Immunohistochemical localization of oxytocin receptors in human brain.

The neuropeptide oxytocin (OT) regulates rodent, primate and human social behaviors and stress responses. OT binding studies employing (125)I-d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin ((125)I-OTA), has been used to locate and quantify OT receptors (OTRs) in numerous areas of the rat brain. This ligand has also been applied to locating OTRs in the human brain. The results of the latter studies, however, have been brought into question because of subsequent evidence that (125)I-OTA is much less selective for OTR vs. vasopressin receptors in the primate brain. Previously we used a monoclonal antibody directed toward a region of the human OTR to demonstrate selective immunostaining of cell bodies and fibers in the preoptic-anterior hypothalamic area and ventral septum of a cynomolgus monkey (Boccia et al., 2001). The present study employed the same monoclonal antibody to study the location of OTRs in tissue blocks containing cortical, limbic and brainstem areas dissected from fixed adult, human female brains. OTRs were visualized in discrete cell bodies and/or fibers in the central and basolateral regions of the amygdala, medial preoptic area (MPOA), anterior and ventromedial hypothalamus, olfactory nucleus, vertical limb of the diagonal band, ventrolateral septum, anterior cingulate and hypoglossal and solitary nuclei. OTR staining was not observed in the hippocampus (including CA2 and CA3), parietal cortex, raphe nucleus, nucleus ambiguus or pons. These results suggest that there are some similarities, but also important differences, in the locations of OTRs in human and rodent brains. Immunohistochemistry (IHC) utilizing a monoclonal antibody provides specific localization of OTRs in the human brain and thereby provides opportunity to further study OTR in human development and psychiatric conditions.

Maternal behavior deficits in nulliparous oxytocin knockout mice.

The first observations of postpartum oxytocin knockout (OTKO) mice found no maternal behavior deficits. However, it is unclear how detailed those observations were. In this study, we compared maternal behavior exhibited by OTKO and wild-type (WT) nullipara toward six 2-4-day-old foster pups during test sessions conducted on 3 successive days. Each day, subjects were placed in a clean cage 30 min prior to introduction of pups which were deposited in a clump adjacent to the middle of a long wall of each test cage. Behavior was measured for 3.5 h after which pups and test subjects were returned to their home cages. On test days 1 and 3, a significantly smaller proportion of OTKO females retrieved pups to a corner of their cage. Also, significantly fewer pups were retrieved to corners by OTKO females. In contrast to most WTs, most OTKO females mothered pups in the center of the cage where they were initially deposited. Pup-licking frequencies were significantly lower in OTKO females. Their self-grooming frequencies also trended toward being lower. Latencies to retrieve and lick pups, latencies to and frequencies of still crouching over pups and proportion of time in nest did not differ between groups. Our findings suggest that OT stimulates a significant proportion of pup-licking in nulliparous mice, a situation similar to lactating rat mothers. Our results also indicate that OT may play a role in the motivation to retrieve pups to a more secure location.

Vasopressin interactions with oxytocin in the control of female sexual behavior.

