INTRACAROTID INJECTION OF ENDOTHELIN-1
FACILITATES THE ACTIVITY OF ROSTRAL VENTROLATERAL
MEDULLARY NEURONS VIA AREA POSTREMA IN RATS^*

LI　DE-PEI，?HE　RUI-RONG^**
(Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017)

ABSTRACT　　To observe the effect of intracarotid administration of endothelin (ET-1) on electrical activity of neurons within rostral ventrolateral medulla (RVLM) region, 87 spontaneous active units were extracellularly recorded in 35 Sprague-Dawley rats with sino-aortic denervation. The results obtained are as follows. (1) Intracarotid administration of ET-1 (0.3nmol/kg) increased the discharge firing rate from 17.8±1.5 to 20.9±1.4 spikes/s (P<0.01) in 30 out of 36 RVLM neurons, while blood pressure and heart rate had no significant change. (2) BQ-123 (0.67nmol/kg), a selective ET_A blocker, completely blocked the facilitatory effects of ET-1 in 11 out of 14 units. (3) In 10 out of 11 units, glibenclamide (3.3 nmol/kg), a blocker of ATP-sensitive potassium channel, had no effect on the action of ET-1. (4) After ablation of area postrema (AP), the facilitatory action of intracarotid administration of ET-1 on 19 units of RVLM was abolished, while in 7 units of sham ablation animals the response of neurons to ET-1 remained unchanged. Taken together, intracarotid-administered ET-1 may act on the ET_A receptors in neurons of AP, thereby resulting in the facilitating effect on RVLM neurons through the efferent projection of AP.
Key words: endothelin; rostral ventrolateral medulla (RVLM); area postrema; spontaneous discharge units; BQ-123

内皮素通过最后区易化大鼠延髓腹外侧头端区神经元活动^*

李德培　何瑞荣^**

摘　要　　在35只切断双侧缓冲神经、 用氨基甲酸乙酯-α-氯醛糖混合麻醉的Sprague-Dawley大鼠, 应用细胞外记录的电生理学方法, 由RM-6000型多道生理记录仪和WS-682G热阵记录器(频响范围0～2.8kHz) 同步记录血压、 心率和单位神经元放电, 观察颈动脉注射内皮素对87个延髓腹外侧头端区(RVLM)自发放电神经元活动的影响。 所得结果如下: (1) 颈动脉注射ET-1 (0.3nmol/kg)时, 36个单位放电中有30个放电频率由17.8±1.5升至20.9±1.4spikes/s(P<0.01), 血压和心率则无明显变化(P>0.05); (2) 在11个放电单位中应用ET_A选择性受体阻断剂BQ-123 (0.67nmol/kg), 可阻断ET-1的上述易化效应; (3) 在10个放电单位中, 应用ATP敏感性钾通道阻断剂格列苯脲 (3.3 nmol/kg) 对ET-1的易化效应无影响; (4)在热毁损最后区(AP)的大鼠, 19个RVLM放电单位对颈动脉内注射ET-1所致的易化效应不再出现; 而在假毁损AP的大鼠, ET-1对7个RVLM放电单位仍有易化效应。以上结果提示: 循环中的ET-1可与AP神经元上的ET_A受体相结合而引起兴奋, 转而经AP 的传出投射再易化RVLM区神经元活动。
关键词: 内皮素; 延髓腹外侧头端区; 最后区; 自发放电单位; BQ-123
学科分类号: Q463

　　Endothelin (ET), ET mRNA and ET receptors are distributed in central nervous system^［1,2］, especially in the brain areas regulating cardiovascular activity. These findings suggest that ET may play an important role in the neural control of a wide range of functions^［3］. A number of studies had described the cardiovascular responses elicited by central administration of ET in conscious or anesthetized rats. Centrally administered ET-1 produced a transient rise followed by a sustained fall in mean arterial pressure (MAP)^［4～6］. A biphasic response in BP, an increase followed by a decrease^［6,7］, had been produced by micropneumophoretic application or microinjection of ET-1 into nucleus tractus solitarius and area postrema (AP) of anesthetized rats. It is recognized that rostral ventrolateral medulla (RVLM) contains vital area subserving cardiovascular control^［8］. Intracisternal injection of ET-1 exerted an excitatory effect on neurons in RVLM^［9］. However, ET is a polypeptide, which contains 21 amino acid residues being incapable of crossing the blood-brain barrier (BBB). There is evidence that circulating ET-1 influences the neuronal function in area postrema as characterized by an unusually weak BBB^［10］. The purpose of present study was to determine whether intracarotid(ia) injection of ET-1 affected the activity of neurons in RVLM, and if so, to define the way by which ET-1 exerts its effect.

