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BrainResearchBUN&Z,Vol. 28, PP. 89-98. 0 PergamonPress pk. 1991.Printedin the U.S.A.

Deficits in NaCl Ingestion After Damage to the Central Nucleus of the Amygdala in the Rat OLIVIER

University

GALAVERNA,’ LAURIVAL A. DE LUCA, JR., JAY SCHULKIN, SHU-ZHEN YAO AND ALAN N. EPSTEIN

ofPennsylvania,

Departments

of Biology and Anatomy, Philadelphia,

Received 4 February

PA 19104-6018

199 1

GALAVERNA, O., L. A. DE LUCA, JR., J. SCHULKIN, S.-Z. YAO AND A. N. EPSTEIN. Deficits in NaCl ingestion after damas?eto the centralouches of fhe umvmfuEu in fhe rut. BRAIN RES BULL 28(l) 89-98. 1992. -These studies examined the NaCl-nnake behaviors of rats with bilateml electrolytic lesions of the central nuclkns of the amygdaia (CeAX). Daily need-free intake of 3% NaCl was abolished by CeAX even in rats in which it had been enhanced preoperatively by a history of repeated sodium depletions but was slightly restored by three successive postoperative sodium depletions. CeAX rats drank water but not 3% NaCl to high doses of DOCA and to the activation of cerebral angiotensin II, and expressed small but reliable salt intake (need-induced salt intake or salt appetite) after postoperativesodium depletions. Other ingestive behaviors (water drinking, intake of food and 5% sucrose) were normal. When given decreasing concentrations of NaCl solution the CeAX rats rejected them until the concentration reached 0.2%. These findings suggest that lesions to the central nucleus of the amygdala produce a global impairment in salt intake behaviors that is possibly due to an alteration in the central processing of the salt taste signal. Amygdala

Brain angiotensin system

Mineralocorticoid

Salt intake

the substrates for these effects or about their mechanisms. The central nucleus of the amygdala (CeA) is an interesting structure for further studies of the neural mechanism of salt intake. It is irn~~t for cardiovascular regulation (20, 24, 32, 35), is unusally rich in angiotensin nerve terminals (21), and contains aldosterone receptors (30,37). Lastly, it is the “head ganglion” in the limbic area of the salt taste afferent system (3,23). Accordingly, we have ablated it electrolytically and, in the following, describe a new syndrome of deficits in the behaviors of salt intake.

SALT intake, the ingestion of sodium-containing commodities, is an innate behavior (11,33) that is essential for sodium homeostasis. Laboratory studies of the rat have identified at least two kinds of salt intake. Animals in sodium balance that are consuming the standard commercial diet which contains more sodium than they need (0.5 to 1.0% NaCI) will drink NaCl solutions across a broad range of concentrations. This is needfree salt intake which can be expressed throughout the postweaning life of the rat and is usually studied as a preference for dilute NaCl solutions (36). Except for its dependence on taste (5), its mechanism remains unknown. The other form of sodium intake is need-induced salt intake or sodium appetite (25). It is a transient intake activated by the synergistic action of aldosterone and angiotensin II (14,31) and can be mimicked by high doses of either aldosterone or angiotensin II acting alone (1, 6, 39, 40). The two hormones have their synergistic effect within the brain because in~acereb~ven~cul~, but not peripheral, blockade of their actions with specific ph~acologic~ ~t~onists abolishes the salt intake that is aroused by sodium depletion (31). The neural circuit underlying the need-induced behavior includes the sensory channel for sodium taste which ascends to the forebrain through the nucleus of the solitary tract and parabrachial nucleus (15,41), and it depends, in ways that are not yet fully understood, on structures in the anteroventral wall of the third ventricle (4, 8, 10, 13) and on the medial nucleus of the amygdala (22, 33, 34). Lesions in the amygdaloid complex both increase and decrease daily need-free NaCl intake (7, 18, 26, 27), but there is no consensus about the subnuclei within the amygdala that are

METHOD Animals and Housing

Adult male Sprague-Dawley rats, weighing 350-400 g at the beginning of the experiments, were used. They were housed in in~vidu~ wire-mesh cages in a temperature- and hu~dity-controlled room on a 14:lO light/dark cycle, fed Purina rat chow pellets (sodium content approximately 0.5%) and given tap water and NaCl solutions in graduated drinking tubes attached to the front of each cage. Surgery and Recovery They received stereotaxically guided electrolytic lesions of the CeA under intramuscular ketamine hydrochloride (40 m&g) and acepromazine maleate (15 mg/kg). A prophylactic dose of gentamicin sulfate was administered intramuscularly immediately

‘Requestsfor reprints shouId be addressed to Olivier Galavema, Leidy Laboratories, Department of Biology, University of PennsyIvama, philadel_ phia, PA 19104-6018.

