Effects of physiological manipulations on locus coeruleus neuronal activity in freely moving cats. II. Cardiovascular challenge

Brain Research, 422 (1987) 24- 31 Elsevier

24 BRE 12897

Effects of physiological manipulations on locus coeruleus neuronal activity in freely moving cats. II. Cardiovascular challenge David A. Morilak*, Casimir A. Fornal and Barry L. Jacobs Department of Psychology, Program in Neuroscience, Princeton University, Princeton, NJ 08544 ( U. S. A.) (Accepted 24 February 1987)

Key words: Cardiovascular system; Locus coeruleus; Noradrenergic neuron; Stress

Several cardiovascular manipulations were examined for their effects on single-unit activity of locus coeruleus noradrenergic (LCNE) neurons in unanesthetized, unrestrained cats: hydralazine (1 mg/kg, i.v.) was administered to present a tonic hypotensive stimulus, and to activate preferentially the neural component of the sympathoadrenal system; hemorrhage was used to decrease blood volume and to activate both the neural and hormonal components of the sympathoadrenal system; intravenous infusion of isotonic saline was used to increase blood volume. LC-NE neurons were activated by hydralazine, in parallel with the sympathetic response (indicated by elevated heart rate and plasma NE). LC-NE unit activity was decreased following a volume load. However, contrary to previous findings in anesthetized animals, hemorrhage had no effect on LC-NE unit activity, but did activate both components of the sympathetic response. It is concluded that: (1) cardiovascular stimuli can influence the activity of LC-NE neurons, though they show less sensitivity to such stimuli than do primary regulatory mechanisms; (2) the response of LC-NE neurons to physiological stimuli can occur independent of changes in behavioral state; (3) these neurons do not appear to play a specific role in cardiovascular regulation, but may respond to physiological challenges in general; (4) finally, in agreement with previous studies, our data show that LC-NE neurons are generally co-activated with the sympathetic nervous system, but also that the two can be dissociated (e.g. hemorrhage).

INTRODUCTION Noradrenergic (NE) n e u r o n s in the locus coeruleus (LC) have been implicated in the response to environmental stressors (see preceding paper). In addition, it has been suggested that these neurons may play a role in the regulation of a n u m b e r of physiological variables. Therefore, in this series of studies, we tested the effects of manipulating several physiological systems on the single unit activity of LC-NE neurons. O u r objective was to determine to what extent the postulated role of these n e u r o n s in the stress response could be generalized to include the response to physiological challenges. In the present paper, we report the effects of cardiovascular manipulations on LC-NE neuronal activity in behaving cats.

Anatomically, LC-NE n e u r o n s project to a number of primary cardiovascular regulatory areas, including the ventrolateral medulla and the dorsal vagal complex, the nucleus ambiguus, and the nucleus tractus solitarius4'3°,35,52,69"7°. In addition, the LC is connected with many areas of the hypothalamus which are involved in cardiovascular function29,3°,35, 38,53.54

The LC has been demonstrated to exert a modulatory influence on a variety of cardiovascular reflexes 5'22'33'50'56'71. Studies utilizing electrical or chemical excitation of LC have suggested that it is a pressor area 1°'2°'47As'57'7°. Additional evidence of a cardiovascular-LC relationship comes from studies of animal models of hypertension. Increased NE turnover and NE receptor function have been demonstrated in LC terminal areas in the spontaneously hypertensive rat (SHR) 26'28'41. Furthermore, in-

* Present address: Department of Medicine, Hinders University Medical Center, Bedford Park, S.A. 5042, Australia. Correspondence: B.L. Jacobs, Program in Neuroscience, Department of Psychology, Green Hall, Princeton University, Princeton, NJ 08544, U.S.A. 0006-8993/87/$03.50(~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)