1　MATERIALS AND METHODS

　　The experiments were performed on 35 male Sprague-Dawley rats weighing300～400g. The animals were anesthetized with urethane (0.5g/kg) and α-chloralose (50mg/kg) intraperitoneally. Supplemental doses of anesthetics were given when required. A midline incision was made on the ventral surface of the neck. The trachea and esophagus were transected in the lower neck and reflected rostrally. The animal breathed spontaneously with room air through a trachea tube and was fixed on a stereotaxic frame (Model 1C Jiangwan) in a supine position. After retraction of the bilateral longus capitis muscles, the basal portion of the occipital bone was carefully cut away from the ventral surface of the rostral medulla. Then, the ventral surface of the medulla was exposed and immersed in warm liquid paraffin. Arterial blood pressure was monitored through a catheter in the left femoral artery, and measured with a pressure transducer (MPU-0.5, Nihon Kohden) and a carrier amplifier (AP-620G, Nihon Kohden). The differential signals of arterial pressure pulse were fed into a heart rate counter (AT-601G, Nihon Kohden). Both BP and HR were recorded with a polygraph system (RM-6000, Nihon Kohden).
1.1　Sino-aortic denervation　　After exposing the bilateral carotid sinus areas, the aortic depressor nerves and carotid sinus nerves were cut under a dissecting microscope. The superior cervical sympathetic trunk and recurrent laryngeal nerves were also cut. Completeness of barodenervation was ensured by the absence of decrease in HR (heart rate, HR) when hypertension was initiated by iv injection of phenylephrine (2～3μg). Single-unit recording of RVLM neurons (vide infra) was carried out at least 2 h after sino-aortic denervation when BP and HR had returned to the baseline level.
1.2　Single-unit recording　　Single-unit recording was made with a glass microelectrode (<1μm tip, DC resistance 5～10MΩ, filled with 2% pontamine sky blue and acetate sodium 0.5mol/L at pH 7.3～7.4). The microelectrode was advanced at the speed of 1μm/s by a micromanipulator (SM-21, Narishige) into the RVLM (stereotaxic coordinates: 1.5～2.2mm lateral to midline, 2.6～3.3mm caudal to interaural line, and 0.35～0.5mm from the ventral surface of medulla oblongata) until the activity of an extracellular single unit was recorded. Signals were amplified and filtered first by a microelectrode amplifier (MEZ-7200, Nihon Kohden) and then by a biophysical amplifier (AB-621G, Nihon Kohden). The amplified bioelectrical signals along with BP and HR were monitored on an oscilloscope and recorded on a polygraph system (RM-6000, Nihon Kohden) with a thermal array recorder (WS-682G, band-pass width 0～2.8kHz).
1.3　Area postrema ablation　　An incision in the dorsal neck muscles was made to expose the atlanto-occipital membrane. The dorsal surface of the caudal medulla was exposed by transecting the atlanto-occipital membrane. The AP was then clearly visible under a dissecting microscope. The AP was ablated by microcautery. Sham ablation was performed by transecting the atlanto-occipital membrane without ablation of AP.
1.4　Protocols　　A stabilized period of 60 min was allowed after completion of the surgery. The microelectrode was inserted into RVLM to record the spontaneous discharge of neurons. After a stable recording was kept for 5 min, drugs were injected into a common carotid artery, then BP, HR and electroneurogram (ENG) of RVLM neurons were simultaneously recorded for 30 min. The experimental animals were divided into the following four groups.
　　Group 1.　The effects of ia injection of ET-1 (0.3nmol/kg 0.1ml) on BP, HR and spontaneous firing rate of RVLM neurons were examined. After the recording was stabilized for 5min, 0.1 ml of ET-1 or saline was administrated. The BP, HR and ENG of RVLM were simultaneously recorded.
　　Group 2．　BP, HR and ENG were recorded following ia injection of ET-1 as group 1 and after administration of an ET_A receptor antagonist BQ-123 (0.67nmol/kg, 0.1ml) by the same route.
　　Group 3． 　BP, HR and ENG were examined following ia injection of ET-1 as group 1 and after administration of a blocker of ATP-sensitive potassium channel-glibenclamide (3.3nmol/kg,0.1ml).
　　Group 4．　BP, HR and ENG were recorded following ia injection of ET-1 after AP ablation or sham ablation.
1.5　Histology　　At the end of the experiment, a deposit of blue dye filled in microelectrode was made by passing a negative direct current (60μA/min). The brains of the animals were quickly removed and fixed in 10% formalin. After 7～10d, frozen brain tissue was sectioned in coronal plane　(40μm). Histological verification was carried out with reference to Paxinos and Watson′s coordinates^［11］. Data from those electrode tips not in the desired area were excluded.
　　In the AP ablation rats coronal sections were cut through the region of the entire dorsal vagal complex, slide-mounted, and stained by cresyl violet. The sections were then carefully examined under a light microscope to determine the completeness and location of the ablation. Only the rats whose brains showed complete ablation of the AP were assigned to be the AP ablation group.
1.6　Statistics　　All data are reported as . Differences between the vehicle and ET-1 were analyzed by paired Student′s t　test, and differences among groups were analyzed by unpaired t　test. Statistical significance was accepted when P<0.05.

