Do neonatal bilateral ibotenic acid lesions of the hippocampal formation or of the amygdala impair HPA axis responsiveness and regulation in infant rhesus macaques (Macaca mulatta)?

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Do neonatal bilateral ibotenic acid lesions of the hippocampal formation or of the amygdala impair HPA axis responsiveness and regulation in infant rhesus macaques (Macaca mulatta)? Anne-Pierre S. Goursaud a,b,d,⁎,1 , Sally P. Mendoza a,c , John P. Capitanio a,c a

California National Primate Research Center, University of California, Davis, CA 95616, USA Department of Psychiatry, University of California, Davis, CA 95616, USA c Department of Psychology, University of California, Davis, CA 95616, USA d Center for Neuroscience, University of California, Davis, CA 95616, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

In response to stressful events, the HPA axis is activated triggering the successive release of

Accepted 2 November 2005

CRF, ACTH, and glucocorticoids. The glucocorticoids in turn provide a negative feedback

Available online 17 January 2006

signal to terminate the stress response. The amygdala and the hippocampus are involved in the regulation of the HPA axis. In rodents, their respective roles have been identified; the

Keywords:

amygdala exerts a stimulatory effect, whereas the hippocampus provides negative

Monkey

feedback control. In primates, however, their regulatory roles are still not well defined.

Plasma cortisol

The present study compared HPA axis responsiveness and regulation in 3- to 5-month-old

Amygdala

rhesus macaques that received neonatal (15 ± 3 days old) bilateral ibotenic acid lesions of

Hippocampus

the hippocampus or amygdala, or sham lesions. Group differences in plasma cortisol

Neonatal lesion

response to separation from the mother and relocation in a novel environment were

Dexamethasone

assessed as well as response to dexamethasone suppression and ACTH challenge. Results revealed that the initial cortisol levels after separation/relocation did not differ between groups. Subjects with hippocampus lesions did not show a suppression of cortisol in response to dexamethasone, suggesting a loss of negative feedback control of HPA regulation. Subjects with amygdala and sham lesions did not differ in response to dexamethasone. Indeed, bilateral neonatal lesions of the amygdala have little impact on HPA axis responsiveness and regulation in contrast to lesions in adult monkeys. Finally, females displayed higher cortisol levels than males, independently of their lesion, indicating that the development of sex differences in the regulation of the HPA axis does not involve the amygdala or hippocampus. © 2005 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Yerkes N.P.R.C., Emory University, 954 Gatewood Road, Atlanta, GA 30329, USA. Fax: +1 404 727 8088. E-mail address: [email protected] (A.-P.S. Goursaud). 1 The first author is now in Psychology Department and Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA. 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.11.027

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1.

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Introduction

The hypothalamic–pituitary–adrenal axis is largely responsible for modulating metabolic, immunologic, and endocrine activity in response to recurrent and unexpected changes in the environment. In response to stressful events, for example, a neuroendocrine cascade is initiated in which CRH is released from hypothalamic neurons, which then triggers release of ACTH by the anterior pituitary, which in turn stimulates glucocorticoids secretion by the adrenal cortex. Given the importance of glucocorticoids in physiological regulation, it is not surprising that the HPA axis is itself tightly regulated by multiple negative feedback systems (Akana et al., 2001; Dallman et al., 1994; Feldman et al., 1995). Research in rats has implicated the amygdala and the hippocampus as two extrahypothalamic CNS sites involved in the regulation of the HPA axis. The role of the amygdala has been shown to be facilitatory. Electrical stimulation of the rat amygdala induces increased secretion of CRH, ACTH, and corticosterone (Feldman and Weidenfeld, 1998; Weidenfeld et al., 1997), whereas amygdala lesions inhibit the hormonal response to a variety of stressful stimuli (Feldman and Conforti, 1981; Feldman et al., 1995). By contrast, the role played by the hippocampus is inhibitory. Electrical stimulation of the rat hippocampus inhibits stress-induced increases of ACTH and glucocorticoids (Dunn and Orr, 1984; Feldman and Weidenfeld, 2001), whereas bilateral lesion of the hippocampus in adults produces hypersecretion of ACTH and glucocorticoids (Feldman and Conforti, 1976, 1980; Sapolsky et al., 1984, 1991). The hippocampus also appears to modulate the negative feedback regulation by glucocorticoids. Lesion of the hippocampus reduces the ability of dexamethasone, a synthetic glucocorticoid, to inhibit stress-induced adrenocortical response (Feldman and Weidenfeld, 1993; Feldman and Conforti, 1980; Margarinos et al., 1987). Microinfusion or implants of glucocorticoid receptor antagonists into the rat hippocampus induce depletion of the CRH content of the median eminence, and hypersecretion of both ACTH and corticosterone following stressful stimuli (Feldman and Weidenfeld, 1999). Thus, whereas the amygdala is involved in stimulation of the HPA axis activity in rats, the hippocampus plays an inhibitory role in its regulation. The precise role of the amygdala and hippocampus in the regulation of the HPA axis in human and nonhuman primates has not been clearly identified. The few studies that have examined CNS regulation of HPA axis activity suggest that like rodents the primate amygdala may not be involved in the regulation of the basal HPA axis activity but may be necessary for the initiation of the HPA axis responses to stress (Mason et al., 1961; Norman and Spies, 1981; Sapolsky et al., 1991). Similarly, most primate studies support the rodent data in suggesting an inhibitory role of the hippocampus in HPA axis function, in basal as well as in some stress responses. Electrical stimulation of the hippocampus in one human patient inhibited stress-induced ACTH and adrenocortical responses (Mandell et al., 1963). Bilateral aspiration lesions of the hippocampal formation and parahippocampal cortex in adult macaques caused transient basal glucocorticoid hypersecretion and dexamethasone resistance with a return to

