Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation

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Autonomic Nervous System Journal of the AutonomicNervous System 50 (1994) 31-43

Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation M i t s u h i k o M i u r a *, K i y o s h i g e T a k a y a m a ,

Junichi Okada

Department of Physiology 1st Division, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi-shi 371, Japan

(Received 20 January 1994;revision received and accepted 14 February 1994)

Abstract

The purpose of this study was to identify the CNS neurons that express Fos protein after repeated activation of the baroreceptor reflex. This was done in Wistar rats anesthetized with urethane and a-chloralose with careful physiological controls. The intact control rat showed few Fos-immunoreactive (ir) neurons, whereas the anesthetized control rat showed many Fos-ir neurons in the CNS from the medulla oblongata to the forebrain. After repeated stimulation of baroreceptors by pressor responses to phenylephrine (dose), we counted the amounts of Fos-ir neurons (response). The correlation coefficient of the dose-response relationship was high, and significant only in the medial part of the nucleus tractus solitarii (NTS) in the medulla and periaqueductal gray (PAG) in the midbrain, whereas it was comparatively high but insignificant in the commissure and lateral parts of the NTS, caudal and rostral ventrolateral medulla, periambiguus nucleus, dorsal and ventral medullary reticular nuclei, lateral parabrachial nucleus, paraventricular nucleus thalamus, and dorsomedial nucleus hypothalamus. No significant correlation was found in the humoral control nuclei in the preoptico-hypothalamic structure. Fos expression was never detected in the sensory neurons in the ganglia petrosum and nodosum, and in the sympathetic preganglionic neurons in the intermediolateral nucleus of the thoracic spinal cord. This study shows that Fos expression in the CNS neurons is induced not only by baroreceptor stimulation but also by anesthesia and/or sham-operation, and that Fos expression in the NTSm and PAG neurons faithfully responds to baroreceptor stimulation. Keywords: c-Fos; Blood pressure; Baroreceptor; Nucleus tractus solitarii; Caudal ventrolateral medulla; Rostral ventrolateral medulla

1. I n t r o d u c t i o n

The baroreceptor reflex pathways have been shown by many electrophysiological and anatomi-

* Corresponding author. Tel.: (81-272) 20-7923; Fax: (81-272)

20-7926.

cal studies [39]. The first-order neurons whose cell bodies exist in the ganglia petrosum and nodosum transmit baroreceptor signals to the second-order neurons in the nucleus tractus solitarii (NTS) [1,2,13,24]. The NTS neurons in turn relay the signal to the third-order neurons in the caudal and rostral ventrolateral medulla (CVLM and R V L M ) [10]. As for ascending pathways, the

0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00032-F

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M. Miura et aL /Journal of the Autonomic Nerl~ous System 50 (1994) .3 1-43

NTS neurons project to the central visceral nuclei in the pons, midbrain, hypothalamus, preoptic area and limbic system [20,31]. As for descending pathways, the glutamatergic and GABAergic neurons in the CVLM and RVLM project to the sympathetic preganglionic neurons of the intermediolateral nucleus (IMLN) of the spinal cord [20,26]. As for intermedullary pathways, barosensitive neurons in the CVLM project to neurons in the RVLM [42]. However, precise location and conformation of second and higher-order neurons within the baroreceptor reflex pathway remains unsettled. Recently, a Fos expression method presented evidence that proto-oncogene c-fos expresses Fos protein rapidly and transiently within neurons after synaptic activation [3,4,34]. Thus, several studies were reported concerning the neuronal expression of c-los after baroreceptor stimulation [7,18,21,33], but they lacked enough consideration on false-positive responses to different stimuli due to anesthesia, surgical operation, light, sound and handling. The purpose of this study was to identify the CNS neurons that express Fos protein after repeated activation of the baroreceptor reflex. To precisely assess the results, we considered the effects of long-term anesthesia and long-term cannulation on Fos expression. Preliminary reports of this study have been presented in abstract form [25].

