Neonatal stress transiently alters the development of hippocampal oxytocin receptors

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 80 (1994) 115-120

Research Report

Neonatal stress transiently alters the development of hippocampal oxytocin receptors Linda R. Noonan *, Jack D. Caldwell, Li Li, Cheryl H. Walker, Cort A. Pedersen, George A. Mason Department of Psychiatry and Brain and Development Research Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Accepted 18 January 1994

Abstract

The development of brain oxytocin (OXT) receptors was examined following the mild stress of daily, 20 min separations of infant rats from their mothers (repeated separation condition) or in undisturbed controls. Changes in OXT receptors were characterized in cell membrane preparations, using the OXT receptor ligand [125I]d(CH2)5[Tyr(Me)ZThraTyr-NH29]-ornithine vasotocin ([125I]OTA), from rats at 4, 8, 14, 22 postnatal days of age or as adults. In the hippocampus of control animals, [125I]OTA binding was highest at day 4 or 8 and declined thereafter. Repeated separation decreased the Bmax of [125I]OTA binding in whole hippocampus at day 8, an effect that did not persist into adulthood. This effect was found to be confined to the rapidly proliferating, dorsal hippocampus. It has been suggested that brain OXT is involved in both affiliative/social and stress-related behaviors. While the specific function of OXT receptors in hippocampus is currently unknown, mild stress to the infant and the disruption of infant-mother contact transiently alters the normal development of this system. Key words: Oxytocin; Receptor; Development; Ontogeny; Hippocampus; Stress; Maternal separation

1. Introduction

Specific receptors for the neuropeptide oxytocin (OXT) have been autoradiographically localized in several discrete areas of the adult rat brain, including olfactory tubercle, ventromedial hypothalamic nucleus, central amygdaloid nucleus, dorsal motor nucleus of the vagus and ventral hippocampus [10,11,30]. Developmentally, specific binding of the O X T receptor lig9 and [ 125 I]d(CH2)5[Tyr(Me) 2 Thr 4 Tyr-NH2]-ornithine vasotocin ([125I]OTA) can be observed in the brain as early as embryonic day 14 [31]. [125I]OTA binding sites in young animals display selectivity and affinity characteristics that are similar to those in the adult [29,31]. Electrophysiological activity indicates that [~25I]OTA binding sites, at least in caudal brain regions, represent functional neural receptors soon after birth [31]. Autoradiographic studies of [125I]OTA binding dur-

* Corresponding author at BDRC CB no. 7250, University of North Carolina, Chapel Hill, NC 27599, USA. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 4 ) 0 0 0 1 8 - U

ing development demonstrate three distinct patterns that are regionally specific [29,31]. In some regions, age-related increases in binding sites are observed beginning soon after birth, while ventromedial hypothalamic and olfactory tubercle binding sites do not appear at all until puberty. The third pattern is characterized by intense proliferation during the first week of life, that then diminishes to low or non-existent levels around weaning. This latter pattern occurs primarily in hippocampus and cingulate cortex. In hippocampus, high concentrations of O X T receptors in the dorsal region can be visualized during the first week of life and subsequently decline. The decline in dorsal receptors is paralelled by an increase in receptors in the ventral region, which persist into adulthood. During early development, even brief separation of the infant rat from the mother is stressful to the infant, as evidenced by an increase in ultrasonic distress vocalizations and hypothalamo-pituitary-adrenal (HPA) axis activation following separation [1,18]. Repeated, short separations during the first 1-2 weeks of life have been shown to permanently affect behavior, peptide and

I 16

L.R. N o o n a n et al. / l)et'elopmental Brain Research 80 (1994) / 15 .. 120

steroid hormone activity, and hippocampal glucocorticoid receptors [19]. Whereas the function of hippocampal oxytocin receptors has not been established, the genesis of the dorsal hippocampal population does coincide with the developmental period of maximal sensitivity to the stress of brief mother-infant separation. These receptors may be more sensitive to environmental manipulation during the period of rapid proliferation that occurs in the first postnatal week. In the present study, we examined the postnatal ontogeny of OXT receptors in developing rats that have been mildly stressed by repeated, daily 20 min separations from the mother.

2. Materials and methods Male and female Sprague-Dawley rat pups were left undisturbed in the nest or separated from the m o t h e r and littermates and placed in a warm incubator (32-33°C) for 20 min per day, and then immediately returned to the mother (repeated separation condition). Separation began on postnatal day 2 and ended at the time of sacrifice by decapitation on postnatal day 4, 8, 14 or 22. Each age and treatment group contained equal numbers of male and female pups. Additional male rats were repeatedly separated or left undisturbed during infancy from postnatal days 2-12. Thereafter all of these animals were undisturbed until weaning on day 23. At weaning, they were housed in same sex groups of 5 - 6 animals. At approximately 50 days of age, the animals were permanently housed in pairs until they were sacrificed as adults (age was approximately 1 year). All animals received ad libitum food and water.