25 creased rate of NE synthesis 3z, and an increase in the length and extent of dendritic arborizations of LCNE neurons 15 have also been observed in the SHR. Some observations, however, have been made which argue against a pressor function for LC. Chemical stimulation of LC-NE cell bodies has been shown to elicit a depressor response, while electrical stimulation by the same investigator was shown to elicit a pressor response 6°, suggesting that the pressor response could be due to current spread or to activation of fibers of passage. A depressor function was also suggested in a study in which experimental hypertension was produced by neurotoxic destruction of LC 42. Electrophysiological recordings of LC-NE neurons in anesthetized rats have indicated that their activity is altered by a number of cardiovascular manipulations. These neurons respond reciprocally to changes in blood volume and arterial blood pressure, and have been postulated to be inhibited by the activity of low-pressure atrial volume receptors 1L14'62. Additional electrophysiological evidence suggesting a cardiovascular role for LC-NE neurons again comes from studies of hypertensive animals. The mean firing rate of LC-NE neurons is reduced in desoxycortone (DOCA)-salt-hypertensive rats 44 and in SHR 44'61. Also, LC-NE neurons in SHR display a reduced sensitivity to acute alterations in blood pressure, and to vagal input 61. On the other hand, recent studies in anesthetized rats have demonstrated little or no effect of blood pressure changes on LCNE unit activity2L65. Thus, the relation of LC-NE neurons to blood pressure remains unresolved. A positive relationship between LC-NE neurons and the neural and/or hormonal components of the sympathetic nervous system (SNS) has also been postulated. A number of investigators have suggested that the LC represents a central branch or analogue of the peripheral SNS 3,23. In support of this, recent electrophysiological studies have shown that, in general, LC-NE neurons and the peripheral SNS respond in parallel to a variety of environmental and physiological stimuli 1'11-14'51. A positive relationship between LC-NE neurons and the adrenal medulla, the hormonal component of the SNS, has also been suggestedlO,16,17,20. In a previous study in behaving cats, we demonstrated a temporal relationship between LC-NE neuronal discharge and the cardiac cycle 39'4°, suggesting

that these neurons can be influenced by the cardiovascular system under basal conditions. The present studies were directed at further elucidating which specific cardiovascular variables could affect the activity of LC-NE neurons. Tonic changes in vascular tone were induced with hydralazine. Blood volume was altered either by hemorrhage or by infusion of isotonic saline. It was predicted, based on existing evidence, that LC-NE neurons should be activated by those stimuli eliciting decreases in blood pressure, blood volume, or vascular tone, and should be inhibited by increasing blood volume. MATERIALS AND METHODS Procedures for microelectrode implantation, single unit recording, neuronal identification, and behavioral state assessment are described in the preceding paper.

Intravenous infusion and blood sampling Intravenous drug injections, infusions, and blood withdrawal were made via a chronic indwelling jugular catheter as described in the preceding paper. Volume manipulations were performed remotely so as not to confound the results by disturbing or arousing the cat. Intravenous catheter patency was maintained by flushing weekly with heparinized saline (1000 IU/ml).

Plasma catecholamine assays Animals were heparinized (1000 IU heparin sodium, i.v.), 1.5-2 h prior to the onset of the experiment. At the time points specified, 1 ml of blood was voided from the venous catheter to clear the line, and 2.5 ml were withdrawn for catecholamine determination. The volume withdrawn was replaced with isotonic sterile saline. Blood samples were centrifuged and plasma was immediately frozen in liquid nitrogen. Samples were then stored at -70 °C until extraction and measurement (typically 1-3 weeks). One ml of plasma samples or standards of known epinephrine (EPI) and NE concentration were used for extraction utilizing acid-treated alumina, with 1 ng of 3,4-dihydroxybenzylamine (DHBA; 100 pl at 10 ng/ml) added to each tube as an internal standard. After elution with 0.1 N perchloric acid containing 100 pmol E D T A , sample catecholamine concentra-

26 tions were measured using high-performance liquid chromatography (HPLC) with electrochemical detection according to the method of Mayer and Shoup 37. Catecholamine concentrations were determined using the ratio of EPI or NE to D H B A peak height, and calculating concentration from a line-ofbest-fit constructed from the peak height ratios of the standards. Recovery values for NE and EPI were 74% and 70%, respectively. Intra-assay and inter-assay coefficients of variations, determined from pooled plasma samples, were 2% and 15% for NE. EPI values, which were often near the detectibility limit, were not obtained from the pooled plasma samples. The mean limits of detectability, defined as the Y-intercept of the regression line, for a total of 8 runs were 67 pg/ml and 139 pg/ml for NE and EPI, respectively.

infusion. During the course of hemorrhage, if an animal displayed cardiac arrhythmias, no more blood was withdrawn and the experiment was terminated.