2　RESULTS

　　Eighty-seven spontaneously active single-units in RVLM were recorded from 35 rats. Mean firing rate of neurons was 17.7±1.5spikes/s. Figure 1 shows the locations of microelectrode tips in RVLM.
2.1　Effect of ET-1 on the discharge rate of RVLM neurons
　　Ia administration of ET-1 (0.3nmol/kg, 0.1ml) evoked changes in firing rate of RVLM neurons: of 36 units, 30 units increased the firing rate from 17.8±1.5 to 20.9±1.4 spikes/s (P<0.01), 5 units showed no significant change and 1 unit decreased the firing rate, while BP and HR were unaffected. Intracarotid injection of saline did not affect BP, HR and the ENG of the RVLM neurons (Fig.2, Table 1).

Fig.1　Localization of microelectrode tips in RVLM
Numbers indicate caudal to interaural line in mm. Coronal sections of medulla are modified from Paxinos and Watson.IO: inferior olive; NA: nucleus ambiguus; PY: pyramidal tract; RM: nucleus raphe magnus; RO: nucleus raphe obscurus; RP: nucleus raphe pallidus; closed circle: units responsive to endothelin; triangle: units not in the nucleus paragigantocelularis lateralis (PGL).

Fig.2　Original tracing showing the effect of intracarotid injection of ET-1 (0.3nmol/kg, 0.1ml) on BP, HR and ENG of RVLM neurons
↓: injection of ET-1
　　Table 1　Effects of intracarotid ET-1 (0.33nmol/kg) on MAP, HR and ENG of neurons in RVLM in anesthetized rats (n=36)

	MAP/kPa	HR/bpm	ENG/spikes^.s^－1
Control	13.8±0.3	381±3.9	17.8±1.5
ET-1	14.1±0.4	382±3.8	20.9±1.4^**

MAP: mean arteral pressure; HR: heart rate; ENG: electroneurogram of neurons in RVLM.^**P<0.01 vs control.

2.2　Blocking action of BQ-123 on the effects of ET-1
　　In response to ia injection of ET-1, 11 out of 14 units increased the firing rate from 16.5±1.4 to 19.2±1.4 spikes/s (P<0.01), 2 units showed no significant change, and 1 unit decreased the firing rate. After ENG returned to the control level, ia injection of BQ-123 (0.67nmol/kg, 0.1ml) had no significant effect on ENG, BP and HR. Five minutes later, ia administration of ET-1 no longer affected the firing rate of the RVLM neurons (Fig.3, Table 2).

Fig.3　Effects of intracarotid injection of ET-1 on BP, HR and ENG of RVLM neurons before and after BQ-123 administration
↓: injection of ET-1; ↓: injection of BQ-123.

2.3　Effect of glibenclimade on the action of ET-1
　　Following ia injection of ET-1, 10 out of 11 units increased the firing rate from 16.8±2.1 to 19.3±2.6spikes/s (P<0.01), and 1 unit showed no significant change. After ENG returned to the control level, ia injection of glibenclamide (3.3nmol/kg, 0.1ml) had no effects on ENG, BP and HR. Five minutes later, ia injection of ET-1 still increased the firing rate of 10 units from 16.8±2.1 to 20.8±1.7 spikes/s (P<0.01) (Table 2).