normal levels within 6–15 months (Sapolsky et al., 1991). Similarly, humans with damage to the hippocampal formation, such as individuals with Alzheimer disease, are dexamethasone resistant (Carroll et al., 1981) and/or transiently hypersecrete glucocorticoids in basal conditions (Buchanan et al., 2004; De Leon et al., 1988). However, one study (Regestein et al., 1986) conducted in monkeys with hippocampus lesions reported diminished plasma cortisol responses to chair restraint, but not in response to shock avoidance. The goal of the present study was to compare HPA axis responsiveness and regulation in 3- to 5-month-old rhesus macaques that received neonatal (15 ± 3 days old) bilateral ibotenic acid lesions to either the hippocampus or the amygdala, or sham lesions. Behavioral studies in nonhuman primates and rodents have shown that damage to the amygdala or to the hippocampus can have different impacts on behavioral stress reactions depending upon the age at which lesions were performed (Bachevalier et al., 1999; Daenen et al., 2002; Daenen et al., 2001; Dicks et al., 1968; Emery et al., 2001; Prather et al., 2001). The behavioral changes observed after early lesions were more pronounced than those observed when lesions were performed in adults. Whereas the amygdala matures very rapidly after birth, the hippocampus and its connections with many cortical areas continue to encounter gradual changes and maturational processes (Alvarado and Bachevalier, 2000; Benes and Farol, 1994; Seress et al., 2001). In addition, data obtained in monkeys (Clarke, 1993) as well as in humans (Goldberg et al., 2003; Lewis and Ramsay, 1995; Watamura et al., 2004) suggest that the HPA axis does not reach its adult-like function until several months after birth (Walker et al., 2001). For instance, cortisol levels are higher in infants than in adults but decrease to adult levels over the first year (Clarke, 1993; Gunnar et al., 1989; Ramsay and Lewis, 1995). The HPA axis response to the stress of inoculation reaches a moderate stability only around the latter half of the first year in humans (Lewis and Ramsay, 1995). The current study was conducted in conjunction with a larger study of biobehavioral characterization of 3- to 4-monthold infant rhesus monkeys (Capitanio et al., 2005, 2006). With respect to the HPA axis, the goals of the larger study were (1) to assess individual differences in the capacity of the HPA axis to respond to a known, potent suite of stressors, namely separation from mother and relocation to an unfamiliar cage (the combination of which we consider to maximally activate the system without in any way causing physical trauma to the monkeys) and (2) to examine individual differences in the susceptibility of young monkeys to pharmacological regulation of the HPA axis. It is important to note that this study did not attempt to measure the magnitude of the response to either separation or novelty. Largely owing to the number of animals (250–350 infants were assessed each year) and to differences in their housing conditions (nursery, with only mothers, small social groups and large social groups) in the larger study, neither basal HPA axis activity nor disturbance control conditions were included in the biobehavioral assessment procedures. The subjects of the current study were evaluated using identical procedures developed in the larger study. Blood samples were collected in order to assess potential differences in the reaction to separation/relocation among lesion groups.