2. Materials and Methods

2.1. General care Experiments were done on 12- to 16-week-old male Wistar rats weighing 280-320 g. To avoid any restlessness of a single rat, pairs of rats were kept in cages. To extinguish Fos expression that might have been present before isolation of the rats, pairs of rats were kept in a dark and soundless room for 12 h. Anesthesia was induced by hatothane and maintained with urethane (initially 600 mg/kg, i.p; after that 60 mg/kg, i.p., every hour) and a-chloratose (initially 60 rag/kg, i.p.; after that 6 rng/kg, i.p., every hour). A bipolar

EEG was recorded between the two sides of the parietal cortex, and the wave frequency spectrum was displayed on a monitor oscilloscope. To maintain optimum anesthesia, a ratio of the sum of the peak frequencies of a and/3 wave against the peak frequency of ~ wave was set at 1 : I [6]. When deep anesthesia happened to induce hypopnea, air containing 30% 0 2 was promptly inhaled. The right femoral artery was cannulated to measure arterial blood pressure and to sample blood, and the right femoral vein to inject drugs. Heart rate was measured with a cardiotachometer triggered by the R-wave of the ECG. Arterial blood pressure, mean arterial blood pressure and heart rate were displayed on a polygraph. Rectal temperature was maintained at 37°C with an infrared heat lamp.

2.2. Experimental procedures Two control experiments and 5 baroreceptorstimulation experiments were carried out. In all experiments, care was taken to avoid Fos expression due to nonspeeific stimulation, i.e., light, sound, odor and pain. In the baroreceptor stimulation experiments, the pressor and reflex bradycardiac responses to an injection of L-phenylephrine hydrochloride (Sigma, i.v., 50 ~ g / k g per 15 s) were observed (pressor test) [38]. To obtain the dose-response relationship, doses of phenylephrine were increased by increasing the frequency of the injections. In order to register the biochemical conditions of the blood, PaCO2, PaO 2 and pH were measured using blood sampled before and just after baroreceptor stimulation, while the concentration of plasma angiotensin II and Arg-vasopressin and osmotic pressure were measured using blood that was sampled just before perfusion. Two hours after the pressor test, the rats were perfused transcardially with a heparinized saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).

2.3. Tissue preparation and histochemistry The ganglia petrosum and nodosum, thoracic cord. and whole brain were removed, fixed 3 h

M. Miura et al. /Journal of the Autonomic Nervous System 50 (1994) 31-43

with the same fixative, soaked stepwise in 5%, 20% and 30% sucrose in 0.1 M PB and frozen. The whole brain was cut into frontal sections at 35/zm from interaural 11.0 mm to - 6 . 0 mm. The thoracic cord was cut at T2_ 3 and T9_10 The ganglia were longitudinally cut. Two sets of alternate sections were processed by the immunohistochemical technique. Sections were preincubated alternately in avidin and biotin solutions, incubated with sheep anti-c-los polyclonal antiserum (OA-11-823, Cambridge Research Biochemicals) at a dilution of 1:2000, incubated in ABC solution (Vector Labs.), and treated with DAB-Nickel solution containing 0.003% H 2 0 2. One set of the alternate sections was counterstained with 0.1% Neutral red, while another set was left unstained. Fos-ir neurons were counted in the counter-stained preparations. Brain histology was checked against the brain atlases of Paxinos and Watson [29] and Swanson [40]. The Fos-ir neurons of the unstained preparations were photographed with Nomalsky's apparatus.

