Examination of the role of adrenergic receptor stimulation in the sensitization of neuroinflammatory-based depressive-like behavior in isolated Guinea pig pups.

Early-life attachment disruption appears to sensitize neuroinflammatory signaling to increase later vulnerability for stress-related mental disorders, including depression. How stress initiates this process is unknown, but studies with adult rats and mice suggest sympathetic nervous system activation and/or cortisol elevations during the early stress are key. Guinea pig pups isolated from their mothers exhibit an initial active behavioral phase characterized by anxiety-like vocalizing. This is followed by inflammatory-dependent depressive-like behavior and fever that sensitize on repeated isolation. Using strategies that have been successful in adult studies, we assessed whether sympathetic nervous system activity and cortisol contributed to the sensitization process in guinea pig pups. In Experiment 1, the adrenergic agonist ephedrine (3 or 10 mg/kg), either alone or with cortisol (2.5 mg/kg), did not increase depressive-like behavior or fever during initial isolation the following day as might have been expected to if this stimulation was sufficient to account for the sensitization process. In Experiment 2, both depressive-like behavior and fever sensitized with repeated isolation, but beta-adrenergic receptor blockade with propranolol (10 or 20 mg/kg) did not affect either of these responses or their sensitization. The high dose of propranolol did, however, reduce vocalizing. These results suggest sympathetic nervous system activation is neither necessary nor sufficient to induce the presumptive neuroinflammatory signaling underlying sensitization of depressive-like behavioral or febrile responses in developing guinea pigs. Thus, processes mediating sensitization of neuroinflammatory-based depressive-like behavior following early-life attachment disruption in this model appear to differ from those previously found to underlie neuroinflammatory priming in adults.

Sensitization of depressive-like behavior is attenuated by disruption of prostaglandin synthesis days following brief early attachment-figure isolation.

Childhood psychological trauma appears to sensitize stress-related neuroinflammatory systems to increase later vulnerability for depression and other stress-related mental disorders. Isolation of guinea pig pups from the maternal attachment figure for 3 h in threatening surroundings leads to a sensitization of inflammatory-mediated, depressive-like behavior and fever during later isolations. A previous study found the non-selective COX inhibitor naproxen administered before the initial isolation moderated depressive-like behavior and its sensitization. Here, we examined effects of naproxen given following early isolation. Male and female guinea pig pups surgically implanted with telemetry devices to measure core temperature were isolated for 3 h on 2 consecutive days near weaning (first isolation Day 20-24). Several days later, they began 4 consecutive days of injection with either saline vehicle or 10 or 20 mg/kg naproxen prior to a third isolation in early adolescence, that is, 10 days after their first isolation. Across the first two isolations, depressive-like behavior and fever sensitized. Both doses of naproxen attenuated depressive-like behavior during the third isolation. Fever was unaffected. Results suggest prostaglandin mediation of sensitization of depressive-like behavioral, but not febrile, responses to subsequent isolation. Findings also support further study of anti-inflammatory treatments to mitigate lasting consequences of early-attachment disruption.

Psychological Stress, Its Reduction, and Long-Term Consequences: What Studies with Laboratory Animals Might Teach Us about Life in the Dog Shelter.

There is a long history of laboratory studies of the physiological and behavioral effects of stress, its reduction, and the later psychological and behavioral consequences of unmitigated stress responses. Many of the stressors employed in these studies approximate the experience of dogs confined in an animal shelter. We review how the laboratory literature has guided our own work in describing the reactions of dogs to shelter housing and in helping formulate means of reducing their stress responses. Consistent with the social buffering literature in other species, human interaction has emerged as a key ingredient in moderating glucocorticoid stress responses of shelter dogs. We discuss variables that appear critical for effective use of human interaction procedures in the shelter as well as potential neural mechanisms underlying the glucocorticoid-reducing effect. We also describe recent studies in which enrichment centered on human interaction has been found to reduce aggressive responses in a temperament test used to determine suitability for adoption. Finally, we suggest that a critical aspect of the laboratory stress literature that has been underappreciated in studying shelter dogs is evidence for long-term behavioral consequences-often mediated by glucocorticoids-that may not become apparent until well after initial stress exposure.