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We hypothesized that (1) cortisol levels during the initial response to separation/relocation would be higher in animals with hippocampal lesions and lower in animals with amygdala lesions relative to sham-lesioned animals, and (2) the response to dexamethasone would be diminished (less or no suppression) in animals with hippocampal lesions as compared to that in animals with amygdala or sham lesions.

2.

Results

Despite the wide age ranges of the animals at testing in the three groups, ANOVA revealed no significant differences in age between the three groups (F = 0.301; df = 1; P = 0.590). Cortisol concentrations were significantly different across the first three samples (Fig. 1, F = 8.024; df = 2; P = 0.001). Post hoc comparisons showed that the mean cortisol level of sample 3 was significantly lower than that of both sample 1 and sample 2 (P ≤ 0.003 for both comparisons). The differences in cortisol levels across the three samples were dependent on lesion condition, however, as demonstrated by a significant lesion × sample interaction (F = 3.669; df = 4; P = 0.014). Further analysis showed that, for the CTL and AMY groups, mean cortisol levels differed significantly between samples (CTL group: F = 4.22; df = 2; P = 0.021; AMY group: F = 9.56; df = 2; P b 0.001). For both groups, the mean levels of cortisol were significantly higher in sample 1 than in sample 3. By contrast in the HIP group, there was no significant difference between samples (F = 1.25; df = 2; P = 0.298). No effect of age was

Fig. 1 – Mean (± SEM) cortisol levels (μg/dl) of each sample for each lesion group. For each group, the first (black) and the second (grey) bars represent the means of the cortisol levels taken the first day, in the morning after separation/relocation (sample 1, S1, N = 8) and in the afternoon (sample 2, S2, N = 8). The third (white) bar represents the mean of the cortisol levels following overnight dexamethasone suppression (sample 3, S3, N = 8) and the fourth (stripe) bar shows the mean of cortisol level taken 30 min after ACTH stimulation (sample 4, S4, N = 6). Asterisks indicate a significant difference between sample 1 and sample 3 (P b 0.05) and pound signs indicate a significant difference between sample 3 and sample 4 (#P b 0.02; ##P b 0.001).

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observed. Finally, as indicated by a significant sex effect (F = 4.838; df = 1; P = 0.042), females had significantly higher mean cortisol levels than males, independently of both the lesion received and the blood sample type. Mean values (±SEM) for females for each of the four samples (S1, S2, S3, and S4 successively) were 79.33 μg/dl (±5.05), 74.82 μg/dl (±4.81), 62.09 μg/dl (±5.20), and 91.01 μg/dl (±8.56), and for males, the values were 73.69 μg/dl (±5.39), 60.13 μg/dl (±3.36), 45.64 μg/dl (±6.78), and 77.10 μg/dl (±6.58). The analysis of sample 4 revealed no significant effect of lesion, age, or sex and also no significant interaction between these factors in response to ACTH challenge. The repeated measure ANOVA revealed that sample 4 cortisol concentrations were higher than were sample 3 concentrations (Fig. 1, F = 164.528; df = 1; P b 0.001). No significant differences between lesion groups were observed (F = 0.003; df = 2; P = 0.997); however, the differences between the two samples were dependent on the lesion condition as demonstrated by a significant lesion × sample interaction (F = 5.373; df = 2; P = 0.017). The cortisol levels increased significantly after ACTH injection within all three groups, but the increase was smaller in the HIP group (CTL and AMY groups: Ps b 0.001; HIP group: P = 0.011).

3.

