Intracranial action of corticosterone facilitates the development of behavioral inhibition in the adrenalectomized preweanling rat

Intracranial action of corticosterone facilitates the development of behavioral inhibition in the adrenalectomized preweanling rat

ELS EV IER Neuroscience Letters 176 (1994) 272-276 LET , , Intracranial action of corticosterone facilitates the development of behavioral inhibiti...

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ELS EV IER

Neuroscience Letters 176 (1994) 272-276

LET , ,

Intracranial action of corticosterone facilitates the development of behavioral inhibition in the adrenalectomized preweanling rat Lorey K. Takahashi*, Hyon Kim Department of Psychiatry, University of Wisconsin Medical School and Middleton Memorial Veterans Hospital, Madison, IV/, USA Received 1 February 1994; Revised version received 16 May 1994; Accepted 21 June 1994

Abstract

We tested the hypothesis that in preweanling rats central administration of exogenous corticosterone (CORT) is sufficient to facilitate the development of behavioral inhibition. 28-gauge cannulae containing varying concentrations of CORT (0, 25, 50 and 100%) were implanted unilaterally into the lateral ventricles of 9-day-old rat pups. After a 24-h postoperative recovery period, pups were adrenalectomized. At 14 days of age, pups were tested for behavioral inhibition which consisted of removing the pup from the nest and exposing it to an unfamiliar adult male rat. Pups implanted with cannulae containing 0, 25 and 50% concentrations of CORT spent significantly less time in freezing postures than pups implanted with cannulae containing 100% CORT. These freezing pups also tended to emit fewer ultrasonic vocalizations than pups in the other three implant conditions, albeit the level obtained was not statistically significant. RIAs indicated that, in general, hormone-filled cannulae produced no detectable concentrations of plasma CORT on the day of the test or on days preceding testing. Results suggest that in the early postnatal period endogenous CORT acts centrally to facilitate the development of neural pathways involved in the ontogenetic expression of behavioral inhibition.

Key words. Preweanling rat; Behavioral inhibition; Freezing; Ultrasonic vocalization; Development; Conspecific threat; Adrenal steroid; Corticosterone

The ability to terminate on-going behavioral activities and assume defensive patterns of behavior when threatened is a characteristic feature of many vertebrate species. Frequently, the response to threat entails a cessation of vocalizations accompanied by defensive immobility or freezing. By engaging in behavioral inhibitory responses, organisms reduce the likelihood of detection and attack from a threatening stimulus [9,23,24]. This laboratory has examined the ontogeny of behavioral inhibition in the rat. We reported that, when rat pups are exposed to a potentially infanticidal adult male rat [25,40], they will freeze and reduce their emission of ultrasounds [35 37]. This ability of preweanling rats to exhibit behavioral inhibition develops near the end of

*Corresponding author. Address: Department of Psychiatry, University of Wisconsin Medical School, 600 Highland Avenue Madison, WI 53792-2475, USA. Fax: (1) (608) 263-0265. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00486-T

2nd postnatal week. Endocrine studies demonstrate that endogenous adrenal steroids facilitate the development of behavioral inhibition [38,41]. That is, adrenalectomy (ADX) performed on 10-day-old pups impairs the development of behavioral inhibition when pups are tested subsequently on day 14. The behavioral deficit in freezing, however, is restored when ADX pups are administered daily systemic injections of exogenous corticosterone (CORT), the major glucocorticoid of the rat. In addition to their well-know involvement in the stress response [3,22,32], glucocorticoids are implicated in the development of the brain [12,19,34]. Glucocorticoids also have numerous developmental effects occurring in the periphery [15,21] that may affect neural development [4]. Isolating the mechanisms involved in the adrenal steroid-induced development of behavioral inhibition requires information concerning the relative contribution of CORT to peripheral and the CNS development. Hence, the previous use of systemic injections o f e x o g e n o u s CORT, which crosses the blood-brain