33

3. Results

3.1. Physiological conditions of rats The control experiments were performed under intact conditions without long-term anesthesia (experiment 1) and intact conditions with long-term anesthesia (experiment 2). Rats were kept in a dark and soundless room for 12 h, after which they were operated on only for arterial cannulation. In experiment 1, the operation was performed under short-term anesthesia. Since Fos expression may begin 30 min after nonspecific stimuli [4], vascular cannulation, measurement of blood pressure and heart rate and sampling of blood were finished 20 min before the onset of perfusion. By contrast, the baroreceptor stimulation experiments (experiment 3-7) were performed under long-term anesthesia. Rats were operated on for arterial and venous cannulation. Since Fos expression may carry on 8 h after nonspecific stimuli due to operation [9], the rats

Table 1 Conditions of arterial blood in seven experiments Experiment L o n g - t e r m anesthesia L o n g - t e r m cannulation Stimulation

1 (- ) (- ) (- )

2 (+ ) (- ) (- )

P a C O z (36-48) a P a O 2 (80-108) b p H (7.26-7.44) a PaCO~ PaO~ pH'

41 102 7.42 -

//(305-317) c A I I (62-173) d A V P (7.8-9.3) e

315 52 10

309 59 15

310 54 11

309 157 7.1

312 32 18

316 83 15

316 104 11

MAP MAP'

115 -

90 -

105 -

105 100

109 105

100 95

110 105

43 91 7.38 -

3 (+ ) (+) 0x 45 99 7.34 -

4 (+ ) (+ ) 1x 47 113 7.36 44 89 7.38

5 (+ ) (+) 2x 44 89 7.38 42 84 7.37

6 (+) (+ ) 3x 46 80 7.39 48 78 7.32

7 (+) (+ ) 5X 42 96 7.39 47 75 7.39

P a C O 2 ( m m H g ) , P a O 2 ( m m H g ) , pH: blood gas and p H before stimulation; PaCO~, PaO~, p H ' : blood gas and p H after stimulation; H ( m O s m / 1 ) : osmotic pressure before perfusion; A I I ( p g / m l ) : concentration of angiotensin II before perfusion; A V P ( p g / m l ) : concentration of arginine vasopressin before perfusion; M A P (mmHg): m e a n arterial pressure before stimulation; M A P ' : m e a n arterial pressure after stimulation. N o r m a l values in p a r e n t h e s e s q u o t e d from; a Mitruka et al. [23]; b Walker [44]; c H o w a r d et al. [12]; d Jacobson et al. [14]; e Fox et al. [8].

34

M. Miura et al. /Journal of the Autonomic NertJous System 50 (1994) 31-43

were kept in a dark and soundless room for 8 h after the operation, and then stimulated. Table 1 shows measured values of arterial blood both in the control and baroreceptor stimulation experiments. PaCO2, PaO2, pH and plasma osmotic pressure were found to be 41-47 mmHg, 80-113 mmHg, 7.34-7.42 and 309-316 mOsm/1, respectively. Angiotensin II (AII) and argininevasopressin (AVP) were found to be 32-157 pg/ml and 7.1-18 pg/ml, respectively. Mean arterial blood pressure (MAP) ranged from 90 mmHg to 115 mmHg. These measured values show that throughout 7 experiments there was no significant disturbance in blood conditions that may influence Fos expression. In the baroreceptor stimulation experiments, the averaged MAP and heart rate (HR) before stimulation were found to be 105 :i: 3 mmHg and 337 + 9 bpm (mean __ S.E.M, n = 5), respectively. Fig. 1 shows that injections of phenylephrine induced an abrupt increase in MAP (baroreceptor stimulation) and a reflex decrease in HR. In a total of 11 cardiovascular responses to phenylephrine, the averaged increase in MAP was 44 + 2 mmHg and the averaged decrease in H R was 48 +_ 3 bpm.