89

GALAVERNA

prior to surgery as well as for three postsurgical days. An insulated tungsten electrode with 0.5 mm exposed at the tip was used to make anodal lesions by delivering a current of 1 mA for twenty seconds with 2 insertions per side of the brain. The coordinates of the bilateral lesions are 2.2 mm and 2.7 mm caudal to bregma, 4.1 mm lateral from the midsagittal sinus, and 7.4 mm in depth from the dural surface. The sham lesions vary only in that a current was not passed through the electrode. The animals used in Experiment 2 were also fitted with a stainless-steel guide cannula (Plastic Products, Roanoke, VA) that opened into the anterior cerebral ventricles. It was permanently attached to the skull by stainless-steel screws and dental acrylic. The cannulation was validated before the experiments by a pulse intracerebroventricular injection (pICV) of angiotensin II (6 ng), after which the animals had to drink at least 5 ml of water in 15 minutes. All of the animals recovered for 2 weeks prior to testing. Some animals with amygdala damage show a mild aphagia and adipsia for the first 3 to 5 days following surgery. During this time their body weights decrease approximately 15 g. As a result, these rats were fed wet mash for several days until normal feeding and drinking resumed. Hormones

and Drugs

EXPERIMENTAL PROTOCOLS

Experiment

1: Daily Need-Free

3% NaCl Intake

Fourteen rats that were expressing high daily need-free intake of 3% NaCl as a result of three sodium depletions prior to surgery were used (see Experiment 4 below for depletion method). Ten received CeA lesions and four were sham operated (sham). Water and 3% NaCl intake were recorded for a period of nine consecutive days prior to surgery and nine consecutive days postoperatively. Experiment

2: Sodium Intake Induced

by Mineralocorticoid

DOCA, a mimic of aldosterone known to induce large NaCl intake (39), was used. Twenty-two CeAX and twelve sham-lesioned rats received daily subcutaneous injections of 5 mg DOCA for three consecutive days. Water and 3% NaCl intake were recorded daily for a period of nine days from three days before the first injection of DOCA to four days after the last DOCA injection. Experiment 3: Sodium Intake Induced Central Angiotensin

Used

ET Al,.

by the Activation of

Angiotensin II, human sequence (Sigma Chemical Co., St. Louis, MO), was dissolved in 0.9% saline. Deoxycorticosterone acetate (Sigma Chemical Co., St. Louis, MO) was dissolved in 100% propylene glycol to a concentration of 5 mg/ml. Renin, purified from hog kidneys by high affinity chromatography as described in Ganten et al. (17), was generously supplied by Professor Detlev Ganten of the German Institute for High Pressure Research. It was dissolved in bovine serum albumin (a 2% solution dissolved in saline) to a concentration of 100 ng/pl. Furosemide was Lasix (American Hoechst Corporation, Sommerville. NJ).

We chose to use pICV renin (100 ng) which reliably induces large NaCl intake dependent on local generation of angiotensin II (2). Renin acts on its substrate angiotensinogen to produce angiotensin I which is then converted into angiotensin II in the brain. Eleven CeAX and thirteen sham-lesioned rats were used in this experiment. All the rats received pICV renin 100 ng (1 ~1); eight sham and five CeAX received 1 pl of vehicle pICV one day before renin. The injections were made around 10:00 a.m. The intake of 3% NaCl and water were recorded every hour for 5 h and at 24 h after each injection.

Blood Chemistry

Experiment

and Hematocrit

Plasma aldosterone concentrations were measured using the Diagnostic Products kit that uses whole plasma (no extraction, no chromatography) and is assayed with lz51 aldosterone and the commercial antibody. The percent cross-reactivity of the aldosterone antibody was as follows: aldosterone, 100%; corticosterone, 0.002%; deoxycorticosterone, 0.006%; estradiol and testosterone were below the detection limit of the assay. Brain Histology

At the conclusion of each experiment the animals (except those that were decapitated for collection of trunk blood) were euthanized and fixed with 10% formalin through a cardiac catheter. The brain was carefully removed and immersed for one week in a 10% formalin solution, after which it was transferred to a 30% w/v sucrose-formalin solution. In the case of the decapitated animals, the brains were removed and allowed to stand for a longer period in the 10% formalin. The fixed brains were then sectioned into slices of 16 micron with a cryostat, and stained with thionin blue to evaluate the anatomical extent of the lesions. Statistical Analysis