Previous studies have found that central administration of arginine vasopressin and arginine vasopressin receptor V1a antagonists respectively inhibited and stimulated receptivity but did not examine effects on other aspects of female sexual behavior. Central oxytocin facilitates both proceptive and receptive components of sexual behavior and diminishes male-directed agonistic behavior. The present study examined i.c.v.-administered arginine vasopressin and V1a antagonist effects on proceptive, receptive and agonistic behaviors, and interactions with oxytocin. In experiment 1, rats were primed s.c. with 2 microg estradiol benzoate x 2 days and with 500 microg of progesterone on day 3. Arginine vasopressin (0.2, 0.4 microg) or normal saline vehicle was administered 5 h after progesterone treatment and sexual and agonistic behavior measured 30, 60 and 90 min later. Compared with saline, both doses of arginine vasopressin significantly decreased lordosis responses to mounting and hop-dart proceptive behavior and trended toward significantly increasing agonistic behaviors. In experiment 2, oxytocin (2 microg) infusion just after arginine vasopressin (0.4 microg) significantly increased lordoses and decreased agonistic behaviors but did not affect hopping and darting. In experiment 3, conducted in ovariectomized rats primed with estradiol benzoate (1 microg/day s.c. x 2 days), i.c.v. infusion of 0.5 and 1.0 microg of the selective V1a antagonist, d(CH2)5Tyr-(Me)arginine vasopressin on day 3 significantly increased lordoses and trended toward increasing hopping and darting 4 and 6 h after i.c.v. treatment. In experiment 4, 1 microg of the selective oxytocin antagonist, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH2(9)]OVT given 1 h before d(CH2)5Tyr-(Me)arginine vasopressin (1 microg) significantly decreased lordoses. Previous studies indicate that arginine vasopressin contributes to light phase inhibition of female sexual behavior. Our findings suggest that arginine vasopressin may exert this effect through interactions that decrease oxytocin stimulation of sexual behavior and raise the question whether sex steroid conditions that stimulate sexual behavior may suppress central arginine vasopressin and V1a receptor activity.

Blink rate in boys with fragile X syndrome: preliminary evidence for altered dopamine function.

BACKGROUND: Dopamine, a neurotransmitter involved in motor and cognitive functioning, can be non-invasively measured via observation of spontaneous blink rates. Blink rates have been studied in a number of clinical conditions including schizophrenia, autism, Parkinsons, and attention deficit/hyperactivity disorder with results implicating either hyper or hypo dopaminergic states. METHODS: This study examined spontaneous blink rate in boys with fragile X syndrome (FXS). Blink rates of boys (4-8 years old) with FXS (n = 6) were compared with those of age-matched typically developing boys (n = 6) during active and passive tasks. Blink rates (blinks per minute) for each task were compared between the two groups. Then, the relation between blink measures and core FXS-related features [problem behaviours, arousal, fmr 1 protein (FMRP)] were examined within the group of boys with FXS. RESULTS: Blink rate in boys with FXS was significantly higher than typically developing boys during passive tasks. Within the FXS group, there were significant correlations between blink rate and problem behaviours and physiological arousal (i.e. heart activity) but not with FMRP. CONCLUSIONS: Observed differences in spontaneous blink rate between boys with and without FXS and the relation between blink rate and physiological and behavioural measures in boys with FXS suggests that further work examining dopamine dysfunction as a factor in the pathophysiology of FXS may be warranted.

Cardiovascular indices of physiological arousal in boys with fragile X syndrome.

In this study, the relationship between physiological arousal, as indexed by heart rate variability, was examined in boys with fragile X syndrome (FXS) and typically developing boys matched on chronological age. In addition, the relationship of heart activity to clinical and molecular factors in the group of boys with FXS was examined. Results suggest that boys with FXS have higher levels of heart activity during the passive phases, as reflected in shorter heart periods. This high level of heart activity appears to be due to increased sympathetic activity and reduced parasympathetic activity. Boys with FXS did not display the expected patterns of heart activity in response to phases of increasing challenge, and sympathetic and parasympathetic systems did not appear coordinated in these boys with FXS. Clinical factors may be related to neural regulation of heart activity while molecular factors do not appear to be.

Brief vs. long maternal separations in infancy: contrasting relationships with adult maternal behavior and lactation levels of aggression and anxiety.

We compared the effects of daily long (3 h), brief (15 min) or no maternal separation (LMS, BMS, NMS) on postnatal days 2-14 on maternal behavior, aggression and anxiety levels during lactation in adulthood. Animals subjected to LMS received less maternal grooming than animals subjected to BMS. Maternal behaviors, including nursing, pup-grooming (PG) frequency and proportion of total grooming (PG+self-grooming) and nest-building during the immediate postpartum period and on postpartum days 2 and 5 were lower in dams with LMS experience compared to dams with BMS experience. LMS dams attacked male rats placed in their home cages less quickly and less often than did BMS or NMS dams. LMS dams also exhibited more anxiety than BMS dams in the elevated plus maze test. Thus, maternal separation during the postnatal period (or associated changes in the amount of maternal grooming received) affected subsequent adult maternal behavior, aggression and anxiety. The mechanism for this remains to be discovered, however, it seems likely to involve alteration of the development of oxytocin receptors in the brain.

