Alterations of locus coeruleus noradrenergic activity in relation to pituitary secretion after hemorrhage in cats

Neuroscience Letters, 161 (1993)85 88 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193l$ 06.00

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Alterations of locus coeruleus noradrenergic activity in relation to pituitary secretion after hemorrhage in cats K.V. T h r i v i k r a m a n * , Paul M. P l o t s k y 1 a n d D o n a l d S. G a n n 2 Department o[' Surgery, Division of Biology and Medicine, Brown University, Rhode Island Hospital, Providence, RI02903, USA (Received 8 December 1992; Revised version received 29 June 1993: Accepted 12 July 1993) Key words: Adrenocorticotropin; Catecholamine; Electrochemistry; Hemodynamic stimulus; Noradrenergic activity; Potentiation; Push-pull perfusion; Vasopressin Noradrenergic (NA) activity in the cat ventral locus coeruleus (vLC), measured either by voltammetry or by push-pull perfusion, increased in response to hemorrhage. This stimulus also elicited plasma adrenocorticotropin (ACTH) and vasopressin responses. Increased vLC NA activity following the initial hemorrhage (InHem) persisted even after plasma hormone levels returned toward prestimulus values. Upon stimulus repetition, the peak increase in vLC NA activity was similar to that observed during lnHem while the hormone responses were of greater magnitude (i.e. potentiated). Hence, it is suggested that the LC may exert a modulatory role in the hemodynamic control of hypothalamic-pituitary axis function.

The locus coeruleus (LC) participates in the modulation of autonomic and visceral stimuli [1, 6, 8]. Although stressors that activate hypothalamic-pituitary-adrenal (HPA) axis also alter LC noradrenergic (NA) activity [9], the role of the LC in the regulation of HPA axis activity is unclear. Studies have established the morphological and functional connectivity necessary for LC participation in the regulation of HPA axis activity [4, 8, 10, 11, 15]. The LC receives hemodynamic input [5, 8], which reliably activates the HPA activity. Furthermore, a correlation between increased ventral LC (vLC) NA activity and pituitary secretory responses has been demonstrated [14, 19, 20]. In this connection, the plasma ACTH response to hemorrhage was attenuated in rats pre-treated with DSP-4 [21], a drug that selectively destroys LC-NA output. In cats, repeated hemorrhage attenuates the discharge characteristics of medullary and hypothalamic neurons responsive to hemodynamic stimuli [23], while the LC firing rate is maintained to sustained hemodynamic stimuli and suppressed to sustained noxious stim-

*Corresponding author. Present address: Stress Neurobiology Laboratory, Department of Psychiatry, P.O. Drawer AF, Emory University, Atlanta, GA 30322, USA. Fax: (1) (404) 727-3233. ~Present address. Stress Neurobiology Laboratory, Department of Psychiatry, P.O. Drawer AF, Emory University School of Medicine, Atlanta, GA 30322, USA. Fax: (1) (404) 72%3233. ;Present address: Department of Surgery University, Maryland School of Medicine, Baltimore, MD 21201, USA.

uli in rats [5]. In contrast to neuronal activity, the plasma ACTH response to repeated hemorrhage is either maintained or potentiated in dogs, cats and rats [7, 13, 22]. The current studies were undertaken in order to examine the relationship between LC-NA tone and HPA axis activity in cats during repeated hemorrhage. Cats were surgically prepared under chloralose-urethane anesthesia with indwelling cannulas as described previously [19]. A carbon paste electrode (n = 26) or push-pull cannula (n = 25) was stereotaxically positioned in the LC [19, 20]. After a 2-h stabilization period, all cats sustained two hemorrhages (20% blood volume over 3 min). The initial hemorrhage (InHem) commenced at 0 min and the repeated hemorrhage (RpHem) occurred at 90 min. Blood was withdrawn via an arterial cannula into a heparinized syringe and retransfused through a femoral vein cannula at 10 min or, in a subset of voltammetric experiments (n = 14), at 30 min. During the stimulus, oxidative current was recorded using computer-controlled normal pulse voltammetry at 1-min intervals [19]. The amplified current during the +230 mV pulse, which was due to oxidation of catecholamines at the electrode surface [2], was used as index of catecholaminergic activity (catechol current) [2, 14, 19]. In push-pull perfusion experiments, the cannula was perfused with a balanced salt solution (21/A/min). Sequential perfusate samples were obtained from LC sites at 5-min intervals throughout the experiment and perfusate monoamine levels were subsequently determined