Presence of mother prompts dissociation of sickness behavior, fever, and hypothalamic gene expression in lipopolysaccharide-injected guinea pig pups.

During infection, sickness behaviors, such as a hunched stance with piloerection, can facilitate host resistance by supporting the generation and maintenance of fever. Fever, in turn, is mediated by hypothalamic neuroimmune signaling. Sickness behaviors, however, can also be influenced by social stimuli. In this study, guinea pig pups were injected with lipopolysaccharide to simulate a bacterial infection and then exposed to a novel, threatening environment while either with their mother or alone. We found that the presence of the mother suppressed sickness behavior, but enhanced fever, and had no measureable effect on gene expression of hypothalamic mediators of fever. This 3-way dissociation induced by the mother's presence is interpreted in terms of the differential adaptive consequences of behavioral and febrile responses for pups in this situation. The results contribute to a growing literature linking immunological and social processes.

Central oxytocin alters cortisol and behavioral responses of guinea pig pups during isolation in a novel environment.

The neuropeptide oxytocin plays key roles in social bonding and stress reduction, and thus appears to be a likely mediator of maternal buffering of infant stress responses. In the guinea pig, the presence of the mother in a threatening environment buffers cortisol elevations as well as active (vocalizing) and passive (e.g. crouching) responses typical of isolation in this species; yet, effects of OT in guinea pig pups under any conditions have not been reported. Here, we examined the ability of intracerebroventricular (ICV) OT to moderate plasma cortisol levels and behavior in guinea pig pups isolated in a brightly lit, novel environment, and the ability of a highly selective OT antagonist (OTA) to reduce buffering by the mother. We found that ICV OT moderated cortisol levels and vocalizations, but increased time spent in the crouched stance, particularly in females. In addition, OT modulated other ongoing behaviors in a sex-dependent fashion. In females, OT reduced duration of walking and rearing, and increased time spent quiet, while in males OT increased duration of rearing. OTA, however, was without effect on cortisol levels or behavior. These findings, including sex differences in response, extend results from other species to the guinea pig. Further, while demonstrating that exogenous OT is sufficient to reduce biobehavioral stress responses typical of isolated guinea pig infants, the results suggest that endogenous OT is not necessary for maternal buffering of infant responses in this species.

Central neuroimmune activity and depressive-like behavior in response to repeated maternal separation and injection of LPS.

Disruption of attachment relations in early life is linked to greater vulnerability to depressive illness at later ages. Evidence suggests this process involves stress-induced activation of central inflammatory factors, though the specific mediators and processes involved are not known. We used a guinea pig model in which effects of maternal separation appear more clearly due to absence of the attachment figure than is the case for other laboratory rodents. Separation in a novel environment on two consecutive days evoked a depressive-like behavioral response that sensitized during a final test 9 days later. At this time, prior separation blunted the response of prostaglandin synthesizing enzymes (COX-2 and mPGES) and chemokines (CXCL-1 and MCP-1) 120 min following injection with lipopolysaccharide and isolation in a novel cage. The blunted response was not associated with a greater plasma cortisol elevation. In addition, injection of saline just prior to isolation at the oldest age elicited small, but significant, elevations in several signaling molecules, particularly at 30 min. These results demonstrate lasting central inflammatory consequences of our separation procedure. However, contrary to expectations, sensitization of depressive-like behavior was not associated with an increase in expression of neuroimmune mediators to inflammatory challenge. Together with earlier findings, the results suggest a multi-step process in which inflammatory response to an initial separation affects downstream mediators to sensitize depressive-like behavior.

Adult males buffer the cortisol response of young guinea pigs: Changes with age, mediation by behavior, and comparison with prefrontal activity.

