3 S H R w e r e 200 _+ 11 m m H g , 137 _+ 9 m m H g , 158 + 10 m m H g and 381 _+ 21 b e a t s p e r min, r e s p e c t i v e l y , while the r e s p e c t i v e values for the 3 W K Y w e r e 130 _+ 8 m m H g , 80 _+ 4 m m H g , 97 _+ 5 m m H g and 333 +_ 10 beats p e r min. T h e s e h e m o d y n a m i c values w e r e significantly h i g h e r in S H R t h a n in W K Y ( P < 0.005 in the case o f systolic, diastolic and m e a n b l o o d pressures and P < 0.025 in the case of h e a r t rate).

General outline of the distribution of HRP-labeled neurons T h e 6 e x p e r i m e n t s m e t the c r i t e r i o n that the b r o w n

Experimental procedure The electrode was positioned rostro-caudally 0.8-1.2 mm rostral to the obex, mediolateraily 2.0-2.2 mm lateral to the midline, and then thrust dorso-ventrally 3.0-4.0 mm from the dorsal surface of the medulla. The SRF vasomotor control neurons were located within these positions. The brain was stimulated in 200 #m steps with a 6 s train of 50 Hz square-wave pulses of width 100 ~m and intensity 10/~A (Fig. 1B top). After setting the electrode at a point at which electrical stimulation evoked a strong pressor response, injection of WGA-HRP was performed while monitoring the pressor response to quisqualate (Fig. 1B bottom).

Histochemistry and statistics After 48 h, the animals were re-anesthetized with pentobarbital sodium (35 mg/kg, i.p.) and perfused transcardially with heparinized saline, followed by 4% carbodiimide and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and then with 4% paraformaldehyde in the same buffer s. Brains were removed and post-fixed in 4% paraformaldehyde for 2-3 h. The entire brain was cut in 40/tm thick serial sections with a vibratome. Alternate sections were treated with tetramethylbenzidine (TMB) followed by diaminobenzidine (DAB) medium containing cobalt acetate to stabilize products of the TMB reactions a2. Next, the sections were incubated in rabbit antiserum to glutamate (1:2500, Immunotech) overnight at 4°C, processed with avidin-biotin peroxidase complex (Vectastain ABC Kit, Vector Labs), reacted with DAB and mounted on slides. For controls, primary antisera to glutamate were replaced by the antisera pretreated with the glutamate/bovine serum albumin conjugate coupled to Sepharose 4B beads (GIu-BSA) 1° which was synthesized according to the method of Seguella et al. 34. The pretreatment of the antisera with GIu-BSA completely abolished the specific immunostaining with the anti-glutamate serum. The brain sections were surveyed under a microscope, and neurons labeled doubly by retrogradely transported HRP and antiserum to glutamate were carefully identified both ipsilaterally and contralaterally to the site of HRP injection. Another series of alternate sections of rostral medulla were processed by the DAB reaction, counterstained with Neutral red (0.1%), and the HRP injection site was then certified to correspond to the SRF in the RVLM. Brain histology was checked against Paxinos' atlas 25. Both single-

DAB

r e a c t i o n p r o d u c t s be c e n t e r e d and a l m o s t c o m -

pletely c o n f i n e d within the S R F site in which stimulation p r o d u c e d a strong p r e s s o r r e s p o n s e (Fig. 1). Fig. 2 shows the l o c a t i o n and c o n f i g u r a t i o n of t h e 3 types o f l a b e l e d n e u r o n s f o u n d in the c o n t r a l a t e r a l S R F o f W K Y rats: (1) a d o u b l e - l a b e l e d n e u r o n , c o n t a i n i n g particulate T M B reaction p r o d u c t s , indicative of r e t r o g r a d e W G A - H R P

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