Central sympathetic mechanisms of blood pressure control in hamsters

Journal of the Autonomic Nervous System

ELSEVIER Journal of the Autonomic Nervous System 48 (1994) 181-186

Short Communication

Central sympathetic mechanisms of blood pressure control in hamsters

Andrei V. Krassioukov *, Lynne C. Weaver a The John P. Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, Ontario N6A 5K8, Canada.

b Department of Physiology, UniL~ersity of Western Ontario. London, Ontario, Canada

(Received 12 October 1993; revision received 10 November 1993; accepted 19 November 1993)

Abstract

The goal of this study was to investigate central vasomotor control of blood pressure in golden hamsters. Electrophysiological experiments demonstrated that tonic and reflex firing of renal nerves was controlled by brainstem and spinal circuits in manner similar to control of these nerves in rats, rabbits and cats. These findings confirmed that autonomic neural circuits for vasomotor control in hamsters are functionally similar to those of other well-studied species.

Key words. Sympathetic control; Glycine; Somato-sympathetic reflex

The organization of sympathetic vasomotor control from the medulla and spinal cord has been investigated extensively in rats, rabbits, cats and dogs and the rostral ventrolateral medulla is known to play an important role in maintaining vasomotor tone [1]. Some sympathetic discharge can also be generated by spinal cord circuits [7,16,18]. Recently we used herpes simplex type 1 virus (HSV-1) to identify preganglionic neurons controlling the kidney in golden hamsters [3]. The study was done in golden hamsters because they

* Corresponding author. Tel.: (519) 663-3776; Fax: (519) 663- 3789.

were much more susceptible to HSV-1 infection than rats and our results suggested that the organization of their central autonomic neurons was similar to that of rats. Although golden hamsters have been used in investigations of control of endocrine function and in a morphological study of sympathetic preganglionic neurons in upper thoracic segments [4,13] information about the functional organization of sympathetic control of blood pressure and renal function in hamsters is not available. This lack of information leads to questions about the relevance of studying central autonomic neurons controlling visceral and vascular function in hamsters. Therefore we did electrophysiological experiments using anesthetized

0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(93)E0149-Y

182 A.V. Krassioukov, L.C. Weaver~Journal of the Autonomic Nervous System 48 (1994) 181-186

hamsters to determine if firing of their renal nerves was controlled by brainstem and spinal circuits in a manner similar to control of these nerves in well-studied species such as rats, rabbits and cats.

Surgical preparation of hamsters. Five adult male Golden hamsters (Mesocricetus auratus) weighing 170-180 g were used in this study. Hamsters were anesthetized with pentobarbital (40 mg/kg, i.p.), followed by propofol (Diprivan, 30 mg • kg-~ • hr-1, i.v.) and artificially ventilated [9]. Arterial blood pressure, arterial pH, p C O 2

and rectal temperature were monitored and maintained within normal parameters during experiments [7]. The hamsters were placed in a stereotaxic frame and a section of the interpari- etal bone was removed to expose the cerebellum overlying the medulla. This exposure was done in preparation for microinjecting the inhibitory amino acid glycine into the rostral ventrolateral medulla. The left renal nerves were exposed in all hamsters via a retroperitoneal approach and prepared for recording multifiber electrical activity as described previously [7,9]. To evoke somato- sympathetic reflexes, the sciatic nerve was iso- lated. The spinal cord was exposed by a dorsal laminectomy and, later in the experiments, was transected at the fifth cervical segment (C5) by an ultrasound scalpel (UZH-201, Medprom, Mos- cow, Russia).