In the guinea pig, the presence of the mother buffers hypothalamic-pituitary-adrenal (HPA) responses of her young during exposure to a novel environment, and can do so even if she is anesthetized. In contrast, under comparable conditions other conspecifics (siblings, other adult females) are less effective or ineffective in doing so. However, we recently observed that an unfamiliar adult male reduced plasma cortisol elevations and increased Fos in the prefrontal cortex of preweaning pups exposed to a novel enclosure for 120min. Here we found adult males buffered the adrenocortical response of preweaning pups at 60 as well as 120min and of periadolescent guinea pigs if exposure was of 120min. Further, because males vigorously engaged in social interactions with the young during exposure, we examined the effect of behavior by comparing the impact of conscious and unconscious (anesthetized) adult males. When tested with a conscious but not unconscious male, pups exhibited reduced plasma cortisol elevations. Pups, particularly females, had greater Fos induction in the prefrontal cortex when with a conscious versus unconscious adult male. Overall, we found that an unfamiliar adult male can buffer the cortisol response of guinea pigs both before and after weaning, though more-prolonged exposure appears necessary in the older animals. Further, unlike buffering by the biological mother, the effect of the male is mediated by behavioral interactions. Thus, the buffering of the infant guinea pig's cortisol response by the mother and an unfamiliar adult male involve different underlying mechanisms.

Early Attachment Disruption, Inflammation, and Vulnerability for Depression in Rodent and Primate Models.

Early experiments in nonhuman primates established the relation between disruption of filial attachment and depressive-like outcomes. Subsequent studies in rats and mice have been instrumental in linking depressive-like outcomes to disturbances in maternal behavior. Another aspect of attachment disruption, absence of the attachment object per se, may be studied more effectively in a different laboratory rodent-the guinea pig. Here, we discuss the rationale for using guinea pigs for this work. We then review guinea pig studies providing evidence for inflammatory mechanisms mediating both depressive-like behavior during separation as well as sensitization of stress responsiveness such as is thought to lead to increased vulnerability to depression at later ages. Finally, we discuss recent complementary work in adult monkeys that suggests cross-species generalizability of broad principles derived from the guinea pig experiments. Overall, the findings provide experimental support for human research implicating inflammatory mechanisms in the development of increased stress responsiveness and vulnerability to depression following attachment disruption and other forms of early-life stress. Specifically, the findings suggest inflammatory mechanisms may set in motion a cascade of underlying processes that mediate later increased stress responsiveness and, therefore, depression susceptibility.

Physiologic, Behavioral, and Histologic Responses to Various Euthanasia Methods in C57BL/6NTac Male Mice.

Rodent euthanasia using exposure to increasing concentrations of CO2 has come under scrutiny due to concerns of potential pain during the euthanasia process. Alternatives to CO2, such as isoflurane and barbiturates, have been proposed as more humane methods of euthanasia. In this study, we examined 3 commonly used euthanasia methods in mice: intraperitoneal injection of pentobarbital-phenytoin solution, CO2 inhalation, and isoflurane anesthesia followed by CO2 inhalation. We hypothesized that pentobarbital-phenytoin euthanasia would cause fewer alterations in cardiovascular response, result in less behavioral evidence of pain or stress, and produce lower elevations in ACTH than would the isoflurane and CO2 methods, which we hypothesized would not differ in regard to these parameters. ACTH data suggested that pentobarbital-phenytoin euthanasia may be less stressful to mice than are isoflurane and CO2 euthanasia. Cardiovascular, behavioral, and activity data did not consistently or significantly support isoflurane or pentobarbital-phenytoin euthanasia as less stressful methods than CO2. Euthanasia with CO2 was the fastest method of the 3 techniques. Therefore, we conclude that using CO2 with or without isoflurane is an acceptable euthanasia method. Pathologic alterations in the lungs were most severe with CO2 euthanasia, and alternative euthanasia techniques likely are better suited for studies that rely on analysis of the lungs.

Maternal separation increases later immobility during forced swim in guinea pig pups: evidence for sensitization of a depressive-like state.

Early-life stress is thought to increase later vulnerability for developing depressive illness by sensitizing underlying stress-responsive systems. Guinea pig pups separated from their mother and isolated in a novel cage for 3 hr exhibit a sensitized depressive-like behavioral response when separated again the following day as well as weeks later. The behavioral response and its sensitization appear to be mediated by inflammatory factors. To determine if this sensitization is specific to the separation response or if it reflects a broader underlying depressive-like state, guinea pig pups that had either been separated for 3 hr or remained with their mothers were observed in the forced swim test the following 3 days. Earlier separation was found to increase the duration of immobility, a measure sensitive to antidepressant treatment. These results support the use of the guinea pig as a model for examining mechanisms of inflammatory-mediated sensitization of depression following stress in early life.