Discussion

Overall, bilateral neonatal lesions of the hippocampus or amygdala had no effect on the cortisol levels following maternal separation and relocation in infant monkeys, contrary to expectations based on data from rodents and studies of adult monkeys. Hippocampal lesions, however, did eliminate the expected suppression of cortisol concentrations by dexamethasone, a result that is consistent with the earlier literature suggesting that this structure is important in negative feedback regulation. The cortisol levels in all groups did increase following the ACTH challenge, suggesting normal functioning of the adrenal gland after both bilateral neonatal amygdala and hippocampus lesions. Finally, our data confirm those of others (Capitanio et al., 2005; Clarke, 1993) that females had higher cortisol levels than did males, independently of the lesion received. This strongly suggests that this sex difference is not mediated by either the amygdala or the hippocampus. The extremely high absolute values of the initial cortisol samples in all groups presumably reflect stress-induced elevations due to the separation from the mother and the relocation to a new environment (Capitanio et al., 2005, 2006; Clarke, 1993; Gunnar et al., 1980; Mitchell and Gomber, 1976), as well as overall higher levels of cortisol in infant monkeys as compared to adults (Capitanio et al., 2005; Clarke, 1993; Mendoza et al., 1978; Shannon et al., 1998; Smotherman et al., 1979). Unfortunately, we were unable to measure baseline HPA axis activity and response to disturbance control conditions, which would have allowed us to directly measure the effect of the lesions on the magnitude of the cortisol response to separation or novelty. Nonetheless, based on the rodent and adult primate literatures (see Introduction), we expected that neonatal lesions of the amygdala would eliminate an important component of the HPA system that

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allows young monkeys to mount a stress response to the psychological stressors experienced and that this would be reflected in lower cortisol levels at the first two blood sample collection times. This was apparently not the case. As shown in Fig. 1, cortisol levels on Day 1 (samples 1 and 2) were nearly identical for the amygdala- and sham-lesioned animals. It is logically possible that the cortisol levels we report, while high, do not reflect activation of the stress response even in the sham-lesioned animals. However, the response to maternal separation and even the response to separation from an inanimate surrogate have long been known to lead to a pronounced increase in cortisol levels in young macaques (Hill et al., 1973). It seems more likely that both the shamlesioned and amygdala-lesioned animals mounted a stress response and that loss of the amygdala did not in any way alter the response. Whether or not the amygdala in infant monkeys is ever involved in activation of the stress response will require further investigation, but at present, we can conclude that it was not a necessary element of the neurobiological apparatus regulating the stress response in the present situation. Loss of the hippocampus likewise did not alter the response to the combined stressors, but it did alter the response to dexamethasone. Both the amygdala- and shamlesioned animals exhibited a substantial suppression of cortisol on the morning following dexamethasone treatment. By contrast, mean values suggest a more moderate suppression in the animals with hippocampal lesions. Statistical evaluation indicated that both the amygdala- and shamlesioned animals exhibited a significant reduction in cortisol levels following dexamethasone treatment in comparison to sample 1 collected at approximately the same time of day on Day 1 of testing, whereas the animals with hippocampal lesions did not show this significant reduction. These results are in agreement with data obtained in adult monkeys (Sapolsky et al., 1991) and rats (Feldman and Weidenfeld, 1993; Feldman and Conforti, 1980) with either hippocampus damage or following pharmacological blockage of the hippocampal glucocorticoid receptors (Feldman and Weidenfeld, 1999). Thus, our results confirm the involvement of the primate hippocampus in negative feedback regulation of glucocorticoids but its precise role cannot be determined by the present study. Indeed, peripheral administration of a high dose of dexamethasone does not define the site of glucocorticoid feedback. Dexamethasone binds preferentially to glucocorticoid receptors (GR, type II corticosteroid receptors) but not mineralocorticoids receptors (MR, type I corticosteroid receptors) (De Kloet et al., 1975; Cole et al., 2000). In contrast to the predominant distribution of GR in the rat hippocampus (and PVN), MR but not GR are abundant in the primate hippocampal formation (Sanchez et al., 2000). By contrast, a very high density of GR is found in the primate hypothalamic PVN, prefrontal and entorhinal cortices, and cerebellar cortex (Sanchez et al., 2000). Moreover, there is some evidence that dexamethasone does not readily cross the blood–brain barrier (De Kloet, 1997; De Kloet et al., 1975) and that dexamethasone exerts its inhibitory influence on the HPA system by activating GR peripherally at the level of the pituitary or adrenal gland (Cole et al., 2000; Miller et al., 1992). Our data suggest that dexamethasone in our subjects did gain access to the brain