are (1) commissure part of the nucleus tractus solitarii (NTSco, Fig. 2A, D), (2) medial part of the NTS (NTSm, Fig. 2B,E), (3) lateral part of the NTS (NTSI), (4) area postrema (AP, Fig. 2C,F), (5) rostral ventrolateral medulla (RVLM, Fig. 3 A,C), (6) caudal v e n t r o l a t e r a l medulla(CVLM, Fig. 3B,D), (7) periambiguus nucleus (pAMB), (8) ventral medullary reticular nucleus (MdV), and (9) dorsal medullary reticular nucleus (MdD). The pontine and midbrain nuclei are (1) lateral parabrachial nucleus (Pbl, Fig. 4C,D), (2) pontine central gray (PCG, Fig. 4B) and (3) periaqueductal gray of the midbrain (PAG, Fig. 4A). The hypothalamic nuclei are (1) dorsomedial nucleus hypothalamus (DMH), (2) tuber cinereum (TC), (3) paraventricular nucleus hypothalamus (PVH) and (4) supraoptic nucleus (SO). The basal telencephalon nuclei are (1) paraventricular nucleus thalamus (PVT), (2) central nucleus amygdala (Ce), (3) subfornical organ (SFO), (4) vascular organ of the lamina terminalis (OVLT), (5) medial preoptic area (MPA) and (6) bed nucleus of the stria terminalis (BST). Only after strong baroreceptor stimulation,

3.2. Distribution of Fos-ir neurons

Phenylephr~ne

Fos-ir neurons were never detected in the intermediolateral nucleus (IMLN) of T 2_3 and T 9_10 spinal segments and in the ganglia petrosum and nodosum, while they were detected widely in the supraspinal nuclei. Table 2 shows the frequency distribution of Fos-ir neurons in the control and baroreceptor-stimulation experiments. Experiment 1 showed few Fos-ir neurons, but experiment 2 showed many Fos-ir neurons in many nuclei. In the baroreceptor stimulation experiments, Fos-ir neurons were detected in 34 supraspinal nuclei in which 22 nuclei are related to the autonomic function (the major group), but t h e o t h e r 12 n u c l e i r e l a t e t o t h e s o m a t i c and other functions (the minor group). The major group consists of 9 medullary nuclei, 3 pontine and midbrain nuclei, 4 hypothalamic nuclei and 6 basal telencephalon nuclei. The medullary nuclei

1rain

15s '~

-

--

HR ~

,,,

-

-

~

.

.

.

.

.

.

.

.

.

.

] 150

1360 B1oocl

sainting Pao284~t,oHg Paco242mmHg pH 7.37 Fig. 1. Example of the pregsor test which induced the baroreceptor r e t d . Prers~r and reflex braOycardiac responses were elicited by two i~ject/onsof plWn~ec4~rine at an interval of 3 rain. Just after stimulation, blood gas and pH showed normal values. AP, arterial blood pressure (mmHg); MAP, mean arterial blood pressure (mmHa~, HR, heart rate (beats per

rain).

M. Miura et al. /Journal of the Autonomic Nervous System 50 (1994) 31-43

35

of the dorsal raphe nucleus (DR), and (3) middle neurons of the intermediodorsal nucleus thalamus (IMD). Nine nuclei of the minor group be-

Fos-ir neurons were found in 3 nuclei of the minor group, i.e., (1) small neurons in the ventral surface of the medulla (vSF), (2) middle neurons

Table 2 Fos-immunoreactive n e u r o n s in the CNS nuclei of Wistar rats in the control and the baroreceptor stimulation experiment r

P<

Experiment Long-term anesthesia Long-term cannulation Stimulation

1 (- ) (- ) (-)

2 (+) (- ) (-)