L.K. Takahashi, H. KimINeuroscience Letters 176 (1994) 272-276

barrier, compromises our ability to discern the potential sites of adrenal steroid action underlying the development of behavioral inhibition. The purpose of the current experiments was to determine whether the administration of exogenous CORT directly into the rat pup's brain is sufficient to facilitate the development of behavioral inhibition, Pups were offspring of Sprague-Dawley female rats (90-120 days old) derived from a stock obtained originally from Sasco (Madison, WI). Rats were maintained on a 12-h light-dark cycle with lights on at 06:00. Pregnant rats were placed in plastic breeding cages (31 cm x 22 cm x 18 cm) with wire-mesh tops. Each cage was provisioned with rat chow, a water bottle and a layer of wood shavings. Breeding cages were checked daily for the presence of pups (day of birth = postnatal day 0). Litters were left undisturbed except for routine cage cleaning. Sexually experienced adult male rats were used as stimulus animals. Adult males were housed singly in hanging cages and maintained on identical light-mlark cycles, Cannulae were cut from 28-gauge (i.d. 0.19 ram) stainless-steel tubing. The tip of each tube was cut square, polished and filled with a mixture of molten CORT and cholesterol. Four concentrations (w/w) of a CORT/ cholesterol mixture were prepared (0, 25, 50 and 100% CORT). After solidifying, the outer tip of the cannula was cleaned with ethanol to ensure that hormone was available only at the surface of the lumen, On postnatal day 9, four male pups were taken, whenever possible, from each litter. Each pup was assigned randomly to one of the four CORT implantation groups (n = 10-11/dose). Pups were anesthetized with methoxyflurane (Pitman-Moore, Mundelein, IL) and placed in a stereotaxic apparatus adapted for neonatal rats (David K o p f Instruments, Tujunga, CA). A hormone-filled cannula was implanted unilaterally into the lateral ventricle using the following flat-skull coordinates: AP = -0.5 mm from bregma, ML - 1.0 mm and DV = -4.0 mm from the skull surface. The cannula was secured to the skull with Caulk-Grip cement. Results of pilot studies revealed successful i.c.v, diffusion of dye using these coordinates. At the conclusion of the operation, pups were returned to the nest box. On day 10, all operated pups were anesthetized with methoxyflurane and bilateral dorsal incisions were made to extract the adrenal glands. After adrenalectomy, pups were returned to the nest box. At 14 days of age, CORT and cholesterol implanted pups were tested for behavioral inhibition. Tests were conducted in a Plexiglas enclosure (26.5 cm x 26.5 cm x 20.0 cm) housed in temperature-controlled incubatot with a glass door. The Plexiglas enclosure was subdivided by a wire-mesh partition positioned with the two ends attached to the mid-portion of two adjacent walls, thereby forming a triangular compartment. The top was