Oxytocin receptors in non-human primate brain visualized with monoclonal antibody.

The identity of putative oxytocin receptors visualized in autoradiographic studies of primate brain is unclear because the ligand used is much less selective in primate than rodent brains. This study tests the feasibility of utilizing a new monoclonal antibody (MoAb) developed against human uterine OTRs to visualize OTRs in primate brain. A block containing ventral hypothalamus of cynomolgus macaque brain, paraformaldehyde, glutaraldehyde-fixed and paraffin-embedded, and positive control tissue (human endometrium) were sectioned at 8 microm and studied with immunohistochemistry. OTRs were located in fibers in septal nucleus and in both cell bodies and fibers of preoptic area. These results indicate that OTRs in primate brain may be visualized with this MoAb, and are located in primate brain sites linked to the regulation of social behavior.

Behavior and autonomic nervous system function assessed via heart period measures: the case of hyperarousal in boys with fragile X syndrome.

Physiological responses may inform us about and help us to interpret behavioral responses. For example, hyperarousal may be a source of behavior problems in children with fragile X syndrome (FXS). To evaluate this approach, we examined heart period data in specific contexts in boys with FXS and in normally developing chronological-age-matched boys. Spectral analysis was used to evaluate the parasympathetic and sympathetic nervous systems' contributions to heart period. Boys with FXS had shorter interbeat intervals, lower parasympathetic activity, and similar sympathetic activity. Also, the groups were differentially responsive to experimental challenge. These results have important implications for our understanding of the basic nervous system dysfunction in FXS and for the strategies likely to be effective in terms of pharmacological intervention with these children. These methods can be applied to a variety of contexts and populations, including children who are sensory defensive, socially avoidant, inattentive, or hyperactive.

Juvenile friends, behavior, and immune responses to separation in bonnet macaque infants.

Individual differences in the response to maternal separation in nonhuman primate infants have been attributed to (among other variables) presence or absence of processes that may model social support in humans. Alternative attachments to other members of the social group buffer the infant against a depressive response to maternal separation. This hypothesis was tested in a group of bonnet macaques by manipulating the presence or absence of alternative juvenile attachment figures (friends) during separation. Infants who retained such attachments showed fewer behavioral evidences of depression when separated from their mothers. These infants without friends also showed changes in lymphocyte activation by mitogens or natural cytotoxicity that were not evident in the infants with juvenile friends. Across all separated infants, natural cytotoxicity was positively correlated with juvenile affiliative behavior directed toward the infants during the separation. These results support the hypothesis that social support, available from alternative attachments, can modulate the response to loss, and can account for some of the individual differences seen in these responses.

Natural cytotoxicity toward K562 cells by macaque lymphocytes from infancy through puberty: effects of early social challenge.

The consequences of a single maternal separation experience followed by reunion at 6-7 months of age was studied in socially housed pigtail (Macaca nemestrina) and bonnet (Macaca radiata) macaques. At 15 months, these subjects were removed from their natal group and placed in same species social groups, consisting of other separated and matched control subjects. Some subjects were followed until they reached an average age of 4 years while remaining in this group. Blood samples were drawn to permit assessment of the ability of peripheral blood lymphocytes to lyse, in vitro, K562 cells. Maternal separation failed to affect lysis of K562 cells acutely, although lysis by matched control subjects appeared to be transiently reduced 2 h after removal of the adult female. A longer-term influence was noted such that lysis of targets in previously separated subjects was greater than that in matched controls. Lysis rose over time regardless of species or experimental condition. A striking internal consistency in the lysis was also noted. Lysis was highly intercorrelated (r's > .60, p < or = .005) within subjects across time during baseline, separation, and reunion phases of the acute social challenge. In addition, there was a tendency for this correlation to hold over the longitudinal phases. Lysis of K562 targets by macaque lymphocytes would appear to possess trait-like stability; however, the range of lysis may be modified by early experiences.