86 by HPLC-EC [19]. During the stimulus, arterial pressure (MAP) and heart rate (HR) were monitored and blood samples were removed for radioimmunoassay of A C T H and AVP [18, 19]. At the termination of the experiment, electrode sites were marked by passing direct current and the cannula sites were marked by Fast green dye injection. Sites were identified histologically and mapped onto brain stem sagittal planes [3, 19, 20]. All cats maintained a stable cardiovascular profile during the course of the investigation. MAP (mmHg) decreased from 132 + 5 to 85 _+ 8 during InHem (n -- 41; prestimulus to the end of blood loss) and from 126 _ 5 to 77 _+ 9 during RpHem. Comparable increases in H R occurred during InHem (211 + 4 to 231 + 4 beats/min) and RpHem (216 + 5 to 234 + 7 beats/min). Significant changes in cardiovascular parameters were only observed at the end of blood loss (ANOVA, P < 0.01 vs. prestimulus). Prestimulus blood gases and pH were similar during both the hemorrhage protocols (data not shown). The InHem resulted in significant elevations in the plasma A C T H and AVP (Fig. 1). In contrast to the constancy of cardiovascular responses, both plasma A C T H and AVP responses were potentiated during RpHem as compared to InHem. The distribution of the 31 recording and 17 perfusion sites is shown in Fig. 2A and Fig. 2B, respectively. Eighteen of the 31 recording sites were located within the boundaries of the LC and the remaining 13 were located outside this region (Fig. 2A). We categorized these sampiing sites based upon the response to InHem. Criteria for inclusion of voltammetric changes required an average change in oxidative current greater than 10 pA during which the first two samples after the onset of hemorrhage were unidirectional [19]. For perfusate monoamines, changes greater than the standard deviation of a comparable concentration of standard were considered as responses [20]. The area encompassing seven contiguous sites in the vLC showing increases in catechol current (Fig. 2A, sites marked by stars) was defined as the active area. A similar active area was evident in our perfusion studies. Differences in the recording and perfusion active areas are attributable to the larger size and sampling volume of the perfusion cannula. Catechol current increased significantly (Table 1, P < 0.01) after InHem at sites in the active area, whereas the increase was not significant after RpHem, The principal metabolite of norepinephrine, 4-hydroxy-3-methoxyphenyl(ethylene)glycol (MHPG), increased during both hemorrhages in the active area. Prestimulus LC-NA activity during RpHem, measured by either voltammetry or perfusion, remained elevated above baseline after InHem. Thus because the catecholaminergic signal remained elevated prior to RpHem, no significant increase

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Fig. 1. Effectof repeated hemorrhageon changes in plasma (A) ACTE and (B) AVP in cats. Values are means + S.E., n = 8. ~P < 0.05 ant bp < 0.01, initial vs. repeated. Hemorrhages(H) separated by 90 min R, retransfusion of the shed blood.

in LC-NA activity in response to R p H e m was observec although response to R p H e m was not significantly different from that observed in response to InHem. At sites surrounding the active area (Fig. 2A,B; fillec circles), catechol current as well as perfusate M H P G levels decreased in response to InHem. No consistent pattern was observed in response to RpHem. The change in oxidative current in this surround was -39 + 12 pA (InHem) versus -1.5 + 12.7 pA (RpHem). Consistenl with the change in oxidative current, perfusate M H P G (pmol/5 min) during InHem decreased from 0.98 + 0.40 (prestimulus) to 0.47 + 0.17 after blood loss; however, during R p H e m it changed from 0.40 + 0.18 to 0.56 + 0.16. Interestingly, these sites exhibited persistent decreases in NA activity after the initial blood loss. At other distant sites, catechol current (Fig. 2A,B; open circles) did not change during either InHem or RpHem. In the current study, we have: (a) observed a longlasting increase in vLC-NA tone following hemorrhage, (b) confirmed the potentiation of plasma A C T H in response to RpHem, (c) extended the finding of elevated

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Fig. 2. Distribution of (A) voltammetric recording and (B) push-pull perfusion sampling sites projected onto sagittal sections of the pons. Sites in the 'active area' are indicated by star while sites in the 'surround' are indicated by filled circles. Those sites 'outside' the principal LC are indicated by open circles. BC, brachium conjunctivum; FTG, gigantocellular tegmental field; LC, locus coeruleus; TRC, tegmental reticular nucleus; V4, fourth ventricle. Area with densest aggregation of neurons is circumscribed.

stress responsive hormone secretion after RpHem to inelude AVP, and (d) demonstrated a high degree of concordance between the NA response patterns as measured by either voltammetric or push-pull perfusion sampling methods. These observations support the hypothesis that NA-activity is specifically increased in the hemodynamically sensitive vLC region after hemorrhage. The peak vLC-NA response was similar during both InHem and

RpHem; however, the magnitude of the prestimulus-topeak NA increase after RpHem was attenuated due to elevated basal levels after InHem. In several individual cats, a significant increase in peak oxidative current during RpHem as compared to InHem was noted in the active area; thus we do not believe that a 'ceiling' effect could account for our observations. Overall, these results imply that a persistent alteration in LC-NA activity occurred in response to the InHem resulting in a long-lasting increase in regional LC-NA tone. Whether these effects may be considered as adaptive characteristics of the LC to blood loss is not known. In conclusion, we suggest that the LC does not directly activate the HPA axis after hemorrhage; rather, it exerts a modulatory role. Increased LC-NA activity during stress is thought to focus ongoing cerebral activity on the immediate challenge, thus promoting efficient sensory processing and/or the organization of appropriate behavioral responses [6, 12, 16]. Thus, the increased vLCNA tone observed after the InHem may serve to channel central processes toward counter-regulatory mechanisms and to reduce intrinsic vLC activity. This is probably mediated via alpha-2 adrenergic receptor activation [17]. The increased vLC-NA tone may then enhance the efficacy of hemodynamic signal processing/transfer in response to RpHem or may 'enable' [16] the actions of other transmitters mediating hemodynamic stimuli. This increased efficacy or enabling of hemodynamic signals could account for the observed potentiation of the pituitary hormone secretion in response to blood loss. Supported by National Institutes of Health Grant DK-26831. TABLE I EFFECT OF REPEATED HEMORRHAGE ON NORADRENERGIC ACTIVITY IN THE ACTIVE AREA OF THE LOCUS COERULEUS IN THE CAT Measurement

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42** 34 0.34** 0.44

Catechol current, oxidative current during the +230 mV pulse; MHPG, 4-hydroxy-3-methoxy-phenyl(ethylene)glycol. *P < 0.01 vs. control; **P < 0.01 vs. initial by Wilcoxon signed-rank test. Catechol current (pA) before hemorrhage (control) and at the end of + 3 rain (blood loss). MHPG (pmol/5 min) was analyzed by HPLC-EC from two sequential 5-rain samples collected before hemorrhage (control) and 5 min through the hemorrhage (blood loss). Values are means _+S.E.; number of sites in parenthesis.

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