Experimental protocols. The renal nerve activity after amplification, was monitored on an oscil- loscope and recorded on magnetic tape [7,9]. Arterial blood pressure and heart rate were recorded and displayed continuously on a Grass polygraph and on magnetic tape. First renal nerve activity was tested for inhibitory responses to activation of baroreceptors. Arterial blood pressure was increased by bolus injections of phenylephrine (1-3/zg, intravenously) to stimulate ca- rotid sinuses and aortic arch baroreceptors in each hamster. Next, glycine (1.0 M, 50-80 nl) was pressure-injected into the rostral ventrolateral medulla unilaterally through glass micropipettes as described previously [9]. These pipettes were positioned stereotaxically in the rostral ventrolateral medulla using modified coordinates deter- mined from the stereotaxic atlas of Paxinos and

Watson [12]. Modified coordinates, proportional to the smaller hamster brain, were calculated by comparing measurements of the skull and brain of the hamsters to those of rats. We used the reference points bregma, lambda and the mid- point of the interaural line in our study as they are commonly used in stereotaxic procedures and they were as easily identified in hamsters as they are in rats.

Sciatic nerves were stimulated with bipolar stainless steel electrodes using single square-wave stimuli (250 /.LA, 1.0 ms, 1.0 Hz) generated by a Grass stimulator (model $44, Grass Instrument Company). Spontaneous discharge of renal nerves, somato-sympathetic reflexes in renal nerves, blood pressure and heart rate were recorded before and after glycine injections and spinal cord transection. At the end of the experiments the brains were removed, fixed, sectioned and stained with neutral red dye for histological verification of the sites of microinjection of glycine into the rostral ventrolateral medulla [9].

Data analysis. Integration of sympathetic dis- charges, averaging of somato-sympathetic reflexes and analyses of cardiac rhythmicity in the renal nerve activity were performed using a program prepared by R.C. Electronics Inc. (Goleta, CA, USA). To determine statistical changes in neural activity, blood pressure and heart rate, a one-way analysis of variance with repeated measures was used, followed by Tukey's test for comparison of means [15].

Characteristics of sympathetic activity in renal nerves and responses to stimulation of arterial baroreceptors. Intravenous injection of phenylephrine (1-3/zg, i/v) increased mean arterial pressure by 66 _+ 9 mmHg and this activation of baroreceptors inhibited sympathetic nerve activity by 75 + 5% (Fig. 1). Electrical discharge of the renal nerve in each hamster was characterized by synchronized bursts of activity when the spinal cord was intact. These bursts of activity showed a strong cardiac-related periodicity consistent with phasic baroreceptor inputs (Fig. 1B-D). Grouped discharge synchronous with cardiac cycle typically occurred during the diastole phase of arterial pressure (Fig. 1C,D). The peak of renal nerve activity occurred approximately 58 _+ 8 ms after

A.V. Krassioukou, L.C. Wea~,er / Journal of the Autonomic Neruous System 48 (1994) 181-186 183

the beginning of systole and the cardiac cycle lasted 140 _+ 5 ms.

Sympathetic responses to blockade of the rostral uentrolateral medulla by glycine. Glycine microinjection into the rostral ventrolateral medulla produced prolonged decreases in blood pressure, heart rate and sympathetic nerve activity in hamsters (Fig. 2A). Nerve activity decreased significantly by 45 + 4% from 39 _+ 2 to 21 +_ 3 txV • s (5 animals, 6 microinjections) after this blockade. Blockade of the ventrolateral medulla decreased mean arterial pressure by 39_+ 3 mmHg (from 115 _+ 3 mmHg) and heart rate was decreased by 13 _+ 6 bpm (from 364 + 8 bpm). These responses

lasted 45 + 4 min after the glycine injection. The sites of medullary injections (Fig. 2B and Fig. 3) were located 150-400 Izm from the ventral sur- face in an area extending from the caudal limit of the facial nucleus to the rostral one-third of the inferior olive and extending medially from the lateral reticular nucleus to the lateral border of the inferior olive. This region has been termed the rostral ventrolateral medulla in rats [1].

Sympathetic responses to cer~'ical spinal cord transection. Transection of the spinal cord increased renal nerve activity and blood pressure transiently and then these variables quickly decreased significantly below control values (Fig.

