The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus

BRAIN RESEARCH ELSEVIER

Brain Research 724 (1996) 186-190

Research report

The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus Michiru Hirasawa, Masugi Nishihara, Michio Takahashi * Department of Veterinao" Physiology, Veterinary Medical Science, Tile Uniuersitv of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan Accepted 27 February 1996

Abstract We recently reported that a train of episodic neural discharges within the ventromedial nucleus of the hypothalamus (VMH) associated with suppression of the circulatory system had been determined by monitoring multiple unit activity (MUA). Abrupt increases in neural activity ( M U A volleys; 1 to 4 min in duration) accompanied transient decreases in heart rate (HR) and blood pressure (BP), and showed circadian rhythm, occurring every 15 to 30 min in the light phase but seldom in the dark phase. The present study was aimed to determine if neurons in the vasomotor area of the rostral ventrolateral medulla (RVL) are involved in this VMH-induced cardiovascular suppression. M U A s of the VMH and RVL were monitored simultaneously with HR and BP in urethane-anesthetized rats. In synchrony with each M U A volley in the VMH, spontaneous activity of RVL neurons significantly decreased, as well as HR and BP. These RVL neurons are most likely vasomotor neurons because M U A of the RVL was attenuated by baroreceptor reflex activation, and electrical stimulation of these cells through the M U A recording electrodes produced pressor responses. These data suggest that VMH neurons that show a train of episodic discharges suppress the circulatory system at least in part by inhibiting the excitability of vasomotor neurons in the RVL.

Keywords: Ventromedial hypothalamic nucleus: Rostral ventrolateral medulla; Multiunit activity; Blood pressure: Heart rate; Rat

1. Introduction

Our recent research revealed characteristic increases in neural activity within the ventromedial nucleus of the hypothalamus (VMH) that were associated with cardiovascular suppression by monitoring multiple unit activity (MUA) [12]. MUA of the VMH showed transient, explosive rises (MUA volleys; 1 to 4 min in duration) occurring frequently in the light phase at 15 to 30 rain intervals but only seldom in the dark phase. Additionally, heart rate (HR) and blood pressure (BP) decreased in a manner synchronized with these MUA volleys. Furthermore, the depressor response was replicated by electrical stimulation through the electrodes that recorded MUA volleys. It is generally understood that the VMH is a sympathoexcitatory nucleus, because stimulation of the VMH induces pressor response accompanied by tachycardia and increases sympathetic nervous activities [4,19,26,27], but our findings give additional support to the idea that sympathoinhibitory neurons co-exist within the VMH [24]. Since blood pressure is recognized to depend mainly on the

* Corresponding author. Fax: (81) (3) 3815-4266. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 3 0 6 - X

excitatory tone of sympathetic nervous system, our results suggest that the sympathoinhibitory neurons are responsible for MUA volleys. However, the pathway by which the VMH exerts this cardiovascular inhibitory effect remains to be elucidated. Hypothalamic control of cardiovascular functions may be mediated by vasomotor cell groups in the medulla, such as the rostral ventrolateral medulla (RVL), nucleus of the solitary tract and nucleus ambiguus [1,3,5,7,13,20,23]. Among these, the RVL is critical for tonic and reflex control of arterial pressure [6,9]. Anatomically, neurons in the RVL, especially the C1 group, project to preganglionic neurons of the intermediolateral columns of the spinal cord, which is the main origin of spinal sympathetic outflow [10,18]. Physiologically, CI area neurons provide an excitatory background to sympathetic preganglionic neurons that contribute to maintenance of vasomotor tone [17]. They also play a crucial role in the baroreceptor reflex control of arterial pressure [21]. Moreover, the hypothalamus is known to exert cardiovascular regulation partially through the RVL [1,3,5,7,20]. The present study was undertaken to examine whether or not RVL neurons are involved in the suppressive effect that VMH neurons have on the circulatory system.