Individual differences in macaques' responses to stressors based on social and physiological factors: implications for primate welfare and research outcomes.

Primates are used extensively in a variety of research settings. Federal regulations in the US mandate that caretakers provide for the 'psychological well-being of laboratory primates'. One of the difficulties in implementing this law has been both in the definition of psychological well-being and in the need to deal with each primate species and, in some cases, age or sex class, uniquely. Non-human primates exhibit distinct individual differences in their behavioural and physiological responses to experimental challenges and caretaking procedures. We have been investigating what factors can predict some of these individual differences, and have found that factors both intrinsic and extrinsic are significant. Extrinsic factors found to predict individual differences in response to stressors include the nature and prior experience with the challenge, the presence of familiar peers and availability of social support. Intrinsic factors include cognitive interpretations of the challenge and temperamental differences in reactivity. These studies highlight the importance of understanding the context and individual psychology of macaques in order to provide laboratory environments conducive to their welfare, and in order to understand the impact experimental and caretaking procedures are likely to have on the health and welfare of our subjects.

Total cortisol, free cortisol, and growth hormone associated with brief social separation experiences in young macaques.

Many behavioral, immunological, and physiological consequences or brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched nonseparated controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables.

Intrinsic and extrinsic factors affect infant responses to maternal separation.

Responses of individuals to the loss of a primary attachment object may be quite variable. In humans, it has been suggested that only about 25% of bereavements result in substantial psychological or medical morbidity (Hamburg et al. 1975). In nonhuman primates, which are used to model responses to separation and loss, a similar estimate of about 25% has also been obtained (McKinney 1985). In addition, there are wide-ranging species differences in vulnerability with regard to the nature and severity of the response to maternal separation and/or loss. All of these findings suggest that there are important processes, intrinsic and/or extrinsic to the individual, that contribute to the probability that a loss will produce a major behavioral or physiological response. We have been systematically examining some of the factors that may account for a portion of this variability in two species of macaques (bonnet monkeys Macaca radiata; and pigtail monkeys, M. nemestrina).

Immune responses following competitive water tests in two species of macaques.

A panel of immune parameters (lymphocyte activation by mitogens, natural cytotoxicity, and differential cell counts) was assessed in socially housed pigtail and bonnet macaques 1 and 2 weeks before, 48 h after, and 1 and 2 weeks after a competitive water test. Species differences were found in both baseline measures and the responses to the test: Immune measures observed during baseline periods were lower in pigtail macaques. Furthermore, only the pigtail macaques showed changes in mitogen activation and cytotoxicity at 48 h post-test. Dominance-related behaviors affected these responses both within and across social groups. The species differences may be accounted for by the differences in the behavioral responses of the two species to the test: Pigtail macaques consistently contested access to the water during the test, whereas bonnet macaques did not. These results suggest that the immune system can be modulated by psychosocial behavioral systems, particularly during times of stress.

Behavioral and immunological consequences of brief mother-infant separation: a species comparison.

The immediate behavioral and immunological consequences of a single 2-week maternal separation experience were studied in socially housed 7-month old bonnet and pigtail macaques. Maternal separation was associated with species dependent behavioral changes. Both species showed significant increases in ingestive behaviors associated with separation. Separated and matched controls showed an increase in disturbance behaviors (vocalization, startles, shaking, temper-tantrums) that subsided after 24-36 hours in control subjects, but continued, albeit with species-dependent patterns, in the separated monkeys. Allomaternal care of the separated bonnet infants but not the pigtail infants was associated with a progressive reduction of these disturbance behaviors during the separation period. Following a period of agitation, pigtail infants showed a depressive phase characterized by slouched, withdrawn postures and reduced motor activity. As a group, the separated infants of both species were not different from controls with respect to lymphocyte activation by mitogens, a measure of immunocompetence. However, when individual behavioral responses were considered, the change in lymphocyte activation during separation was significantly related to behavioral responses which reflected disturbance, such that the change in lymphocyte activation following in vitro stimulation with the mitogens phytohemagglutinin and Concanavalin A (markers of the immunocompetence of T lymphocytes) was related to levels of vocalization and time spent in slouched postures. The activation of B lymphocytes by pokeweed mitogen was not influenced by the separation experience nor was it associated with specific behavioral responses to separation. The importance of assessing the affective consequences of stressor is discussed.