Physiological, Behavioral, and Histological Responses of Male C57BL/6N Mice to Different CO2 Chamber Replacement Rates.

Rodent euthanasia with CO2 by using gradual displacement of 10% to 30% of the chamber volume per minute is considered acceptable by the AVMA Panel on Euthanasia. However, whether a 50% to 100% chamber replacement rate (CRR) of CO2 is more painful or distressful than 10% to 30% CRR is unclear. Therefore, we examined physiological and behavioral parameters, corticosterone and ACTH levels, and lung histology of mice euthanized at CRR of 15%, 30%, 50%, or 100%. Adult male C57BL/6N mice were euthanized at different CO2 CRR as physiological parameters were recorded telemetrically. Video recordings were reviewed to determine when the mouse first became ataxic, when it was fully recumbent (characterized by the mouse's nose resting on the cage floor), and when breathing stopped. Overall, CO2 euthanasia increased cardiovascular parameters and activity. Specific significant differences that were associated with 50% to 100% compared with 15% to 30% CO2 CRR included an increase in systolic blood pressure per second from initiation of CO2 until ataxia, a decrease in total diastolic blood pressure until ataxia, and a decrease in total heart rate until ataxia, immobility, and death. All physiological responses occurred more rapidly with higher CRR. Activity levels, behavioral responses, plasma adrenocorticotropic hormone and corticosterone levels, and lung pathology were not different between groups. We found no physiological, behavioral, or histologic evidence that 15% or 30% CO2 CRR is less painful or distressful than is 50% or 100% CO2 CRR. We conclude that 50% to 100% CO2 CRR is acceptable for euthanizing adult male C57BL/6N mice.

Effects of repeated petting sessions on leukocyte counts, intestinal parasite prevalence, and plasma cortisol concentration of dogs housed in a county animal shelter.

OBJECTIVE: To describe changes in WBC counts, plasma cortisol concentration, and fecal parasite shedding of dogs housed in an animal shelter and determine the effects of daily petting sessions on these variables. DESIGN: Hybrid prospective observational and experimental study. ANIMALS: 92 healthy dogs newly arrived to an animal shelter and 15 healthy privately owned dogs (control group). PROCEDURES: Blood and fecal samples were collected from shelter dogs 1, 3, and 10 days after arrival and from control dogs once. A subset of shelter dogs (n = 15) was assigned to receive 30 minutes of petting daily. Plasma cortisol concentration was measured, CBCs were performed, and fecal samples were evaluated for parasite ova. RESULTS: For shelter dogs, total leukocyte, neutrophil, and lymphocyte counts increased significantly between days 1 and 10, with less consistent increases in monocyte count and neutrophil-to-lymphocyte count ratio. Parasite shedding was unaffected by duration of shelter stay but was greater for shelter versus control dogs. For shelter dogs, plasma cortisol concentration decreased with time and was higher than that of control dogs on each day. Total leukocyte, neutrophil, and monocyte counts and neutrophil-to-lymphocyte count ratios were also higher for shelter versus control dogs. Petting sessions resulted in a decrease in plasma cortisol concentration but in no other variables. CONCLUSIONS AND CLINICAL RELEVANCE: Large increasing immunologic responses, heavy parasite shedding, and high but decreasing plasma cortisol concentration were identified in shelter dogs. Daily 30-minute petting sessions affected only cortisol values, so the clinical importance of petting for immunologic and other health outcomes remains unclear.

Naproxen attenuates sensitization of depressive-like behavior and fever during maternal separation.

Early life stress can increase susceptibility for later development of depressive illness though a process thought to involve inflammatory mediators. Isolated guinea pig pups exhibit a passive, depressive-like behavioral response and fever that appear mediated by proinflammatory activity, and which sensitize with repeated separations. Treatment with an anti-inflammatory can attenuate the behavioral response during the initial separation and separation the following day. Here we used the cyclooxygenase inhibitor naproxen to examine the role of prostaglandins in mediating the depressive-like behavior and core body temperature of young guinea pigs during an initial separation, separation the next day, and separation 10 days after the first. The passive, depressive-like behavior as well as fever sensitized with repeated separation. Three days of injection with 14 mg/kg of naproxen prior to the initial separation reduced depressive-like behavior during all three separations. A 28 mg/kg dose of naproxen, however, had minimal effect on behavior. Fever during the early separations was moderated by naproxen, but only at the higher dose. These results suggest a role of prostaglandins in the behavioral and febrile response to maternal separation, and particularly in the sensitization of depressive-like behavior following repeated separation.