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M. H irasawa et al. / Brain Research 724 (1996) 186-190

2. Materials and methods

2.1. Animals All experiments were done using adult male Wistarlmamichi rats weighing 375-500 g. Animals were housed in a constant temperature (23 ___2°C) animal room illuminated between 05.00 and 19.00 h and provided with rat chow and water ad libitum. 2.2. Monitoring of MUA, blood pressure and heart rate All rats received implantations of two separate electrode assemblies in the VMH and RVL for recording MUA as previously reported [14]. Briefly, the electrode assembly consisted of two 75-/xm teflon-insulated platinum (90%)iridium (10%) wires incased in a stainless steel guide tube (650 /xm in diameter). The impedance of each platinumiridium electrode measured at 1 kHz was 50-100 k O . Animals were anesthetized by urethane (1.0-1.2 g / k g , i.p.) and implanted with electrodes unilaterally to the left side of the VMH and ipsilateral RVL according to the stereotaxic atlas of the rat brain by Paxinos et al. [15]. After fixing the electrodes to the skull with anchor screws and dental cement, rats were placed in a supine position on a heated operating table. The electrodes were connected to a buffer amplifier where signals were passed through to a biophysical amplifier (Nihon Kohden, AVB-21) with low and high cutoff frequencies of 500 Hz and 3 kHz, respectively, and amplified signals were displayed on an oscilloscope (Nihon Kohden, VC-11). Neural spikes were distinguished by their amplitude, and the number of spikes was counted and integrated for 1 s with a pulse counter (Nihon Kohden, MET-1100). Outputs were recorded as a histogram on a thermal array recorder (Nihon Kohden, RTA1200M). A polyethylene catheter inserted into one femoral artery and connected to a pressure transducer (SPECTRAMED) served for monitoring pulsatile arterial blood pressure (BP; mmHg). The phasic signal from the transducer was simultaneously fed to a heart rate counter (Nihon Kohden, AT-601G) for registering heart rate (HR; beats/min). These measurements were also displayed on the thermal array recorder. Another catheter was placed in a femoral vein for drug administration. 2.3. Characterization of RVL neural activity The electrophysiological properties of medullary vasomotor neurons have been described in several previous studies [8,21]. Activity of sympathoexcitatory neurons in the RVL was inhibited immediately by baroreceptor reflex activation, and stimulating them induced pressor response. RVL neurons from which MUA were recorded were verified by whether or not they possessed these physiological characteristics. Response of these neurons to hypertension was examined by a peripheral vasoconstrictor drug,

phenylephrine (10 m g / k g i.v., Sigma) [25]. The ability of these neurons to induce pressor effect was assessed by applying electrical stimulation with positive-negative biphasic rectangular pulses (duration: 200 /xs, intensity: 20-40 /xA, frequency: 100 Hz) through the MUA electrode implanted into RVL for 10 s with an electric stimulator (Nihon Kohden, SEN 7203). BP and HR were recorded continuously throughout the stimulation.

A

A6.7 A6.2 A5.7 B

~

-2.8

~~)A

-3.0

~ A

-3.3

Fig. 1. (A) Location of the MUA electrode tips in the hypothalamus. Solid and open circles represent the recording sites of MUA volleys which did or did not accompany suppression of MUA in the medulla, respectively. Abbreviations: DMH, dorsomedial nucleus of the hypothalamus; fx, fornix; LH, lateral hypothalamic area; OT, optic tract; VMH, ventromedial nucleus of the hypothalamus. (B) Location of the MUA electrode tips in the medulla. Solid and open circles represent the recording sites of MUA which was or was not suppressed in association with MUA volleys in the VMH, respectively. Abbreviations: NA, nucleus ambiguus; NTS, nucleus tractus solitalis; RVL, rostral ventrolateral medulla; STN, spinal trigeminal nucleus.

M. Hirasawa et a l . / Brain Research 724 (1996) 186-190

188

200

jl

VMH

phenylephrine

A

80

(]mpulses/sec)

VMH lOO 1 (impulses/sec) ~,,u..,,,..a.aai~l~ o I

0

RVL

l°°1 0.a

HR

(mmHg)

..........

RVL lOO 1 (impulses/see)

3ool

(beats/min) 150 u * ~ " ~ ~ ' ~

BP

o

1

2503507- - - - " - ~ - - - W ~

12°1

12o

0J

0

HR

5min

~llllah~a~l

,.~ildlu~

lat~~,,~k~,~ll~.