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open except for the area above the triangular compartment which housed the rat pup. The enclosure was placed on a cardboard floor that was changed after every test. The ultrasound detector was positioned directly above the Plexiglas enclosure. Ambient temperature in the incubator varied from 34 to 35°C which corresponds to the thermoneutral range for 14-day-old rats [7]. Immediately before testing, an adult male rat was anesthetized with sodium pentobarbital (50 mg/kg i.p.) and placed into the large compartment of the test apparatus. The adult rat was anesthetized to eliminate any possibility that behavioral differences among treatment groups would arise from differences in adult behavior toward pups. The 14-day-old pup was then placed in the adjacent triangular compartment. Behavioral tests were 10 rain in duration and conducted in the first half of the light cycle. During testing, the number of ultrasonic vocalizations and duration of freezing were recorded [36]. Ultrasounds were detected with headphones attached to the socket of a Mini-2 bat detector (Bat Conservation International, Austin, TX) tuned to 40 kHz. The bat detector transforms the ultrasound into the audible range of humans. Each discrete vocalization was recorded using a counter, except those produced infrequently during face washing. The duration (in s) of freezing was recorded using a stopwatch. Freezing was recorded whenever the pup assumed an immobile posture with the head in a stationary position elevated above the floor. After behavioral testing, the pup's rectal temperature was measured using a microprobe (IT-21; Physitemp, Clifton, N J) attached to a BAT-12 digital thermometer (Physitemp), with a resolution of 0.1°C. The probe was inserted into the rectum to a depth of 10 mm and held in position until the temperature stabilized which occurred within a 10-s period. Rectal temperature was recorded because ultrasonic vocalizations are reported to vary with body temperature [1,6]. After rectal temperatures were obtained, pups were decapitated and trunk blood collected in ice-chilled microcentrifuged tubes containing EDTA. Tubes were centrifuged in an Eppendorf microcentrifuged (Brinkman Instruments, Westbury, NY) for a duration of 3 rain. Plasma was aliquoted and stored at -70°C until the time of assay for CORT. The cannula was removed t¥om the brain and the lumen examined using a microscope for the presence of hormone. Duplicate plasma samples were analysed for CORT using an [12sI] CORT kit (Diagnostic Products, Los Angeles, CA). All samples within an experiment were measuredin one assay. The antiserum exhibits a cross-reactivity to 11-deoxycorticosterone of < 2.9%. Cross-reactivity to other adrenal steroids was <0.9%. The detection limit of the assay was 1.5 ng/ml. The intraassay and interassay coefficients of variation were 4.0 and 4.8%, respectively. One-way ANOVA procedures was used to assess the

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differ significantly from levels produced by cholesterolfilled cannulae (0% group). Rectal temperatures did nol differ significantly across groups, k~-. = 0.45, P > 0.05 (mean group range = 35.9-- 36.2°C). With the exception of only three pups, concentrations of plasma CORT in all groups were below the level of detection of the assay (< 1.5 ng/ml). Two pups in the 25% CORT group had plasma concentrations of 9.3 and 12.3 ng/ml and a pup in the 50% CORT group had a plasma value of 16.5 ng/ml. Inspection of cannula tips indicated that although hormone was occasionally recessed in the lumen, all cannula contained hormone. It should be noted that the plasma concentration of CORT detected in those few pups on day 14 is below the endogenous basal range of 20-25 ng/ml [39]. Studies have shown that CORT pellets prepared in a similar manner and implanted s.c. in the nape of the neck of A D X adult rats initially release a high amount of CORT [20]. Therefore, hormone-filled cannulae may have initially released a high amount of CORT that crossed the blood-brain barrier into the periphery where it may influence behavioral development. To assess this possibility, male pups were implanted unilaterally with cannulae containing cholesterol, 25 or 100% CORT as described previously. Pups were A D X on day 10 and tru k

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Fig. 1. Mean number of ultrasonic vocalizations(panel A) and mean duration of freezing(panel B) by 14-day-oldADX rats with i.c.v implants containing varying concentrations of CORT. Behavioralscores were taken during a 10-rain exposure period to an unfamiliar anesthetized adult male rat. *P < 0.05, **P < 0.01, significantlydifferentfrom 100% CORT group, n = 10-11/group.

overall significance of the various dependent measures, Posthoc comparisons were conducted using the Duncan's multiple range test. Because o f heterogeneity of variance, a square-root transformation, (x) ~2 + (x + 1) v2, was performed on the number of ultrasounds before analysis. However, the nontransformed mean and S.E.M. values are presented in the figure, Pups in the 100% CORT group emitted few ultrasonic vocalizations (Fig. 1A). However, ultrasound production did not vary significantly among the four test groups, F3,37 = 0:91, P > 0.05. Implantation of a unilateral 28-gauge cannula containing 100% C O R T into the lateral ventricle of an A D X pup was highly effective in facilitating the development of freezing, F3,37 = 6.38, P < 0.01 (Fig. 1B). In contrast, pups implanted with 25 and 50% CORT-filted cannulae exhibited low levels of freezing. In these pups, the duration of freezing did not