A 350

HR (bpm)

3O0

1 2 3 B

AP (mmHg)

C AP

(mmHg)

RNA (~v)

2O0

120 8O

0 1 2 3 t

4 rain

D 120 t

0j 100 04

Fig. 1. Effect of stimulation of arterial baroreceptors and evidence of cardiac rhythmicity in renal sympathetic nerve activity. (A) Changes in arterial pressure (AP) and heart rate (HR) after a bolus intravenous injection of phenylephrine (1.0 ~zg). The top trace illustrates HR, the lower trace shows AP. The time of injection of phenylephrine is shown by a closed arrow. The numbered open arrows above the recording of H R show time of recording of renal nerve activity (RNA). (B) R N A before (1), during (2) and after (3) activation of the baroreceptors. Numbers correspond in time to the numbered arrows above the heart rate record. Calibration for B: 50/zV, 100 ms. (C) A single trace of AP and R N A recorded simultaneously. Grouped discharge in the nerve is synchronized with pressure pulse during the diastolic phase. (D) AP and R N A and averages of these two signals triggered by the arterial pressure pulse in a hamster . Averaged R N A obtained from 25 consecutive cardiac cycles. The R N A consistently occurred at the beginning of the diastolic phase of AP. The trigger for the averages is shown by the interrupted line. Time bars at the bot tom on C and D are 50 ms.

184 A.V. Krassioukol', L.C. Weauer /Journal of the Autonomic Neruous System 48 (1994) 181-186

2C). By 60 min after spinal cord transection renal nerve activity had stabilized and mean discharge of renal nerves in five hamsters was decreased by only 11 _+ 5%. Before transection in these five hamsters renal nerve activity was 38_+ 3 ~zV.s and 60 min after transection this discharge was 35 _+ 2 pN • s. Mean values of arterial blood pressure and heart rate before transection were 87 _+ 4 mmHg and 364 + 8 bpm. After transection blood pressure was 76 + 3 mmHg (supported by phenyl-

ephrine infusion at 0.1-0.2 ~zg/min in three of five hamsters) and heart rate was decreased significantly to 342 _+ 6 bpm.

Somato-sympathetic reflexes in hamsters. Re- flexly evoked potentials with a long latency (medullary component of the somato-sympathetic reflex) ranging from 89-104 ms were elicited in the renal nerve of the hamsters (n = 5) by stimulation of the sciatic nerve (Fig. 2D,1). The early reflex-potential (spinal component of this reflex)

A B

RNA 2'21 (~V.s)

1.2 HR

(bpm)

AP 160~ (mmHg)

80

0 I 2

t P~lyclno Into RVUVl

60 61 rain

C D 2.4 ~r

(~.V-s) \~.~ .~.~...,,.~'~ 1 114

(bpm) 2 ~ . ~ 320 J

164:]~ nn _

(rnmHg]

0 1 2 60 61 min t Spinal cord Transection

Fig. 2. Responses to blockade of the rostral ventrolateral medulla by glycine and to transection of cervical spinal cord. (A) Sympathetic responses to blockade of the rostral ventrolateral medulla by glycine. Effect on integrated renal nerve activity (RNA), heart rate (HR) and arterial blood pressure (AP). Each data point for R N A represents average discharge during a 10-s interval. Right panel: recordings of RNA, H R and AP 60 rain after glycine injection. Time of glycine injection is indicated with an arrow. (B) Medullary sites of glycine injection. Triangles represent injection sites from which responses in renal nerve activity, arterial pressure and heart rate were elicited. NTS (nucleus tractus solitarius); Arab (nucleus ambiguus); IO (inferior olivary nuclei): 7 (facial nucleus). Calibration is 1 mm. (C) Effect of cervical spinal cord transection on RNA, AP and HR. Right panel: recordings of RNA, H R and AP 60 rain after spinal cord transection. The time of spinal cord transection is indicated with an arrow. Format is the same as that of A. (D) Changes in the bulbar component of the somato-sympathetic reflexes in the renal nerve to stimulation of sciatic nerve (250 ~A, 1 ms, 1 Hz) after spinal cord transection. 1 - control recording of the somato-sympathetic reflex, 2 - absence of reflex after transection. Arrow shows time of stimulation of the sciatic nerve. Calibrations for D: 200 /zV, 20 ms.