Table 2　Pretreatment with BQ-123(0.67nmol/kg) and glibenclamide(3.3nmol/kg) on the effects of intracarotid injection of ET-1 on MAP, HR and ENG of RVLM neurons

	n	MAP /kPa	HR /bpm	ENG /spikes^.s^－1
Control	11	13. 6±0.6	385±4.5	16.5±1.4
ET-1	11	14.2±0.5	386±2.4	19.2±1.3^**
BQ-123+ET-1	11	13.8±0.6	386±5.1	15.6±1.7
Control	10	13.9±0.8	381±3.5	16.8±2.1
ET-1	10	14.5±0.4	383±4.5	19.3±2.6^**
Gli+ET-1	10	13.6±0.2	384±2.8	20.8±1.7^**

^**P<0.01 vs control.

2.4　Effect of ia ET-1 after AP ablation
　　Figure 4 is the microphotograph showing the ablation of AP. In AP-ablated animals, BP and HR were decreased just after the ablation, but could return to the control level 1～1.5h later. The firing rate of 19 units in 6 rats with AP ablation showed no significant changes before and after ia injection of ET-1 (17.2±1.6vs 17.0±2.8 spikes/s, P>0.05) (Fig.5). BP and HR were unaffected. While in 7 discharge units of 3 rats with AP sham ablation, ia injection of ET-1 was still able to increase the firing rate from 16.9±2.2 to 20.3±3.2 spikes/s (P<0.01).

　　Fig.4　Microphotograph showing the ablation of area postrema(AP)
A: Before ablation.B: After ablation.NTS: nucleus tractus solitarius.

Fig.5　Effects of intracarotid injection of ET-1 on BP, HR and ENG of RVLM neurons after AP ablation
A. ET-1 before AP ablation.B. ET-1 after AP ablation. ↓: injection of ET-1.

3　DISCUSSION

　　The present study showed that intracarotid administration of ET-1 could increase the firing rate of the most neurons in RVLM, while the blood pressure(BP) and heart rate(HR) showed no significant changes. Pretreatment with a specific bloker of ET_A receptors BQ-123 abolished the response induced by ET-1, indicating that the effect is mediated by ET_A receptors.
　　Since systemic endothelin can not cross the blood-brain barrier which excludes most circulating peptides from reaching the CNS neurons, now a question arises: by which way does ET-1 exert its effect on RVLM neurons? It is well established that circulating peptides may act on the circumventricular organs(CVO) which are characterized by an unusually weak BBB. The AP is a midline CVO located on the floor of the fourth ventricle and rich in binding site for ET-1^［12］. It has been reported that systemically applied ET-1 could influence the activity of neurons in AP^［10］. As fibers from AP project to RVLM^［13,14］, so it may be supposed that the facilitatory effect of ia injection of ET-1 on RVLM neurons is indirectly produced by AP activation. In order to prove the hypothesis, the AP had been carefully ablated. The results showed that AP ablation did eliminate the effect of ET-1, while in the sham ablation animals facilitatory effect of ET-1 remained unchanged. The above results suggest that intracarotid-administered ET-1 binds to ET_A receptors in the AP, which results in the facilitation of the RVLM neurons via the AP efferent.
　　The change in firing rate of RVLM neurons induced by ia administration of ET-1 did not seem to be secondary to hemodynamic alterations, because BP and HR were not affected by ia injection of ET-1 in the present experimental condition. Some studies reported that activation of sympathetic nervous system might account for the cardiovascular effects induced by centrally administered ET-1^［15,16］. However, in these experiments, ET-1 was applied intracerebroventrically and inevitably acted on a variety of brain areas. Thus it was difficult to determine the exact site on which ET-1 exerted its action. Whereas in the present study, a precise site was demonstrated on which ET-1 acts.
　　It had been demonstrated in our laboratory that ET-1 exerted inhibitory effect on carotid baroreflex and sinus nerve activity, which might be blocked by ATP-sensitive potassium channel blocker-glibenclamide^［17,18］. However, the fact that ia injection of glibenclamide did not affect the central action of ia injection of ET-1 suggests that ATP-sensitive potassium channel is not involved in the central effect of ET-1. In summary, intracarotid-administered ET-1 may act on the ET_A receptors in AP, and the resultant activation of AP exerts a facilitatory effect on RVLM neurons.

^*国家自然科学 (No.39770280) 基金资助
^**联系作者
^*Supported by the National Natural Science Foundation of China (No.39770280)
^**Correspondence to Prof.HE Rui-Rong. Tel: 86-311-6044121, Ext. 5566. Fax: 86-311-6048177.
E-mail: syho@sjz.col.com.cn

作者单位：河北医科大学基础医学研究所生理室, 石家庄 050017

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Received 1998-08-07　　Accepted 1998-09-21