Statistical analyses All analyses were performed as described in the preceding paper.

Drugs Drugs used in these studies were: clonidine HC1 (Sigma); 3,4-dihydroxybenzylamine HBr (Sigma); (-)-epinephrine-(+)-bitartrate (Sigma); hydralazine HCI (Sigma); (-)-norepinephrine HCI (Sigma); All substances were dissolved in 0.9% sterile saline, and all concentrations and doses are expressed as the salt. RESULTS

Hydralazine Hydralazine The effects of hydralazine (1 mg/kg i.v.) on LC-NE single unit activity was examined. This dose of hydralazine was chosen because it produces a prolonged decrease in vascular tone 19,31,59, a marked reflex increase in sympathetic outflow 27'34'64, and a modest decrease in mean arterial pressure e7'55'68. Three 1min samples of unit discharge and heart rate were recorded as baseline, and then at 15, 30, 45, 60 and 120 rain following drug administration.

Blood volume changes Volume expansion. Total blood volume was estimated at 60 ml/kg 2. Volume was expanded by a total of 15% with intravenous infusion of sterile isotonic saline administered at a rate of 2.25 ml/kg/min. The volume load was administered in two steps of 7.5% each, separated by 10 min. The mean unit discharge rate was determined for 10 min immediately preceding the infusion (baseline), for the 10 min following a 7.5% load, and for 10 min following 15% volume expansion. Hemorrhage. Venous blood was withdrawn at a rate of 2.25 ml/kg/min, in steps of 7.5% of the estimated total blood volume, up to a maximum of 30%. Three 1-min samples of unit discharge were obtained immediately prior to hemorrhage (baseline) and following each 7.5% withdrawal step. Following the last withdrawal, blood was reinfused, and unit activity was measured immediately, and 15 min following re-

A group of 7 cells meeting all of the criteria for LCNE neurons (see first paper in this series) was recorded in 6 cats in response to administration of hydralazine. As a group, these cells showed a significant increase in firing rate after hydralazine (F5.30 = 3.58, P < 0.02). On an individual cell basis this effect was seen in 6 of the 7 cells. Baseline quiet waking (QW) firing rate of these 6 cells was 0.43 + 0.13 spikes/s, and this was significantly elevated from 15 to 60 min following hydralazine (F5.25 = 3.79, P < 0.02). The maximal level of unit activity, which occurred at 30 min, was 0.78 + 0.17 spikes/s. Unit activity returned to near-baseline by 2 h (Fig. 1). Heart rate recorded simultaneously with unit activity increased from a QW baseline of 144 + 10 b.p.m, to a maximum 217 + 8 b.p.m, by 15-30 min following hydralazine. Plasma catecholamines were measured in 6 cats without simultaneous unit recordings (Fig. 1, bottom). (The heart rate response to hydralazine in this group of animals was similar to the heart rate response in animals used for unit recordings). Plasma NE levels were consistently elevated following hydralazine administration, increasing from a baseline 906 + 235 pg/ml to a maximum 2355 + 57 pg/ml (F4,20 = 4.03, P < 0.02). Plasma EPI, on the other hand, showed only a moderate, non-significant increase, from 446 + 133 pg/ml to 677 + 126 pg/ml (F4.20 = 1.13). These baseline values are within the range reported in a number of other studies 6'8'58. While the period of elevated neuronal discharge

27 the E E G . This is in a g r e e m e n t with on-line polygraphic and behavioral observations.

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A group of 7 cells meeting all of the criteria for LCN E neurons were r e c o r d e d in 3 cats during b l o o d volume expansion. A s a group, these cells significantly decreased their firing rate after 15% volume expansion (F2,12 = 10.58, P < 0.005), with all 7 o f these cells showing a significant decrease in rate. O n l y one of these 7 cells showed a significant reduction after a 7.5% volume load. A s a group, these L C - N E neurons had a Q W firing rate of 1.39 + 0.24 spikes/s, and this decreased to 1.08 + 0.25 spikes/s after 15% (Fig. 2). Power spectrum analyses of E E G r e c o r d e d in 3 cats, not being used for simultaneous unit recordings, revealed that volume load had no effect on E E G . This is in support of on-line polygraphic and behavioral observations.