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(beats/min)150-'j 200

Fig. 2. Representative examples of M U A in the VMH and RVL, recorded simultaneously with HR and BP from two individual rats.

BP (mmHg)

2.4. Histological verification After completion of each experiment, anodal direct current (100 /xA, 15 s) was passed through the electrode tip to localize the M U A recording site, and then the animal was perfused with 10% formalin transcardially. The brain was removed and stored in 10% formalin for several days, then transferred to 10% formalin-10% sucrose solution and stored at least overnight before cutting serial coronal sections of 50 ~ m . Sections were evaluated under a light microscope to localize the position of the electrode tip.

5rain

B

50-

e 25 i 0 --25

o

-50

2.5. Statistical analysis -75 Changes from baseline in MUA, HR and BP were calculated by using the integrated means of 30 s just before and during volleys or treatments. Data were expressed as mean _+ standard error of the mean (S.E.M.) and analyzed by the paired t-test.

3. Results In the present study, 12 rats were confirmed to exhibit episodic M U A volleys in the VMH. M U A recording sites in the VMH are depicted in Fig. 1A. All of them showed

vMH

RVL

Fig, 3. (A) Representative effects of intravenous injection of phenylephrine (arrow) on MUA in the VMH and RVL, HR and BP. (B) Summary of changes in MUA in the VMH and RVL following phenylephrine injection, expressed as percentage change from preinjection value (mean_+ S.E.M., n = 5). * Significantly different compared with preinjection value ( P < 0.05).

decreases in HR and BP coincident with the appearance of MUA volleys in the VMH under anesthesia, as previously reported [12]. Another MUA electrode assembly was successfully implanted within the RVL (Fig. 1B) in 9 of these rats, and synchronized with MUA volleys in the VMH,

Table 1 Changes in M U A of the VMH and RVL, HR and BP during appearance of M U A volleys in the VMH

M U A in the VMH ( i m p u l s e s / s ) M U A in the RVL ( i m p u l s e s / s ) HR ( b e a t s / m i n ) BP ( m m H g )

n a

Control b

During MUA volley ~

8 8 8 8

24.5 _+ 17.0 24.4 _+ 12.7 302.9 ± 27.9 76.4 _+ 9.03

74.4 _+ 43.8 * * 10.7 4__9.78 * * 282.5 ± 17.3 * 65.9 _+ 2.33 * *

a Number of animals used. Means of 2 to 8 data obtained from each rat were calculated and used for statistical analysis. b Integrated means of 30 s just before MUA volleys. c Integrated means of 30 s during MUA volleys, at the point of maximal response. P < 0.05, * * P < 0.01 vs. control.

M. Hirasawa et al. / Brain Research 724 (1996) 186-190

electrical stimulation

A

20#A

HR

300

3 0 # A 40#A

-I ~,~-~--,~,~.,~..~o~,

(beats/min) 150 J

/

BP

1

;--J

(mmHg) 0 lmin B

20

O

E • BP

~ i

nlaR

20gA

30gA 4dgA

Fig. 4. (A) Representative effects of electrical stimulation of the RVL through the MUA electrodes on HR and BP. Stimulation was applied at bars depicted at the top of the trace. Note that sweep speed is different from that in Fig. IA and Fig. 3A. (B) Summary of changes in HR and BP by electrical stimulation of the RVL, expressed as percentage change from prestimulation value (mean_+ S.E.M., n = 5). * Significantly different compared with prestimulation value ( P < 0.05).

suppression of MUA in the RVL was observed in 8 of them. Representative examples of changes in MUAs in the VMH and RVL, HR and BP are indicated in Fig. 2. During the appearance of MUA volleys in the VMH, neural activity of the RVL, as well as HR and BP, were decreased. Table 1 shows changes in these parameters in the 8 rats. While MUA of the VMH increased, MUA of the RVL, HR and BP significantly decreased. The characteristics of RVL neurons from which MUA was recorded were further examined in 5 out of these 8 rats. An intravenous injection of a pressor agent, phenylephrine, suppressed MUA of the RVL (Fig. 3A), while it did not affect MUA of the VMH significantly. Fig. 3B summarizes changes in MUAs of both VMH and RVL induced by phenylephrine in these 5 rats. Electrical stimulation of RVL neurons through the MUA electrode resuited in a pressor response (Fig. 4A). BP increased proportionally to the intensity of stimulation (20, 30, and 40 /xA) applied to the RVL, while HR did not change significantly (Fig. 4B).