3 (+ ) (+ ) 0×

4 (+ ) (+ ) 1x

5 (+ ) (+) 2x

6 (+ ) (+ ) 3x

7 (+ ) (+ ) 5x

NTSco NTSm NTSI AP CVLM RVLM pAMB MdV MdD Pbl PCG PAG DMH TC PVH SO PVT Ce SFO OVLT MPA BST

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0 0 0 0 0

4 81 3 3 0 0 0 0 0 273 14 182 6 31 0 0 16 86 76 221 180 563

14 121 8 9 50 45 0 0 0 101 48 143 58 76 0 0 89 0 0 0 8 54

35 265 4 18 48 39 3 2 0 177 119 159 126 471 305 39 246 74 19 56 106 9

47 273 11 6 141 16 8 2 2 318 196 243 81 175 2 3 38 1 4 3 8 0

76 487 12 8 84 75 29 5 4 168 108 382 141 206 38 12 236 116 335 158 43 3

204 816 57 47 465 155 95 21 63 392 60 555 330 695 40 18 671 194 81 71 13 22

0.84 0.90 * 0.71 0.58 0.74 0.66 0.80 0.78 0.69 0.79 0.17 0.90 * 0.79 0.63 - 0.13 0.18 0.69 0.79 0.49 0.58 - 0.10 - 0.62

0.08 0.04 0.2 0.4 0.2 0.3 0.2 0.2 0.2 0.2 0.8 0.04 0.2 0.3 0.9 0.8 0.2 0.2 0.5 0.4 0.9 0.3

vSF DR IMD Sp5C Sp5I DC VCP IC APN EW SC isl

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 32 31 64 13 27 0 42

0 0 0 6 1 17 0 154 14 69 0 68

0 0 0 0 12 8 68 134 92 24 1 11

0 0 0 1 8 0 0 0 0 0 0 851

0 0 0 11 14 10 29 36 44 115 0 111

10 105 141 23 51 11 11 285 38 123 0 180

0.65 0.65 0.65 0.65 0.79 - 0.39 - 0.01 0.10 0.06 0.45 - 0.25 0.22

0.3 0.3 0.3 0.3 0.2 0.6 0.9 0.8 0.9 0.5 0.7 0.8

r, correlation coefficient of regression line of the dose-response relationship (Experiments 3-7); P < , level of significance. Abbreviations according to Paxinos and Watson [29] and Swanson [40]; AP, area postrema; APN, anterior pretectal nucleus; BST, bed nucleus of the stria terminalis; Ce, central nucleus amygdala; CVLM, caudal ventrolateral medulla; DC, dorsal cochlear nucleus; D M H , dorsomedial nucleus hypothalamus; D R , dorsal raphe nucleus; EW, Edinger-Westphal nucleus; IC, inferior colliculus; IMD, intermediodorsal nucleus thalamus; isl, island of Calleja; MdD, dorsal medullary reticular nucleus MdV, ventral medullary reticular nucleus; MPA, medial preoptic area; NTSco, commissure part of the nucleus tractus solitarii; NTSI, lateral part of the NTS; NTSm, medial part of the NTS; OVLT, vascular organ of the lamina terminalis; PAG, periaqueductal gray; pAMB, periambiguus nucleus; Pbl, lateral parabrachial nucleus; PCG, pontine central gray; PVH, paraventricular nucleus hypothalamus; PVT, paraventricular nucleus thalamus; RVLM, rostral ventrolateral medulla; SC, superior colliculus; SFO, subfornical organ; SO, supraoptic nucleus; SP5C, caudal spinal trigeminal nucleus; SP5I, interpolar spinal trigemial nucleus; TC, tuber cinereum; VCP, ventral cochlear nucleus; vSF, ventral surface of the medulla.

36

M. Miura et al. /Journal of the Autonomic Neruous System 50 (1994) 31-4.3

Fig. 2. Photomicrographs of Fos-immunoreactive neurons observed in experiment 5. A and D, neurons in commissure part of the nucleus tractus solitarii (NTSco). B and E, neurons in medial part of the nucleus tractus solitarii (NTSm). C and F, neurons in the area postrema (AP). Areas in A, B and C (scale bar: 200 ~ m ) indicated by arrows are magnified in !), E and F (scale bar: 50 ~zm). Abbreviations: cc, central canal; ts, solitary tract.

¢ig. 3. Photomicrographs of Fos-immunoreactive neurons observed in experiment 5. (A and C) Neurons m rostral ventrolateral medulla (RVLM). (B and D) Neurons n caudal ventrolateral medulla (CVLM). Areas in A and B (scale bar: 200/xm) indicated by arrows are magnified in C and D (scale bar: 50/~m).