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Fig. 1. Effects of hydralazine (1 mg/kg, i.v.). A: unit response of the 6 LC-NE cells showing a significant change after hydralazine (solid line), and of simultaneously recorded heart rate (dashed line). Note the generally parallel responses, except at 120 min, where heart rate was still elevated, but unit discharge rate was not different from baseline. B: changes in plasma EPI (solid line) and NE (dashed line) in response to the same dose of hydralazine (n = 6). Plasma NE was significantly elevated at all time points, while plasma EPI showed no change. Time B = baseline. All values are mean +S.E.M. * Statistical significance was determined at P < 0.05.

Hemorrhage A group of 5 cells meeting all of the criteria for LCN E neurons were r e c o r d e d in 4 cats during hemorrhage. B l o o d was withdrawn in steps of 7.5% of total b l o o d volume up to 30% of e s t i m a t e d total volume. F o r one cell, only 7.5% could be withdrawn; for one cell 15% was taken; for two cells 22.5% was withdrawn; and for one cell, a total 30% (18 ml/kg) of blood volume was r e m o v e d . The baseline Q W firing rate of these 5 cells was 0.55 + 0.14 spikes/s. A n a l y s e s revealed that hemorrhage had no effect on the discharge rate of L C - N E

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corresponded well to the p e r i o d of elevated heart rate and plasma N E (Fig. 1), only two cells showed their maximal discharge rate coincident with the m a x i m u m heart rate response. F u r t h e r m o r e , the duration of the elevation in neuronal activity did not match that of the cardiac and sympathetic activation. Both heart rate and p l a s m a N E were still significantly elevated at 2 h, while L C - N E unit activity had returned to baseline. Power spectrum analyses of E E G r e c o r d e d in 4 cats not being used for simultaneous unit recordings, revealed that hydralazine h a d no significant effect on

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creased from 214 + 20 pg/ml to 448 + 50 pg/ml. Power spectrum analyses of E E G recorded from 4 cats, 2 of which had simultaneous unit recordings, and 2 of which were also used for plasma catecholamine measurements, revealed that hemorrhage had no effect on EEG. This supports the polygraphic and behavioral observations•

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Fig. 3• The effect of hemorrhage on the activityof a representative LC-NE neuron. This cell, taken to 30% hemorrhage, typifies the lack of effect on the activity of all cells tested• Upper traces = Schmitt-triggeroutput of unit discharge. Lower traces = EKG. Bar = 5 s. neurons. For the 5 cells taken to 7.5% hemorrhage, the firing rate was 87% of baseline (F1,4 = 0.79). For the 4 cells taken to 15%, firing rate was 82% of baseline (F2,6 = 2.83). For the 3 cells taken to 22.5% hemorrhage, discharge rate was 103% of baseline (F3,6 = 0.96). The lack of change for the cell taken to 30% blood withdrawal is shown in Fig. 3. Baseline heart rate during these recordings was 177 -4- 14 b.p.m. Heart rate increased slightly following the first two withdrawal steps, reaching a maximum of 196 + 11 b.p.m, after 15% hemorrhage, and decreased to a minimum 124 + 13 b.p.m, following hemorrhage beyond 15%. These responses suggest that the stimulus was potent enough to influence the activity of peripheral cardiovascular receptors, including the atrial type-B volume receptors and the ventricular C-fiber receptors 7'45'63. Plasma catecholamine measurements, taken in two cats not being used for unit recordings, indicate that hemorrhage activated both components of the SNS. After 22.5% hemorrhage, plasma NE increased from a baseline 226 + 46 pg/ml to 527 + 52 pg/ml, and plasma EPI in-

