Neuroscience Letters, 116 (1990) 210-215 Elsevier Scientific Publishers Ireland Ltd.

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NSL 07065

The central amygdala is involved in the conditioned but not in the meal-induced cephalic insulin response in the rat B. Roozendaal, W.P. Oldenburger, J.H. Strubbe, J.M. Koolhaas and B. Bohus Department of Animal Physiology, University of Groningen, Centrefor Biological Sciences, Haren (The Netherlands) (Received 18 January 1990; Revised version received 24 April 1990; Accepted 24 April 1990)

Key words: Central amygdaloid nucleus; Electrolytic lesion; Conditioned cephalic insulin response; Meal-induced cephalic insulin response; Glucose. The central nucleus of the amygdala (CEA) is considered to be involved in the regulation of autonomic correlates of fear. Its involvement in the control of autonomic functions other than elicited by fear has received little attention. The effects of a bilateral electrolytical lesion of the CEA on feeding related insulin responses have been analyzed in male Wistar rats. The cephalic phase of the insulin response is a vagally mediated elevation of plasma insulin concentration during the first minute after meal onset, before any increase in plasma glucose can be noticed. This response can also be entrained to environmental stimuli. The insulin response elicited under these conditions is due to conditioning. CEA lesioning abolished the conditioned insulin response but not the early insulin elevation during the presentation of food. The CEA lesion failed to affect plasma glucose levels in both the meal-induced and conditioned test situations, To our knowledge this is the first study that shows that the CEA is also involved in the organization of conditioned metabolic endocrine responses.

Electrical and chemical stimulation of the central nucleus of the amygdala (CEA) in awake, freely moving animals produces gastric ulcer formation and stress-like changes in arterial blood pressure, heart rate, respiratory rate, and plasma catecholamines [1-3]. Conversely, lesioning of the CEA selectively disrupted the expression of autonomic responses in conditioned stress situations, e.g. the bradycardiac response elicited as a consequence of a previous stress experience [8]. Furthermore, CEA lesions attenuated the formation of stress-induced gastric ulcers [2]. These studies

Correspondence." B. Roozendaal, Dept. of Animal Physiology, University of Groningen, Centre for Biological Sciences, P.O. Box 14, 9750 AA Haren, The Netherlands. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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demonstrate the involvement of the CEA in the modulation of the conditioned autonomic stress response. Several lines of investigation suggest that also autonomic responses other than those elicited as a consequence of previous stressful events can be modified through learning or experience. Pavlov has originally shown that dogs secrete saliva upon presentation of stimuli predicting food, while others have reported that e.g. insulin secretion [11] takes place at the same time. Such an insulin response is also elicited during the first minute after the start of a meal, before any increase in plasma glucose can be noticed. This cephalic phase insulin response is a vagally mediated autonomic endocrine reflex that is activated by the presence of food in the oropharyngeal region, and not by the absorptive consequences of incoming nutrients [10]. Under stringent feeding regimens the insulin response can also be entrained to arbitrary environmental stimuli that reliably predict the presentation of food [7]. This indicates that the insulin response elicited under these conditions is due to conditioning. The present study was designed to investigate whether the CEA plays a role in the organization of this conditioned response as well. Thirty-four young adult (8-10 weeks old) male Wistar rats, weighing 260-320g, were used. During the experiment they were housed individually in perspex cages (25 x 25 x 30 cm) with a sawdust covered solid floor at a standard room temperature of 20 +__2°C in a light controlled room (light on from 06.00 to 18.00h). Water was available ad libitum. Standard food was available in metal food hoppers. Access to food could be restricted by a horizontally sliding door in front of the hopper. Dooropening and closing was activated by a programmed clock. Starting after catheterization, food was available twice a day for a period of 2 h (from 10.30 to 12.30h and from 22.30 to 00.30 h). After five days the food availability was reduced to 75 min (from 10.45 to 12.00h and from 22.45 to 00.00 h). Surgery was performed under ether anesthesia. All rats were provided with a silicon heart catheter through the jugular vein [9]. This method allows blood sampling in unanesthetized and undisturbed, freely moving rats. One week was allowed after surgery for recovery and habituation to the sampling procedure. One week after surgery, basal plasma levels of immunoreactive insulin (IRI) and glucose were determined. Blood samples of 0.25 ml were withdrawn unless otherwise stated. Baseline samples were taken at - 5 (0.5 ml), - 1.5 and 0 min before the opening of the sliding door. The time when the rat started to eat was considered as t = 0 min. Additional blood samples were withdrawn 1, 2, 3, 4 (0.5 ml) and 5 min after the start of the meal (meal-induced test situation). The loss of blood was compensated by transfusions of heparizined blood, both during the baseline samples (at - 1 0 (0.5 ml), - 5 (0.5 ml) and - 1.5 min (0.25 ml)) and after termination of the test (1.5 ml). Donor blood was obtained from undisturbed rats with permanent heart catheters. The samples were transferred immediately to chilled (0°C) centrifuge tubes containing 5/~1 of a heparin containing solution (500 U/ml) as anticoagulant and centrifugated. Plasma was stored at - 2 0 ° C . Two days later, the experiment was repeated, but no food was offered after opening of the doors (conditioned test situation). The sound of the opening of the door served as the conditioning stimulus. In this situation the