Upon sacrifice, brains were placed on icc, blocked caudally using the caudal end of the cortex as a landmark and dissected into hippocampus, cortex and remaining forebrain (minus olfact(ns bulbs) and then frozen at - 7 0 ° C until assay. Addition~d hippocamr~i were dissected into dorsal and ventral portions. For this di,~section, the dorsal one-third (by length) was designated as dorsal, a!~d the, ventral two-thirds was designated as ventral hippocampus. This dissection was based upon the autoradiographs of dorsal and ventral popul~ttions of hippocampal O X T receptors from Tribollet el al [31]. The tissue was homogenized in 1).32 M sucrose containing l mM H ) T A and centrifuged at 800× g for 1() rain to remove nuclei and cell debris. The supernatant was centrifuged at 48,000~: g l~n 21) mii~. The pellet was resuspended in 40 mM Tris buffer-HCI (pH 74) and recentrifuged. The final pellet containing celt m e m b r a n e s was resus, pended in the 40 mM Tris-HCl buffer containing BSA and MnCI:. O X T receptors were characterized using the specific O X T receptor ligand, [12Sl]d(CHz)5[Tyr(Me)2Thr4Tyr-NH~]-ornithinc vasotocin ([125I]OTA). Aliquots of cell m e m b r a n e preparations pooled within each treatment group were incubated with ~)l.-2.fl nM concentrations of [12slIOTA for 1 h at 37°C to obtain saturation binding data. M e m b r a n e s from individual rats were incubated under the same conditions with a single concentration of [z:51]OTA ((~.2 nM). Separation of bound and free ligand was accomplished by dilution and washing with cold assay buffer by vacuum filtration. Non-specific binding (about 30% at 0.2 nM [12511OTA) was determined in the presence of a 500-fold excess of unlabeled OXT. Saturation binding data were analyzed using the iterative curve fitting program, In Plot (Graph P A D Software Inc., San Diego, CA). For the developmental assessment of O X T receptors, binding data from individual animals were analyzed for each age and treatment condition (n = 6 / g r o u p ) with the following exceptions: whole hippocampus from two animals was pooled at Day 4, dorsal or ventral hippocampus were pooled from 6 animals at Day 4, 4 animals at Day 8, 3 animals at Day 14, and 2 animals at Day 22 to obtain enough tissue for a single sample. Binding data from developing

Hippocampus ',,

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Fig. 1. [I:5I]OTA binding characteristics in hippocampus are shown across development. Bmax peaked at day 8, the same age that repeated separation was efficatious in decreasing the Bmax of O T A binding when compared with undisturbed animals. K D values were equal across ages.

L.R. Noonan et aL / Developmental Brain Research 80 (1994) 115-120 animals were analyzed by ANOVA followed by Tukey HSD test. Binding data from individual adult animals (12 animals per condition) were analyzed using Student's t-test. Comparisons were considered significant when P < 0,05.

20

15

10

3. Results

The binding characteristics of [125I]OTA in hippocampus, cortex and remaining forebrain of infant, pre-weanling and weanling rats are presented in Fig. 1 and Table 1. In all three brain areas, [t25I]OTA binding peaked at Day 4 or 8 and declined thereafter. These age-related changes were accomplished through an increase in receptor number (Bmax), with no change in affinity (KD). No differences attributable to the sex of the animal were observed. Repeated separation decreased [125I]OTA binding (Bmax) in the hippocampus of 8-day old infant rats compared to undisturbed infants of the same age. This effect did not persist as there was little evidence for treatment differences at Days 14 and 22. Cortex and remaining forebrain did not exhibit consistent treatment differences in binding characteristics at any age. K D values in all three brain regions were 0.l nM and were unaffected by repeated separation at any age. Fig. 2 shows [125I]OTA binding from individual animals at the 0.2 nM concentration. These data confirm our observations of repeated separation effects in infant hippocampus. ANOVA revealed a significant interaction of age and treatment (F3.40 = 8.2, P < 0.0003); at the younger ages repeated maternal separation and reunion decreased binding, but this effect was no longer evident at Days 14 and 22. A Tukey post-hoc test was only significant for the treatment difference at Day 8 (P < 0.0002). In cortex, treatment differences were inconsistent. Repeated separation decreased binding at Day 4 and increased binding at Day 8, as evidenced by a significant age by treatment interaction (F3.40 = 19.8, P < 0.000001) and Tukey tests (Day 4, P < 0.0003; Day 8, P < 0.0002). There were no statistically significant treatment differences in remaining forebrain, although