C.

I 4~

Fig. 4. Photomicrographs of Fos-immunoreactive neurons observ~ :in e x p ~ f h ~ 5 (A) Nem~ons in periaqueductal gray (PAG) 0 f midbrain. (B) Neurons in pontme :entral gray (PCG). (C) Neurons in caudal part of lateral parabrachial nucleus (Pbl). (D) Neurons in rostral part of PbL Abbreviations: AQ, cerebral aqueduct; mtV. nesencephalic trigeminal tract; LL, lateral temniscus; scp, superior cerebeliar peduncle:; V4, fourth ventricle. Scale bar in A: 200 p.m.

t~

M. Miura et aL /Journal of the Autonomic Nervous System 50 (1994) 31-43

long to the sensory nuclei responding to teleceptive and exteroceptive stimuli. It is probable that somatic sensation associated with involuntary

teleceptive and exteroceptive stimuli may generate Fos expression in the caudal and interpolar spinal trigeminal nuclei (Sp5C and Sp5I), dorsal

B

A

R ~ ~ +O.S R

+1.1 +0.5

+1.1

O

0

-0.1

-0.5 p

-0.8 3 2. i

6]

39

-O.2

A

M

~

-O.S

-0.8

2 3mm

Fig. 5. Distribution of Fos-immunoreactive neurons in nucleus tractus solitarii (NTS), area postrema (AP), periambiguus nucleus (pAMB), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). (A) Anesthetized and sham-operated rat (experiment 3). (B) Anesthetized, operated and stimulated rat (experiment 7). Stimulation consisted of 5 times repeated injections with phenylephrine. Each dot represents 2 labeled neurons. Numbers on the right of sections indicate distance in mm from obex (ahead of obex, + ; obex, 0; behind obex, - ;). LRt, lateral reticular nucleus; Rf, retrofacial nucleus.

40

M. Miura et al. / Journal of the Autonomic Nen,ous System 50 (1994) 31-43

cochlear nucleus (DC), posterior ventral cochlear nucleus (VCP), inferior colliculus (IC), anterior pretectal nucleus (APN), Edinger-Westphal nucleus (EW), superior colliculus (SC) and islands of Calleja (isl).

3.3. Dose-response relationship in experiments 3 - 7

After repeated activation of the baroreceptor reflex by pressor response to phenylephrine (dose), amounts of Fos-ir neurons were counted in 34 nuclei (response). The correlation coefficient of the dose-response relationship was high and significant only in the NTSm and PAG, while it was comparatively high but insignificant in the NTSCO, NTSI, CVLM, RVLM, pAMB, MdV, MdD, Pbl, PVT and DMH.

3.4. Distribution of Fos-ir neurons in the N T S and AP

The NTS neurons are regarded as the secondorder neurons in the baroreceptor reflex pathway, and now we have found that the NTSm neurons responded proportionally to the intensity of baroreceptor stimuli. Fig. 2 shows that Fos-ir neurons were diffusely distributed bilaterally in three parts of the NTS, NTSm at the highest frequency, NTSco at intermediate frequency, and NTSI and AP at the lowest frequency. Few Fos-ir neurons in the AP were localized mainly on the borderlines of the NTS and AP. Fig. 5 shows topological distribution of Fos-ir neurons in the lower medulla, based on the observations of the nonstimulated control rat (experiment 3, Fig. 5A) and the stimulated rat (experiment 7, Fig. 5B). It should be noted that baroreceptor stimulation induced many Fos-ir neurons throughout the NTS, but few Fos-ir neurons were still present in the NTS even without stimulation. Along the rostroeaudal coordinate, the distribution of Fos-ir neurons was most dense at the level of the AP ( - 0.2 and - 0.5 in Fig. 5B) and steeply decreased outside the AP, forming Gaussian distribution.