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each sampling day) to evaluate peripheral concentrations of CORT on days preceding the behavioral test. As in the previous experiment, all animals with the exception of two d a y - l l pups had plasma levels of CORT below the detection limit of the assay. One pup was in the 25% treatment group (14.3 ng/ml) and the other pup was in the 100% group (41.7 ng/ml). The current work is the first to our knowledge to directly examine the effects of central administration of exogenous CORT on rat behavioral development occurring during the preweaning period. In this study, i.c.v. implantation of 28-gauge cannulae containing 100% CORT promoted freezing without detectable concentrations of plasma CORT on the day of the test. Even on days before testing, the majority of hormone-filled cannulae did not produce an augmented release of CORT into the peripheral circulation that acted on peripheral target organs. Although these experiments do not exclude a contribution of peripheral mechanisms acting through the spinal cord and producing secondary effects on neural pathways, current results suggest strongly that in rat pups a central action of CORT is sufficient to facilitate the development of behavioral inhibition. A clear implication of these results is that during development endogenous CORT influences the development of specific neural pathways mediating freezing. The duration of freezing in rats with cannula containing 100% CORT is similar to levels reported in pups with intact adrenal glands and in ADX pups treated daily with 3.0 mg/kg of exogenous CORT [38,41]. This consis-

L.K. Takahashi, H. Kim/Neuroscience Letters 176 (1994) 272 276

tency in the duration of freezing suggests that there may be a species typical or strain typical [29] level of freezing induced by CORT action during this period of development. Studies incorporating an increased range of doses of CORT, measurement of the amount of CORT released by the cannula and CORT receptor-binding studies are needed to obtain further information on the CORT-induced neural development of freezing. On the basis of previous research, the action of exogenous CORT on freezing appears to be primarily developmental rather than modulatory, as demonstrated in the adult rat [29]. That is, behavioral deficits are clearly observed only when adrenal steroids are removed before, but not after, the development of freezing [38], These results suggest that after development, neural systems regulating the occurrence of freezing are no longer highly vulnerable to an acute absence of CORT. In further support of a neurotrophic effect of CORT, studies demonstrate that exogenous CORT treatments must cornmence before postnatal day 14 in order for ADX pups to develop the freezing response [38]. Although the current work supports strongly the view that a central action of CORT is involved in the development of freezing, the role of CORT, whether developmental or modulatory, in suppressing ultrasonic vocalizations in a threatening context is less clear. Previous studies using systemic injections of various doses of exogenous CORT also fail to implicate a major role of glucocorticoids in dampening ultrasound emission in freezing rats, albeit ADX rats without CORT treatment have elevated levels of vocalizations and do not freeze [38,41]. Perhaps both central and peripheral stressinduced concentrations of CORT may be necessary to significantly suppress vocalizations in freezing rats. AIternatively, or in addition, adrenal medullary catecholamines released in response to autonomic activation may play a role in modulating ultrasound emission under s o m e stressful situations. A relation between peripheral catecholamines and ultrasound production per se, however, does not appear to exist [cf. 16]. Current results are of particular relevance for studies concerned with the development of the hippocampus which contains a high density of adrenal steroid recept o r s [18,33]. Both mineralocorticoid and glucocorticoid

receptors are present in the hippocampus from the I st postnatal week, albeit their regional distribution pattern and binding differ during ontogeny [17,26-28,30,42]. Importantly, hippocampal dentate granule cells, which contain both adrenal steroid receptor types [17,27,28], exh i n t marked postnatal development during the preweaning period [5,8,31]. Studies demonstrate that postnatal neurogenesis and cell survival in the dentate gyrus are regulated by adrenal steroids [13,14]. In addition, behavioral studies implicate the involvement of the hippocampus in controlling some forms of response suppression or inhibition [2,10,1 l]. Taken together, a strong possibil-

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ity exists that adrenal steroid-induced maturation of the hippocampal system mediates the age-dependent appearance of freezing. The research was supported in part by N I M H Grant MH-43986 and by the Wisconsin Research Foundation. We thank Jennifer S. Willborn for technical assistance.

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