and did in some way interact, directly or indirectly, with the hippocampus to induce its effects. The most likely explanation is that GR are present in the hippocampus of the young monkeys and that activation of these receptors induces an inhibitory action on the HPA axis leading to attenuated levels of plasma cortisol in all of our subjects with an intact hippocampus. Other routes of influence are also conceivable. For example, it is possible that GR in the frontal lobe are responsible for detecting high levels of glucocorticoids and initiating a dampening of the HPA system through neural circuitry involving the hippocampus. Clearly, more research is needed before we can claim confidently that the hippocampus of young monkeys is rich in GR and that dexamethasone introduced peripherally can gain access to hippocampal GR, but our results indicate that it is a possibility and that such research is thus necessary. In response to the ACTH challenge after the overnight dexamethasone treatment, the sham-lesioned animals showed an increase in cortisol levels similar to that observed in rats (Cole et al., 2000) and in normal rhesus monkey infants (Capitanio et al., 2005). The subjects with amygdala lesions and those with hippocampal lesions showed a similar increase in cortisol levels as compared to the sham-lesioned animals suggesting that neither lesion affected the ability of the adrenal gland to respond to ACTH. Finally, a main sex difference in overall cortisol levels was observed, independently of the lesions received. The females had higher cortisol levels than the males. Similar results have been found by others for young rhesus monkeys (Capitanio et al., 2005; Clarke, 1993) as well as for adults. For instance, introduction of adult monkeys to a strange group or introduction of a new aggressive male into a natural troop induced a greater cortisol increase in females than in males (Alberts et al., 1992; Scallet et al., 1981). Adult female monkeys also displayed a greater CRF-induced cortisol secretion than adult males (Lyons et al., 2000). Thus, it appears that similar to what has been observed in rodents (Handa et al., 1994; Patchev and Almeida, 1998; Young, 1996) a sex difference exists in the regulation of the primate HPA axis. Little is known about this sex difference in primates, although in rodents it has been suggested that gonadal steroids may exert an early influence on developing neural systems that regulate HPA axis activity (Patchev and Almeida, 1998). If such an influence also exists in primates, our present results strongly suggest that it does not involve the amygdala and the hippocampus.

4.

Experimental procedure

4.1.

Subjects and housing

The subjects were 24 3- to 5-month-old infant rhesus macaques (mean age (days) ± SEM: 115.29 ± 3.83), 10 males and 14 females (Macaca mulatta). They were born at the California National Primate Research Center (Davis, CA) and reared with their mother in individual standard cages (61 cm W × 66 cm D × 81 cm H) that permitted auditory and olfactory but no visual contact between animals. The neonates were assigned to one of three groups: AMY group (N = 8, 3 males and 5 females) received bilateral ibotenic acid lesions of the amygdala, HIP group (N = 8, 3 males and 5 females)

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received bilateral ibotenic acid lesions of the hippocampal formation, and CTL group (N = 8, 4 males and 4 females) received a sham lesion. All lesions were performed 15 days (±3 days) after birth. It should be noted here that since all lesions were performed very early after birth and approximately at the same age for all subjects, the age at testing, from 3- to 5-month-old, reflects also approximately the time elapsed between lesions and assessment of the HPA axis regulation carried out in the present experiment. All animal housing, rearing, and experimental procedures were in accordance with institutional guidelines. 4.2.

Rearing experience

The subjects were assigned to one of four cohorts. Each cohort consisted of 6 mother–infant pairs. Within a cohort, two infants were from group AMY, two infants from group HIP, and two infants from group CTL. In addition, the age and the sex of the neonates were balanced within each cohort insofar as possible; owing to birth seasonality, however, cohort 4 contained 1 male and 5 females. The subjects were allowed daily socialization (3–6 h per day, five days a week) in large enclosures with the other members of the cohort. Beginning at 1.5 months of age, one adult male per cohort was introduced into the enclosure for socialization purposes. Each adult male monkey remained within the same cohort throughout the study. 4.3.