3.5. Distribution of Fos-ir neurons m the ('VLM and R V L M

The CVLM and RVLM neurons are regarded as third-order neurons in the baroreceptor reflex pathway. Fig. 3 shows a group of Fos-ir neurons in the RVLM and CVLM that are bilaterally distributed along the ventrolateral surface of the medulla. Similarly to the distribution of Fos-ir neurons in the NTS, baroreceptor stimulation induced many Fos-ir neurons throughout the CVLM and RVLM, but few Fos-ir neurons were still present even without baroreceptor stimulation (see Fig. 5). Table 2 shows that Fos-ir neurons in the CVLM and RVLM did not increase as the stimuli increased stepwise from experiment 3 (stimulation 0 x ) to experiment 6 (stimulation 3 x ), but abruptly increased in experiment 7 (stimulation 5 × ). Along the rostrocaudal coordinate, the distribution of Fos-ir neurons was most dense at the level of the AP ( - 0 . 2 and -0.5 in Fig. 5B) and at the level of the retrofacial nucleus (Rf;+ 1.1 in Fig. 5B), forming distribution with dual peaks.

3.6. Distribution of Fos-ir neurons m the PAG. PCG and Pbl

Fig. 4 shows the typical topology of Fos-ir neurons in the PAG, PCG and the Pbl. Fos-ir neurons were scattered around the cerebral aqueduct in the PAG (A), concentrated in the regions medial to the mesencephalic trigeminal tract in the PCG (B), and distributed widely in the Pbl from the dorsal regions of the superior cerebellar peduncle (C) to the mesencephalic tegmentum (D). Since we now obtained evidence that Fos-ir neurons in the PAG increased proportionally as baroreceptor stimuli increased, we surveyed Fos-ir neurons in the PAG in comparison with neurons in two neighboring nuclei, the PCG and t h e Pbl. The postsyaaptic order of these neurons in the baroreceptor reflex pathway is unknown, but the linearity of the dose-response relationship was completely maintained in the PAG neurons (r =

M. Miura et al. /Journal of the Autonomic Nervous System 50 (1994) 31-43

0.90, P < 0.05), partially in the Pbl neurons (r = 0.79, P < 0.2), but not at all in the PCG (r = 0.17, P < 0.8).

4. Discussion

Several investigators surveyed the neuronal expression of c-los m R N A or Fos protein after baroreceptor stimulation. Rutherfurd et al. electrically stimulated the axotomized carotid sinus nerve (CSN) and aortic depressor nerve (ADN) of rats anesthetized with barbiturate [33], Erickson et al. electrically stimulated the axotomized CSN of rats anesthetized with urethane and achloralose [7], Li et al. stimulated baroreceptors with phenylephrine injections in rabbits anesthetized with Saffan [18], McKitrick et al. electrically stimulated the axotomized A D N of rats anesthetized with urethane [21]. They found the neuronal expression of Fos within the NTS, AP, CVLM and RVLM of the medulla, and McKitrick et al. further detected Fos-ir neurons in the pAMB, MdV, MdD, Pbl, locus coeruleus, pontine reticular field, A5 region of the pons, periventricular hypothalamus, PVH, SO, SFO, MPA, Ce, OVLT, BST and islands of Calleja. In these studies, however, the false-positive effects of axotomy [5,35], mishandling of animals [37] and anesthesia [16] on the neuronal expression of Fos were not elucidated. In this study, we found Fos-ir neurons in many supramedullary nuclei, not only in the baroreceptor stimulation experiments but also in the control experiments. Since we avoided axotomy and mishandling of animals, the false-positive effects may be ascribed to the anesthesia. Although the effect of anesthesia has never been studied systematically, we strongly suggest that anesthetic drugs may induce the neuronal expression of Fos by unknown mechanisms. So as to differentiate the effects of baroreceptor stimulation from the anesthetic effects, we used the correlation coefficient of the dose-response relationship as an indicator. The correlation coefficient was significantly high in the NTSm ( P < 0.04), supporting the theory that the NTSm is a site of second-order neurons of the baroreceptor reflex pathway. The topology of the Fos-ir neu-