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Fig. 1. Coronal section of the rat brain at 6.7 rnm rostral to interaural showing a representative lesion (black) and the total area covered by the lesions (dotted) (n = 10).

time point the rat moved his head into the food hopper was considered t = 0 min. The blood samples were taken with the same schedule as described for the mealinduced test situation. Following these tests lesioning of the CEA was performed. The animals were anesthetized with ether and placed in a David-Kopf stereotaxic apparatus. Bilateral electrolytical lesions aimed at the CEA were made (coordinates: 6.7 mm rostral to interaural, 4.1 mm lateral to the midline and 7.0 mm below dura, with the incisor bar 3.3 mm below the interaural line) [6]. The lesions were made with an anodal current of 1.25 mA during 5 s. In the sham-operated controls the electrode was lowered 5.0 ram, but no current was passed. On the 6th post-operative day the meal-induced and two days later the conditioned tests were repeated, using the same blood sampling schedules as before the lesion. Plasma glucose was determined in aliquots of 10/~1 by the ferricyanide method of Hoffman (Technicon autoanalyzer TMII). Rat plasma insulin (IRI) was estimated by means of a sensitive and specific radioimmunoassay kit (Novo, Denmark). Guinea pig serum M8309 served as antiserum. Duplicate assays were performed on 25/tl samples. The bound and free 125I-labeled insulin was separated by means of a polyethylene glycol solution (23.75% wt/wt). After the experiments, the CEA-lesioned animals were deeply anesthetized with sodium-pentobarbital (90 mg/kg, i.p.) and perfused intracardially with saline, followed by a 4% formaldehyde solution. The brains were fixed in 4% formaldehyde for at least 24 h. Frozen sections of 40/tm were cut and the lesion place was examined on unstained sections. The data were evaluated for statistical significance using a two-way ANOVA with repeated measures. Post-hoc analysis was performed with the Dunnett's test. Differences between absolute insulin and glucose levels of two groups were calculated with the Mann-Whitney U-test (two-tailed, corrected for ties) to test differences between

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animals. Differences within animals were calculated with the Wilcoxon matchedpairs test (two-tailed). A probability level of P < 0.05 was taken as statistical significance for all tests. The data from 19 animals were analyzed, 10 of which were CEA-lesioned and 9 sham-lesioned. A total of 15 animals was left out from the analysis because of an incomplete set of data due to clogging of the catheter, infection, or improper CEA lesioning. In the remaining lesioned animals the CEA damage as determined at 6.7 mm rostral to interaural was restricted to the CEA and reached a minimum of 50% (Fig. 1). When food was presented to the unoperated animals, blood glucose rose progressively with time (F4,68= 48.02; P < 0.001), the first noticeable rise in blood glucose occurred between 2 and 3 min after the start of a meal (Fig. 2). Plasma insulin also rose progressively with time (F4,72=4.93; P < 0 . 0 5 ) , but an elevation in insulin could be determined already in the first minute (Fig. 2). When no food was offered

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Fig. 2. Changes in plasma concentration of lmmunoreactive insulin (mU/1) and glucose (mg/dl) during 5 min after meal onset (meal-induced test situation) or after opening of an empty food hopper (conditioned test situation) in control ( • - - • ; n = 19), followed by central amygdaloid ( • - - - 0 ; n = 10) or sham lesions ( 0 - - 0 ; n=9).

214 TABLE I BASAL INSULIN AND GLUCOSE LEVELS Data are mean _+ S.E.M. Conditioned

Pre-operated (n= 19) CEA-lesioned (n = 10) Sham-lesioned (n=9)

Meal-induced

Insulin (mUd)

Glucose (mg/dl)

Insulin (mU/l)

Glucose (mg/dl)

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102± 1 103_+2 102_+3

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The central amygdala is involved in the conditioned but not in the meal-induced cephalic insulin response in the rat.

The central nucleus of the amygdala (CEA) is considered to be involved in the regulation of autonomic correlates of fear. Its involvement in the contr...
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