4. D i s c u s s i o n

We previously observed a series of MUA volleys of the VMH showing a circadian rhythm, i.e., appearing fre-

189

quently in the light phase but only seldom in the dark phase [12]. The present study demonstrated that spontaneous neural activity of the RVL declined in synchrony with the appearance of these MUA volleys. Furthermore, MUA volleys in the VMH also were associated with decreases in HR and BP, confirming our previous findings [12]. These causal relationships were compatible with the idea that VMH-induced suppression on the circulatory system is mediated by RVL neurons. Since direct projection from the VMH to the RVL is not known, other brain regions may relay this pathway. In this study spontaneous electrical activity of RVL neurons which correlates with MUA volleys in the VMH also was suppressed by hypertension induced by phenylephrine injection, indicating they were baro-sensitive. Further, electrical stimulation of these RVL neurons through the MUA recording electrode induced hypertension in a current intensity-dependent manner. These observations suggested that the RVL neurons from which MUA were recorded were vasomotor neurons that control sympathetic cardiovascular outflow [8,21]. The RVL in the rat is known to contain several groups of bulbospinal cells with different chemical properties. Among these, C1 neurons, phenylethanolamine N-methyltransferase (PNMT)-immunoreactive cells [2,16], are suggested to provide tonic and reflex drive to preganglionic neurons that are designated as the main origin of the spinal sympathetic outflow [10,16]. Taking these facts into account, MUA of the RVL in the present study may comprise the activity of C1 neurons. Activity of VMH neurons responsible for MUA volleys was not effectively affected by arterial baroreceptor excitation, while RVL neural activity was significantly suppressed. Our previous study indicated that the appearance of MUA volleys preceded decreases in BP and HR, and that electrical stimulation applied to these MUA volley-exhibiting neurons in the VMH replicated the BP fall [12]. Together with these previous observations, the present study suggests that MUA volleys in the VMH is a consequence of changes in neither the circulatory system nor the neural activity of the RVL. A group of VMH neurons that evokes sympathetic vasodilation, i.e., an atropine-sensitive decrease in vascular resistance of the skeletal muscle, has been reported [11,22]. This neural activity which increases muscular blood flow is considered essential in situations requiring sudden muscular effort such as in the defense reaction. If MUA volleys recorded in this study represent neural activity of these cholinergic vasodilating neurons, they should have appeared more during the dark phase or during defense reaction when more muscular movements were required. However, this neural activity was almost exclusively observed during the light phase, or resting period for rats [12]. Therefore, it is unlikely that the VMH neurons that induced depressor response in this study are cholinergic vasodilating neurons.

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M. Hirasawa et al. / Brain Researeh 724 (1996) 186-190

In c o n c l u s i o n , t h e p r e s e n t s t u d y s u g g e s t s that V M H n e u r o n s c h a r a c t e r i z e d b y d i u r n a l e p i s o d i c d i s c h a r g e s inh i b i t t h e c i r c u l a t o r y s y s t e m , at l e a s t in part, b y s u p p r e s s i n g t h e e x c i t a b i l i t y o f v a s o m o t o r n e u r o n s in t h e R V L . T h e p a t h w a y a n d n e u r o t r a n s m i t t e r s b y w h i c h i n f o r m a t i o n is

[12]

[13]

r e l a y e d to t h e R V L r e m a i n to b e e l u c i d a t e d . [14]

Acknowledgements [15] T h i s s t u d y w a s s u p p o r t e d in p a r t b y J S P S F e l l o w s h i p s for Japanese Junior Scientists and Grants-in-Aid from the

[16]

Ministry of Education, Science and Culture, Japan.

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