41

rons in the NTSm is similar to that obtained by the experiments of the aortic nerve tracing [1] and the carotid sinus nerve tracing [13]. Compared with the Fos-ir neurons in the NTSm, the Fos-ir neurons in the AP were few and mostly scattered on and near the borderline of the AP and NTS. Since the correlation coefficient of the dose-response relationship in the Fos-ir neurons of the AP was low and insignificant, we suggest that the AP neurons may not be involved in the baroreceptor reflex. The pAMB is known as the site of the cardioinhibitory preganglionic neurons of the vagus efferent [28], and the CVLM and RVLM as the sites of sympathetic premotor neurons [32,39]. The correlation coefficients of the dose-response relationship in these nuclei are comparatively high but insignificant. This may reflect the fact that they are third-order neurons to which baroreceptor afferents do not project directly. Since distribution of the Fos-ir neurons in the CVLM and RVLM was similar to that of the glutamatergic and GABAergic neurons projecting to the IMLN of the thoracic cord [26], we suggest that the Fos-ir neurons in the CVLM and RVLM may be glutamatergic or GABAcrgic. Unexpectedly the correlation coefficient of the dose-response relationship was significantly high in the PAG. This fact is consistent with several anatomical studies which showed that the PAG receives afferent projections from the NTS [31]. Recently, Nakai and Maeda showed that the PAG have a cerebrovasodilator action as part of their defence reaction [27]. Therefore, it is likely that hypertension associated with defence reaction may induce cerebrovasodilation by the ascending baroreceptor pathway via the NTS and PAG. In baroreceptor stimulation experiments, we found Fos-ir neurons in many autonomic nuclei of the hypothalamus and basal telencephalon, i.e., the DMH, TC, PVH, SO, PVT, Ce, SFO, OVLT, MPA and BST. So far, evidence that the NTS projects to these nuclei was obtained by autoradiographic study [31], the Phaseolus vulgaris lectin tracing study [41] and immunohistochemical study [11]. However, we found that the correlation coefficients in the dose-response relationship were not significant in these nuclei, and

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M. Miura et al. /Journal of the Autonomic Nervous System 50 (1994) 31 .-43

many Fos-ir neurons were still present even without baroreceptor stimulation. Therefore, it is undetermined whether or not the Fos-ir neurons in these nuclei are induced by baroreceptor stimulation. In addition to the above-mentioned ascending projection from the NTS, the hypothalamus possibly receives humoral control input descending from the SFO and OVLT [15,22]. Concerning interrelation between the cardiovascular control input and humoral control input, several lines of evidence accumulated: (1) the NTS projects to the SFO [45]; (2) the SFO directly projects to the OVLT, SO and PVH [19]; and (3) the PVH projects to the SFO and OVLT [17]. However, we failed to establish linear relationship between the baroreceptor stimulation and neuronal Fos expression in the SFO and OVLT under normal concentration of plasma angiotensin II [30,36] and normal plasma osmolality [43]. Fos-ir neurons appeared in the pain sensory nuclei (SP5C and SP5I), auditory sensory nuclei (DC, VCP and IC), visual sensory nuclei (APN, EW and SC) and olfactory sensory nucleus (islands of Calleja). Since Fos-ir neurons in these nuclei also appeared in the anesthetized a n d / o r sham-operated experiments, we suggest that they may be induced by stimulation due to anesthesia and involuntary sensation. Fos-ir neurons were never found in the primary, sensory neurons of the ganglia petrosum and nodosum and preganglionic neurons of the IMLN. However, it is possible that oncogenes other than c-fos may express in these neurons. In conclusion, by the neuronal Fos expression method, we detected Fos-ir neurons in the CNS nuclei after repeated baroreceptor stimulation, and found that the relationship between the stimulus intensity and Fos expression is significantly linear only in the neuronal Fos expression in the NTSm and PAG.

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