Surgical procedures

4.3.1. Presurgical preparations Each infant was temporarily separated from its mother for a 30- to 60-min period, three times (postnatal days 3–5, 7–9, and 10–12) before the actual surgery (postnatal days 12–18) in order to familiarize the mother with the separation procedure and to increase the likelihood of her acceptance of the infant after surgery. During each separation, the infant's head was shaved and scrubbed with Betadine and 70% ethanol. 4.3.2. Magnetic resonance imaging-guided bilateral ibotenic acid lesion procedures The surgical procedures were as reported by Bauman et al. (2004). In brief, prior to surgery, the infant was anesthetized with ketamine hydrochloride (15 mg/kg i.m.) and medetomadine (0.3 mg/kg), then, was placed in an MRI-compatible stereotaxic apparatus (Crist Instruments Co., Damascus, MD, USA). The animal's brain was imaged using a General Electric 1.5 T Gyroscan magnet; 1.0-mm-thick sections were taken using a T1-weighted Inversion Recovery Pulse sequence (TR = 21, TE = 7.9, NEX 3, FOV = 8 cm, matrix = 256 × 256). The MRI scans obtained were exported to the Canvas graphics program (Deneba Systems, Miami, FL, USA) to evaluate the location of the amygdala or of the hippocampus and to calculate the stereotaxic coordinates for each injection of ibotenic acid. Throughout surgery, a stable level of anesthesia was maintained with a combination of isoflurane (1.5%) and i.v. infusion of fentanyl (7– 10 mg/kg/h) and the monkey's vital signs were monitored. A midline incision was made and craniotomies were performed to expose the brain over the predicted location of the amygdala or hippocampus. A tungsten microelectrode was lowered at the predicted location of the middle amygdala or hippocampus to record extracellular activity and to confirm the dorso-ventral extent of the structure. The electrode was then withdrawn and the bilateral ibotenic acid injections were made simultaneously at multiple sites within the amygdala or hippocampus using two 10-μl Hamilton syringes (26-gauge beveled needles). At each site, 0.5 to 1.5 μl of ibotenic acid (Biosearch Technologies, Novato, CA, USA, 10 mg/ml in 0.1 M phosphate-buffered saline) was injected at a rate of 0.2 μl/min. The total amount of ibotenic acid injected ranged from 7 to 12 μl per amygdala and from 5.5 to 7 μl per

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hippocampus. After injections, the dura was replaced and sutured when possible, the craniotomy was filled with Gelfoam (Pharmacia and Upjohn, Peapack, NJ, USA), and the fascia and skin were sutured in two separate layers. The sham lesions consisted of the same surgical procedures except that after incision of the dura above the suspected position of the amygdala for 4 monkeys and above the suspected position of the hippocampus for the 4 others, the dura was immediately sutured, the craniotomy filled, and the fascia and skin layers sutured. All surgical procedures were performed under an approved University of California, Davis Institutional Animal Care and Use Protocol and strictly adhered to the NIH guidelines on the use of nonhuman primate subjects. 4.3.3. Postsurgical procedures After surgery, the infants were returned to their mothers and were monitored continuously for at least 24 h. No medical complications occurred and all mothers accepted their infants with no conflict. 4.4.

Lesion assessment

4.4.1. Magnetic resonance imaging-based lesion assessment Ten days after surgery, the AMY and HIP subjects received a second MRI scan. A General Electric 1.5 T Gyroscan magnet allowed us to obtain 1.5-mm-thick sections of the brains using a T2-weighted Inversion Recovery Pulse sequence (TR = 4000, TE = 102, NEX 3, FOV = 8 cm, matrix 256 × 256). These T2-weighted images were obtained to confirm the accuracy of the lesions and to assess potential extra damage. As expected, the hyperintense signals caused by the lesion-induced edemas on the T2-weighted images were clearly focused in the amygdaloid complex or in the hippocampal formation (see Bauman et al., 2004, for details and images). 4.4.2. Histological lesion evaluation One animal with an amygdala lesion was sacrificed at 1.5 years old for health issues thus enabling histological evaluation of the lesion (see Bauman et al., 2004, for detailed description). It was observed that the actual region of damage was mostly confined to the amygdala with only little collateral damage in the superior temporal sulcus, ventral claustrum, and the most rostral portion of the hippocampal formation. In addition, the damaged area was more confined to the amygdala than suggested by the extent of the edema observed in the postlesion MRI hyperintense T2 signal. This histological analysis provides reassurance that much of the amygdala (or hippocampus) was indeed lesioned. 4.5.

Experimental procedures

The 24 subjects were tested when they were 3 to 5 months of age within cohorts, alongside normal infants of similar age participating in the larger study, in a total of 8 sessions involving six to eight animals. Each testing session occurred over a 24-h period (Table 1). Between 0830 and 0930 h on the first day (Day 1), the infants were removed from their mother and transferred to an individual standard cage (same dimensions as their living cage), in another building. A series of behavioral assessments (see Capitanio et al., 2006, for a description) were performed which are incidental to the present report. Briefly, during the behavioral assessment part, the infants were observed in their new cage and were exposed to several testing situations including viewing a video of an adult male rhesus macaque displaying nonsocial and aggressive behavior (video playback test), and a live human “intruder” presenting a frontal or profile face to the animal. The infants were fed with Monkey Chow (Ralston-Purina, St. Louis, MO) and fresh fruits. Water was always available. At the end of