A. 1A Krassioukot,, L. C. Wearer/Journal of the Autonomic Nerrous System 48 (1994) 181-186 185

was not apparent in our recordings. The long latency medullary component of the somato-sympathetic reflex was abolished completely by spinal cord transection at the C5 level in these five hamsters (Fig. 2D,2).

Although the organization of sympathetic vasomotor control from the medulla and spinal cord has been investigated extensively in different species such as rats, rabbits, cats and dogs [1,16,17], we present the first evidence of such an electrophysiological study in hamsters. Similari- ties between the functional organization of sympathetic circuits in hamsters and rats were pre- dicted by the correspondence of some aspects of the anatomical organization of these neurons in these two species. The location of central and peripheral autonomic neurons may not necessar- ily have functional significance but a comparison of the distributions of pre- and postganglionic neurons in the spinal cord nuclei and ganglia of rats and hamsters yields very similar results. For example, preganglionic neurons contributing to the greater splanchnic nerve of rats and hamsters are located mostly in the eighth to twelfth thoracic spinal cord segments and these neurons are found mostly in the intermediolateral cell column and more medial nucleus intercalatus and rarely are found in the central autonomic nucleus [3,8].

A minor difference between rats and hamsters is the relative prevalence of preganglionic neurons in the lateral funiculus of hamsters in comparison to the paucity in rats [3,8]. The renal postganglionic neurons of both rats and hamsters are located mostly in the twelfth and thirteenth thoracic paravertebral ganglia in contrast to renal neurons of cats that are found mostly in the celiac ganglia [2,3,10]. These anatomical distributions of sympathetic neurons demonstrate that these neurons are organized similarly in rats and hamsters. However, this information alone was inadequate to conclude that control of the cardio- vascular system in these two species is accom- plished similarly.

Our electrophysiological study demonstrated that control of sympathetic discharge, arterial pressure and heart rate in hamsters was almost identical to that of rats. Firing patterns character- istic of postganglionic sympathetic nerves such as the well-known cardiac-related rhythm [6,11] were clearly apparent in the discharge of the renal sympathetic nerve of the hamster. Since activity of renal nerves in hamsters was always inhibited by stimulation of baroreceptors, these nerves were considered likely to contain mostly vasomotor fibres as do renal nerves of other animals [5,10]. Decreases in renal nerve activity, blood pressure

Fig. 3. Site of glycine injection into the hamster medulla. (A) Printed image of a section of the medulla showing the electrode track dorsally (left side) and the dye-marked microinjection of glycine into the RVLM (arrow). Image was produced using a computerized imaging system (Imaging Research, St. Catherines, Canada). (B) Magnified area of brain section from box in A. Arab, nucleus ambiguus; py, pyramidal tract; 4V, 4th ventricle. Calibration for A is 400 ~m and for B is 100 p~m.

186 A.V. Krassioukov, L. C Weaver/Journal of the Autonomic Nervous System 48 (1994) 181-186

and heart rate after microinjection of the inhibitory amino acid glycine into the rostral ventrolateral medulla caused responses in hamsters comparable to those in rats [9]. Therefore, our results demonstra ted that the 'vasomotor centre ' of the hamster is in the rostral ventrolateral medulla, like that of many other species [1]. Re- flex control of sympathetic nerves from somatic nerve stimulation in hamsters also followed the same pathway that it does in cats and rats [14]. Finally the spinal networks that can drive renal sympathetic nerve activity in spinal rats [7,16] were also present in hamsters. Sympathetic responses to transection of the spinal cord in hamsters were almost identical to the responses ob- served in rats; after an initial brief period of instability, discharge persisted at a level only slightly lower than it had been before transection. These results demonstrate that central autonomic circuits in hamsters function in a manner very similar to those of rats. As the rat is widely accepted as a suitable experimental animal for studies of central autonomic control of the circu- lation and of visceral function, the hamster also may be considered an appropriate animal for studies of sympathetic control of the cardiovascu- lar system.