Behavioral and autonomic responses to peer separation in pigtail macaque monkey infants.

Brief maternal separations of young nonhuman primates have been used extensively to study the behavior and physiology of attachment, loss, and bereavement. The physiological responses to the loss of alternative attachment figures, such as peers, is less well documented in nonhuman primates. This study examined both autonomic and behavioral responses of peer-reared pigtail macaque infants to separation. Eight infants were removed from their mothers at birth and reared in four peer pairs. At 6 months of age, each monkey was implanted with a multichannel biotelemetry device which transmitted heartrate, body temperature, EEG, EMG, and EOG. Blood was collected twice weekly for immunological assessment. Behavioral and physiological data, including sleep, were collected for 1 week of baseline, 2 weeks of separation, and 1 week of reunion. Behavioral and physiological results indicated agitation but not depression following separation from their peer attachment figures. We found reduced mitogenic responses to pokeweed consequent to peer separation, suggestive of altered B-cell function. REM variables were the only sleep measures affected by the separation, and were suggestive of agitation but not depression.

Comparison of the physical characteristics of grooming in two species of macaques (Macaca nemestrina and M. radiata).

Examined social grooming in groups of bonnet and pigtail macaques to test hypothesis that the physical aspects of grooming (body sites, postures, methods) evolved in more aggressive species of primates to serve social functions (proximity maintenance and tension reduction). Both species used social presents to direct grooming to particular sites, and used grooming to other sites to terminate interactions, thus regulating proximity with grooming to certain body sites. The 2 species differed in method of grooming. Pigtails primarily stroked (method used for tension reduction); bonnets picked or pick-stroked (method used for hygiene). Pigtail but not bonnet grooming method regulates tension reduction. Pigtails groomed in a social context, whereas bonnets groomed in solitary context. The pigtail results are similar to those found with rhesus monkeys. Different aspects of grooming serve different functions.

Maternal separation in bonnet monkey infants: altered attachment and social support.

Maternal separation in young pigtail (M. nemestrina) monkeys results in an agitation-depression reaction that is a useful animal model of the behavioral and physiological consequences and separation and loss experiences in humans. In this study, 5 social-group-living bonnet (M. radiata) monkey infants were separated from their mothers for 4 days, and behavioral, physiological, and sleep-pattern changes were monitored. Behavioral and physiological changes were consistent with an agitation reaction. There was evidence of depression in both behavioral and physiological measures; these changes were not as pronounced as in pigtail infants, however. All infants were adopted by another adult female during separation. Our findings suggest that, in bonnet monkey infants, adoption by a female within the social group ameliorates the response to loss.

On the physiology of grooming in a pigtail macaque.

Several functions of social grooming have been proposed, including building affiliative bonds between group members, and reducing tension or anxiety. In this case study, heart rate (HR) of a single adult female pigtail macaque living in a social group was monitored in several behavioral contexts to examine the relationship between grooming and HR level, an indicator of autonomic nervous system activity. The subject was implanted as a part of another study with a multichannel telemetry system which monitored several physiological variables. Physiological and behavioral data were collected over a nine day period. Heart rate during agonistic interactions was significantly higher than during other categories of behavior, and sleep HR significantly lower. When the subject received grooming from others, HR was significantly lower than during self grooming or initiating grooming to others, as well as during other behaviors. HR showed significant declines during bouts of receiving groom (up to 20 bpm), but not during bouts of initiating or self grooming. Following aggression, HR decline was greatest while receiving grooming. There were also HR differences for methods of grooming: stroking but not other methods related to decreases in HR.