Table 1 [125I]OTA binding characteristics in cortex and forebrain Day 4

Day 8

Day 14

Day 22

Bmax KD Bmax KD Bmax KD Bmax KD Cortex Undisturbed Repeated separation Forebrain Undisturbed Repeated separation

117

27.9 25.7

0.1 0.1

25.3 0.1 24.8 0.1

18.6 0.1 22.4 0.1

14.3 0.1 11.6 0.1

18.0 0.1 19.4 0.1

28.1 0.1 25.1 0.1

23.8 0.1 21.6 0.1

12.1 0.1 12.3 0.1

Bmax is expressed as fmol OTA/mg protein. K D is expressed as nM concentration.

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Fig. 2. [1251]OTA binding characteristics in three brain regions are shown for 6 animals per age and treatment. * Significantly different from undisturbed animals, Tukey post hoc test, P < 0.05.

there was a trend toward an age × treatment interaction ( F 3 , 4 0 - - 2 . 6 , P < 0.07) similar in direction to the effect found in cortex. In Fig. 3 the effects of repeated maternal separation on the dorsal and ventral populations of [125I]OTA binding sites are shown at Days 4, 8, 14 and 22. For the dorsal hippocampus, ANOVA revealed a significant main effect of repeated separation (F1,39 = 4 . 6 , P < 0.05). [lZSI]OTA binding was decreased by repeated separation (Tukey HSD test, P < 0.05); an effect that was evident at Day 4 and 8. An age-related decline in [125I]OTA binding in dorsal hippocampus after Day 8 was also evident. In the ventral hippocampus, there were no significant effects of repeated separation at any age. [125I]OTA binding in whole hippocampus of adult male rats was 3.5 + 0.2 fmol O T A / m g protein in undisturbed controls and 4.0 + 0.3 fmol O T A / m g protein in repeated separation animals (data not shown). Repeated maternal separation did not significantly affect binding in the hippocampus 022 = 1.57, P > 0.05), confirming our observation that the effect of this manipulation is limited to approximately the first week of postnatal life. Binding in adults was lower than that of the developing animals shown in Fig. 2, further confirming the ontogenetic trend for peak [~25I]OTA bind-

1t 8

L.R. Noonan et al. / Decelopmental Brain Research 80 (1994) 115- 120

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Fig. 3. Repeated separation decreased OTA binding in dorsal hippocampus, ANOVA, P < 0.05, an effect that is most evident at Day 4 and 8.' Ventral hippocampus was not significantly affected by treatment.

ing in hippocampus during the first week of life, with a gradual decline as the animal grows to adulthood.

4. Discussion

In examining the normal ontogeny of [125I]OTA binding (undisturbed pups) it was evident that binding in membrane preparations from whole hippocampus, cortex and remaining forebrain was highest during the first 8 days of postnatal life, and declined thereafter in the pre-weanling and weanling age animals. In hippocampus, this trend continued with a further decrease in [125I]OTA binding between weaning and adulthood. All ontogenetic and treatment differences that we observed in developing animals appear to reflect changes in binding site number, not affinity, since Ko's were similar across all ages. The developmental pattern of early, intense increases in binding that we observed is in general agreement with previous autoradiographic studies using the same ligand [29,31]. One previous study also included a Scatchard-type analysis of homogenate binding in the cingulate cortex of 10-dayoold pups [29]. KD values for day 8 animals in the current study and day 10 animals in the previous study are identical (0.1 vs. 0.099 + 0.021 nM, respectively). How-