Acknowledgements

This research was supported by grants from the Heart and Stroke Foundation of Ontario and the Medical Research Council of Canada/Cana- dian Hypertension Society. L.C. Weaver is the recipient of a Career Investigator aw~ard from the Heart and Stroke Foundation of Ontario and A.V. Krassioukov holds a Fellowship from the Medical Research Council of Canada/Canadian Hypertension Society. The authors are grateful to Robert Atkinson and Barbara Atkinson for assis- tance with preparation of this manuscript.

References

[l] Calaresu, F.R. and Yardley, C.P., Medullary basal sympathetic tone, Annu. Rev. Physiol., 50 (1988) 511-524.

[2] Chevendra, V. and Weaver, L.C., Distribution of splenic,

mesenteric and renal neurons in sympathetic ganglia in rats, J. Auton. Nerv. Syst., 33 (1991) 47-54.

[3] Dehal, N.S., Dekaban, G.A., Krassioukov, A.V., Picard, F.J. and Weaver, L.C., Identification of renal preganglionic neurons in hamsters using transynaptic transoprt of herpes simplex type 1 virus, Neuroscience, 56 (1'~93) 227-240.

[4] DonCarlos, L.L. and Finkelstein, J.A., Hypothalamo-spi- hal projections in the golden hamster, Brain Res. B011., 18 (1987) 709-714.

[5] Dorward, P.K., Burke, S.L., Janig, W. and Cassell. J., Reflex responses to baroreceptors, chemoreceptor and nociceptor inputs in single renal sympathetic neurones in the rabbit and effects of anaesthesia on them, J. Auton. Nerv. Syst., 18 (1987) 39-54.

[6] Gebber, G.L., Central oscillators responsible for sympathetic nerve discharge, Am. J. Physiol., 239 (1980) HI43- H155.

[7] Hayes, K., Yardley, C.P. and Weaver, L.C., Evidence for descending tonic inhibition specifically affecting sympathetic pathways to the kidney in rats, J. Physiol., 434 (1991) 295-306.

[8] Hong, Y. and Weaver, L.C., Distribution of immunoreac- tivity for enkephalin, substance P and vasoactive intesti- nal peptide in autonomic nuclei of middle and lower thoracic spinal cord of rat: relationship with splanchnic sympathetic preganglionic neurons, Neuroscience (1993) (in press)

[9] Krassioukov, A.V., Gelb, A.W. and Weaver, L.C,, Action of propofol on central sympathetic mechanisms controlling blood pressure, Can. J. Anaesth., 40 (1993) 761-769.

[10] Meckler, R.L. and Weaver, L.C., Characteristics of ongo- ing and reflex discharge of single renal and splenic sympathetic postganglionic fibers in cats, J. Physiol. (Lond.), 396 (1988) 139-153.

[11] Ninomiya, I., Akiyama, T. and Nishiura, N., Mechanism of cardiac-related synchronized cardiac sympathetic nerve activity in awake cats, Am. J. Physiol., 259 (1990) R499- R506.

[12] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1986, 237 pp.

[13] Reuss, S., Johnson, R.F., Morin, L.P. and Moore, R.Y., Localization of spinal cord preganglionic neurons inner- vating the superior cervical ganglion in the golden hamster, Brain Res. Bull., 22 (1989) 289-293.

[14] Sato, A., The spinal and supraspinal somato-sympathetic reflexes. In: F.F. Kao, K. Koizumi and M. Vassalle (Eds.), Research in Physiology, Aulo Gaggi Publisher, Bologna. 1971, pp. 507-516.

[15] Snedcor, G. and Cochran, W.G., Statistical Methods, The Iowa State University Press, IA, 1993, 503 pp.

[16] Taylor, R.F. and Schramm, L.P., Differential effects of spinal transection on sympathetic nerve activities in rats, Am. J. Physiol., 253 (1987) R611-R618.

[17] Weaver, L.C. and Stein, R.D., Effects of spinal c~rd transection on sympathetic discharge in decerebrate-un- anesthetized cats, Am. J. Physiol., 257 (1989) R1506- R1511.

Documents

Central sympathetic mechanisms of blood pressure control in hamsters