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Table 1 – Schedule of the experimental procedures Time Day 1 Separation from mother and relocation in individual cage Behavioral observations Day 1 (not relevant to the present study) Blood sample #1 Behavioral tasks (not relevant to the present study) Blood sample #2 + dexamethasone injection Day 2 Behavioral observations Day 2 (not relevant to the present study) Blood sample #3 + ACTH injection Blood sample #4 Reunion with mother

830–930 945 1100 1130 1600

730 830 900 1030–1130

the overnight assessment period (between 1030 and 1130 h on Day 2), infants were reunited with mothers in their living cage. 4.6.

Blood sampling, dexamethasone and ACTH administration

One infant at a time was removed from its individual cage by an experienced technician and was brought to an adjacent room where it was hand-restrained while blood was collected via femoral venipuncture. This procedure took 1–2 min per subject and thus in a maximum period of 12–16 min after the first entrance of the technician in the room, the blood samples for all subjects were collected. Preliminary analysis of the data revealed no effect of the sampling order on cortisol levels. A total of four blood samples were collected per subject during the 24-h assessment period (see Table 1 for schedule details). The first blood sample was collected on Day 1 at 1100 h, and the second sample was collected at the conclusion of behavioral testing on Day 1 at 1600 h and was immediately followed by an injection of dexamethasone (0.5 mg/kg). The third blood sample was collected at 0830 h on Day 2 and was immediately followed by an ACTH injection (2.5 IU/kg). The fourth blood sample was collected 30 min after the injection of ACTH. Because ACTH was unavailable near the end of this experiment, the fourth cohort did not receive the injection of ACTH and thus, the last blood sample was not collected for six subjects. Immediately after collection, the blood samples (1 ml for sample 1 and 0.5 ml for each subsequent sample) were transferred to EDTA tubes. Samples were then centrifuged at 4 °C at 3000 rpm for 10 min. The plasma fraction was extracted and stored at −80 °C until assayed for cortisol concentration by RIA (Diagnostics Products Corp., Los Angeles, CA). The intra- and inter-assay coefficients of variation were 7.9% and 5.8%, respectively. 4.7.

Data statistical analysis

A one-way ANOVA was first performed in order to verify that the mean age of the subjects at testing did not differ between lesion groups. The cortisol levels from samples 1, 2, and 3 were analyzed together since we had data for all 24 subjects. A repeated-measure ANOVA with lesion (CTL, AMY, HIP) and sex as the between-subject factors, age as a co-factor, and sample (1, 2 and 3) as the repeated measure was performed. Probabilities, P values, were calculated using Huynh–Feldt correction for degrees of freedom of the repeated measures (Huynh and Feldt, 1976). When an overall F was significant, tests of simple main effect within each lesion and/ or each sample were done and follow-up contrasts were evaluated

with Bonferroni corrected t tests. Cortisol concentrations from sample 4 were analyzed separately from the 3 other samples (owing to the reduced sample size [N = 18]) using ANOVA with lesion and sex as the between-subject factors and age as a cofactor. Cortisol response to ACTH injection after pretreatment with dexamethasone was assessed with a repeated-measure ANOVA with lesion (CTL, AMY, HIP) as the between-subject factor and sample (3 and 4) as the repeated measure with the Huynh–Feldt correction for degrees of freedom of the repeated measures. The six subjects, two in each group, which did not receive the ACTH injection, were removed from this analysis. Follow-up contrasts were evaluated with Bonferroni corrected t tests.

The level of significance was set at P ≤ 0.050 in all analyses.

Acknowledgments This study was supported by National Institute of Mental Health Grant R37 MH/HD57502 to David G. Amaral and a Resource Research Project (John P. Capitanio, PI) on Grant RR00169 to the California National Primate Research Center. The authors wish to thank David G. Amaral for providing the monkeys involved in this study and for his support to AnnePierre S. Goursaud during her postdoctoral training in his laboratory. The authors also wish to thank Laura Del Rosso, Carmel Stanko, and Greg Vicino, for their technical assistance with data collection and care of the animals during the study as well as Nicole Maninger who performed the plasma cortisol assays. This study was conducted at the California National Primate Research Center, Davis, CA. The C.N.P.R.C. is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. All procedures used in the present study were carried out in accordance with guidelines of the Public Health Service, USA.

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