ever the Bma x values in the current study were much lower than those of the previous study (25.3 vs. 152 1.45 fmol O T A / m g protein, respectively). This can be explained by the higher amount of protein found in our samples, since we used whole cortex and only cingulate cortex was used in the previous study. Repeated separation from the mother for 2(1 rain per day resulted in a decrease in [~25t]OTA binding in the hippocampus of 8-day-old rats relative to undisturbed controls, an effect demonstrated by our saturation binding data and data from individual animals obtained using a single concentration of [t25I]OTA. This treatment effect coincided with the highest number of [125I]OTA binding sites during development. The effect of repeated separation was transient, as it was no longer evident at Day 14, Day 22 or in adult male rats. Separation-induced changes in [t25I]OTA binding in infant rats were further limited anatomically to the dorsal hippocampus during the first 8 days of postnatal development. This dorsal population of binding sites rises precipitously during the first week of life and then declines to levels that are undetectable by autoradiography at Day 19 and in the adult [29,31]. In the present binding study we observed detectable OTA binding in dorsal hippocampus at Day 22, though it was significantly less than at Day 8 and showed no effect of separation. It appears that some dorsal hippocampal binding sites do remain into adulthood, albeit at low levels. Neuronal activation has been reported to occur following OXT injection in the CA t field [22] and in dorsal hippocampus [16] of the adult rat. Significant separation-induced changes were also observed in cortex, another brain area that expresses transient OTA binding sites during infancy, although these changes were inconsistent. Analysis of binding in samples from cerebral cortex of individual animals suggested that repeated separation decreased binding at Day 4 and increased binding at Day 8; however, treatment effects were not evident from analysis of saturation binding data, at any age. This is in contrast to the consistent effects in hippocampus, where both types of binding data indicated that repeated separation decreased OTA binding. In the adult rat, OXT has been shown to influence behavioral and endocrine responses of two types that may be relevant to the infant separation paradigm employed in this study. OXT promotes social or affiliative responses of animals by increasing sexual [2,3,6] and maternal [25] behaviors. The concomitants of ejaculation (human) [7], uterine contraction during parturition and milk ejection also involve OXT. It has been demonstrated that OTA binding parameters differ in several mouse and vole strains based upon their degree of social, parental and sexual affiliation [13]. Additionally, low doses of OXT facilitate social recognition of juvenile rats by adult males [27]. OXT is also a stress

L.R. Noonan et al. / Developmental Brain Research 80 (1994) 115-120

hormone. In adult rats, O X T has been shown to increase peripherally in response to stress and to participate in H P A activation by promoting CRF-induced A C T H release [12]. Centrally, O X T increases the stress-related behaviors of grooming [9,21], locomotion in the open field [8] and hot-plate analgesia [5]. Similar effects of O X T may exist in the developing rat. Whereas infant rats do not normally exhibit maternal or sexual behavior, there is some evidence that early social and stress-related responses are influenced by OXT. Infant and preweanling rats treated with i.c.v. O X T exhibit an increase in licking of other infant rats [26]. The differently affiliative vole strains that exhibit different patterns of O X T receptor binding as adults also exhibit dicotomous responses to maternal separation as infants [13]. Moreover, central O X T administration decreases separation-induced ultrasonic vocalizations in infant rats [13]. The stress-related behavior of grooming is increased by intracisternal administration of O X T in infant rats as young as 5 days of age [26]. Also, a single intracisternal administration of O X T in the 3- to 4-day-old rat produces an increase in noveltyinduced grooming when the animal is an adult [23]. Infant H P A axis responses to maternal separation also a p p e a r to involve OXT. We have observed that O X T is released in small amounts peripherally in response to the stress of a single separation from the mother (unpublished observations). A single maternal separation facilitates OXT-induced corticosterone release in l 1to 12-day-old rat pups, whereas repeated maternal separations reduce OXT-induced corticosterone release at the same age (unpublished observations). The maternal separation procedure used in this study causes several changes in the infant rat's environment. During separation the infant rat is isolated from the mother, nest and littermates, and the tactile and olfactory stimulation these provide, and presented with the novelty of experimenter handling, transport and housing during separation. Although loss of the warmth provided by the mother has been suggested to be an important determinant of separation effects in previous studies, [20] body t e m p e r a t u r e was probably not a factor in the current study because the pups were housed in a warm incubator during separation. Food (milk) deprivation was probably unimportant as well, since separation was limited to 20 min. Environmental changes may persist following separation, as rat mothers alter their maternal behavior toward separated pups [15]. The repeated nature of the separations may produce habituation to some of these stimuli, a process that could also alter brain development. The function of transient binding sites in the developing brain is not well understood, although it appears to be a common p h e n o m e n o n in neuropeptide receptor ontogeny [14,17,24,28]. Even less clear are the long-term consequences of transiently disrupting neu-

119

ropeptide receptor proliferation during development, as we have done in the current study. No consequence for brain function and behavior would result if the O T A binding sites that are decreased by separation represent the common developmental p h e n o m e n o n of overproduction of neurons or receptors that are later lost due to p r o g r a m m e d cell death or failure to form synapses. Alternatively the transient nature of some of these binding sites suggests that they may have an organizational or trophic function in the development of the hippocampus, similar to what has been suggested for the structurally related peptide, argininevasopressin [4]. It is interesting to speculate that even brief separations of the infant from the mother, a manipulation that involves both stressing the infant and the disruption of a social bond, may have effects on oxytocinergic activity that subsequently influence the expression of social- or stress-related behaviors or endocrine function.

Acknowledgements This research was supported by grants to L.R.N. from N I M H 46442-01, The Foundation of H o p e for T r e a t m e n t and Research in Mental Illness and the National Alliance for Research in Schizophrenia and Depression. Thanks to Mrs. Betsy Shambley for help in the preparation of this manuscript.

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12t)

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