The results are presented ANOVA tests were performed level was set at pcO.05.

as means & SEM. Appropriate on all the data and significance

4: Sodium Intake Induced

by Sodium Depletion

The natruiretic agent, furosemide (Lasix), combined with removal of ambient sodium, produces an endogenous increase in plasma aldosterone and angiotensin II and a reliable NaCl appetite (28,29). Seven CeAX and six sham-lesioned rats were housed in metabolic cages. After a period of 1 week to record basal intake the animals were challenged with 4 sodium depletions separated by 1 week during which daily water and 3% NaCl intakes were recorded. Each depletion consisted of 2 SC injections of 5 mg of furosemide separated by 2 hours. Prior to the time of the first injection the cages were washed to remove ambient sodium, and the animals were provided with water and sodium-deficient powdered food (Teklad #81263) for the next 24 hours. The animals were introduced to this food, given together with the normal chow, one day prior to the depletion in order to avoid neophobia. Urine samples were collected 2 hours after the first injection (i.e., just prior to the 2nd injection), 2 hours after the 2nd injection, and at the end of 24 h. After 24 h, for the first 3 sodium depletions the animals were given access to the 3% NaCl solution in 0.1 ml-graduated burettes. Their water and salt intakes were recorded at 15 min, 30 min, 1 h, 2 h and 24 h, and urine was collected at 2 and 24 h. One week before, and between each sodium depletion, daily water and need-free salt intakes were recorded. At the end of the last SOdium depletion and before any access to the NaCl solution, the animals were decapitated. Trunk blood was collected to determine aldosterone concentration by radioimmunoassay.

AMYGDALA

AND SALT INTAKE

GALAVERNA

ET AL.

FIG. 2. Control lesions: (A) Unilateral lesions outside the CeA; (B) Unilateral lesions that involved part of the CeA; (C) Bilateral lesions outside the CeA.

Experiment 5: Taste ~espo~i~~en~ss to Other NaCl Solutions and to 5% Sucrose A) Other NaCl solutions. Because the CeAX rats appeared to consistently avoid the 3% NaCl solution, they were offered NaCl solutions of decreasing concentrations. Eight CeAX and four sham were given, in addition to water, five different concentrations of NaCl solutions for two weeks each. All the animals had the same solution at the same time in descending order of concentrations. The concen~ations were: 3%. 1.5%. 0.75% and

0.2%. The positions of the bottles were switched daily in order to account for side preferences among the animals. Intakes of water and NaCl solution were recorded daily. In addition, four CeAX and two sham-lesioned rats were given the 0.75% solution as the unique available fluid for ten days and the intake recorded daily. B) 5% Sucrose solution. Six CeAX and six sham-lesioned rats, four CeAX and two sham-lesioned rats from Experiment .5A were offered a choice of water and a 5% sucrose solution for five days. Every day the water and sucrose intakes were recorded and the position of the bottles was switched.

TABLE 1 TABLE 2

MEAN AMOUNT OF SODIUM LOSS IN mEq DURING EACH 24 H OF SODIUM DEPLETION (DEP.)

Ce AX

1st dep. 2nd dep. 3rd dep. One-way ANOVA

2.52 ir 0.25 2.71 ” 0.10 2.61 2 0.14 0.7, n.s.

Sham 2.12 2 0.27 2.42 + 0.23 2.80 2 0.50 0.4. n.s.

MEAN DAILY INTAKE OF WATER AND 5% SUCROSE (AVERAGED OVER 5 CONSECUTIVE DAYS)

t-Test 0.3. n.s. 0.2, n.s. 0.8, n.s.

CeAX Sham r-Test

Water

Sucrose 5%

6.9 -t 2.9 12.4 t 2.5 0. I. n.s.

94.7 f 3.8 100.5 i_ 2.0 0. I, n.s.

_---_

AMYGDALA AND SALT INTAKE

f

post-op

100 90 c

pm-op

post-op

FIG. 3. Average daily intake of need-free 3% NaCl (top) and of water {bottom), using 9 consecutive days prior to surgery (prz-op) and 9 consecutive days postoperatively (post-op), at least 3 days after the validation of the lesions. Values are mean + SEM of $0 rats which received CeA lesions and 4 rats that received sham lesions. The large salt intake of these 14 animals before surgery is the result of a history of several sodium depletions. Paired f-test within group, pre-op vs. post-op, *p

Deficits in NaCl ingestion after damage to the central nucleus of the amygdala in the rat.

These studies examined the NaCl intake behaviors of rats with bilateral electrolytic lesions of the central nucleus of the amygdala (CeAX). Daily need...
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