In accord with studies done in anesthetized rats ]4,62, we have shown that increases in blood volume produce decreases in LC-NE unit discharge. However, the sensitivity of LC-NE neurons to this manipulation was less than that observed in anesthetized rats, since volume changes of as little as 0.5 ml (approximately 2% total blood volume) influenced unit discharge in those studies. Even greater discrepancies between the present data and those in anesthetized rats were revealed by the response to hemorrhage. Contrary to previous studies, removing from 15 to 30% estimated total blood volume had no effect on LC-NE unit activity in conscious cats, while hemorrhage of as little as 2% activated LC-NE neurons in anesthetized rats n. Although this discrepancy may simply represent a species difference, a more likely possibility concerns the effects of anesthesia. Anesthesia has been shown to alter the tonic discharge rate and the response characteristics of monoaminergic neurons, including NE neurons 25,49. Likewise, anesthesia alters the response characteristics of the cardiovascular system, both in terms of receptor functioning and the ability of the system to adequately compensate for a given stimulus, thus altering the nature and effectiveness of the stimulus itself 9A8"24'36"46. For instance, hemorrhage produces greater falls in blood pressure under anesthesia 9'66'67. In addition, a different set of secondary effects may be elicited under anesthesia. For example, hemorrhage stimulates vasopressin release, and vasopressin has been shown to produce opposite cardiovascular effects when administered centrally to anesthetized vs conscious animals 72. Moreover, these secondary factors may have direct effects on LC-NE neurons (e.g. vasopressin has been shown to excite LC-NE neurons in anesthetized rats43). What general conclusions can be drawn on the ba-

29 sis of these data? These studies represent the first direct investigation of the influence of cardiovascular manipulations on the activity of L C - N E neurons in unanesthetized and unrestrained animals. The first two questions addressed, and the most basic issues examined, were whether L C - N E neurons respond to such manipulations, and whether they do so independent of changes in arousal. L C - N E neurons showed significant increases in tonic firing rate in response to hydralazine, and they showed significant decreases in rate after blood volume expansion. Behavioral observations, polygraphic measures, and power spectrum analyses of E E G all showed that no change in behavioral state or level of arousal could account for the observed changes in L C - N E unit activity. The third issue addressed in these studies concerns the role of L C - N E neurons in cardiovascular regulation. These data suggest that L C - N E neurons are not directly involved in the regulation of the cardiovascular system, but that they may serve a more general modulatory function. For instance, the lack of response to hemorrhage suggests that not all cardiovascular stimuli are capable of eliciting L C - N E neuronal responses. Furthermore, L C - N E neurons displayed, in general, less sensitivity than primary cardiovascular regulatory mechanisms. For example, L C - N E neurons were inhibited by 15%, but not by 7.5% blood volume expansion. Thus, the activity of these neurons does not appear to be greatly influenced by low pressure cardiac receptors. Finally, the duration of the L C - N E neuronal response did not match that

REFERENCES

of the cardiac and sympathetic responses after hydralazine. The fourth issue addressed in these studies was whether L C - N E neuronal activity was coupled with the sympathetic response to cardiovascular stimuli. In this regard, we employed hemorrhage and hydralazine administration as manipulations which produce reflex activation of the SNS, and volume load as a stimulus which decreases sympathetic outflow. LCN E neuronal activity was significantly increased following hydralazine administration, and decreased following volume load, but was not significantly altered following hemorrhage. These studies indicate that L C - N E neurons are co-activated or de-activated with the SNS in response to some manipulations (e.g. hydralazine and volume load), however, there are situations in which sympathetic activation occurs without a concomitant increase in L C - N E unit activity (e.g. hemorrhage). ACKNOWLEDGEMENTS Excellent technical assistance was provided by U. Saini and R. Harris. Thanks also to Dr. Ron Notvest and colleagues of Ayerst Laboratories for use of their facilities and assistance with the power spectrum analyses of E E G . This research was supported by an NSF Predoctoral Fellowship to D . A . M . , by N I M H Grant MH23433 and U.S. Air Force Grant A F O S R 85-0034 to B.L.J., and by a grant from the Campbell Institute for Research and Technology.

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Note added in proof In preliminary studies, sodium nitroprusside and phenylephrine hydrochloride (5-15 ~g/kg, i.v.) were administered to produce transient periods (30-90 s) of hypo- and hypertension, respectively. A brief increase in LC-NE unit activity was observed during and immediately following injecion of both drugs, and was associated with behavioral activation. Perhaps more importantly, we found no evidence for changes in LC-NE unit activity during periods of drug action associated with sustained hypotension or hypertension. Thus, in agreement with previously publised studies in anesthetised rats 21 •65 we were unable to detect any effect of alterations in blood pressure on LC-NE unit activity independent of changes in behavioral arousal.