Selective social buffering of behavioral and endocrine responses and Fos induction in the prelimbic cortex of infants exposed to a novel environment.

In mammals, the presence of the mother can reduce or "buffer" stress responses of her young in threatening conditions. We compared the effect of the mother, a familiar littermate, and an unfamiliar adult male on three classes of response shown by guinea pig pups in a novel environment: short latency active behaviors, particularly vocalizing; slower developing passive behaviors that appear mediated by inflammatory mechanisms; and hypothalamic-pituitary-adrenal activity. We also examined Fos induction in the prelimbic cortex, a region hypothesized to mediate buffering effects. Only the mother significantly suppressed all classes of behavior. The greatest selectivity was observed for passive behavioral responses. Contrary to expectations, the adult male reduced plasma cortisol levels of pups as effectively as did the mother. The presence of the male also resulted in increased Fos induction in the prelimbic cortex and high levels of social interaction. Maternal buffering was not associated with prelimbic activity. These results confirm the ability of the mother to reduce active behavioral and HPA responses and suggest a specific maternal buffering effect on the later developing passive behavioral responses. The findings also demonstrate an unexpected ability of adult males to reduce HPA responses and raise the possibility that different social partners buffer HPA activity through different underlying processes.

Sociality and sickness: have cytokines evolved to serve social functions beyond times of pathogen exposure?

During pathogen exposure or some forms of stress, proinflammatory processes induce an array of motivated and behavioral adjustments termed "sickness behaviors". Although withdrawal from social interactions is a commonly observed sickness behavior, the relation between social behavior and sickness is much more complex. Sickness can suppress or stimulate social behavior. Sickness can serve as a social cue. Stressors that are social in nature can induce sickness behaviors, and sickness behavior can be readily suppressed by meaningful social stimuli. The nature, context, and timing of these effects together suggest that cytokine-induced behavior may play a role in mediating social interactions in various non-pathological conditions.

Maternal inhibition of infant behavioral response following isolation in novel surroundings and inflammatory challenge.

During isolation in a novel environment, guinea pig pups gradually begin to display passive behavior that appears to be mediated by proinflammatory activity, that is, "sickness behavior.". Administration of substances that increase proinflammatory activity [corticotropin-releasing factor (CRF), lipopolysaccharide (LPS)] prior to isolation induces passive behavior from the beginning of the isolation episode. Here, we show that reunion with the mother in the novel environment rapidly and potently suppresses the passive behavior of isolated pups (Experiment 1); inhibits the passive behavior of pups administered CRF (10 µg, subcutaneous; Experiment 2); and inhibits the passive behavior of male, though not female, pups administered LPS (250 µg/kg, intraperitoneal; Experiment 3). Together these findings suggest that the presence of the mother either recruits other processes that moderate the impact of proinflammatory processes on brain mechanisms mediating the passive response or initiates compensatory mechanisms that counter the effect of proinflammatory activity. Further, the results suggest that for physically ill animals of social species, the adaptive advantage that accrues from maintaining normal social interactions may sometimes outweigh the advantage gained by engaging in sickness behavior.

Human interaction moderates plasma cortisol and behavioral responses of dogs to shelter housing.

Housing in an animal shelter is a stressful experience for dogs. This study examined the effects of different forms of human interaction on concentrations of circulating cortisol and stress-related behaviors of dogs within 40 h of admittance to a county animal shelter. Blood samples were collected before and after 30-min sessions in a secluded area in which dogs received one of three forms of human interaction: exposure to a passive human, petting, or play. Controls were either exposed to the secluded area alone, or remained in the general housing area. There was a substantial and near uniform reduction in plasma cortisol concentrations from pretest to post-test in all three conditions receiving human interaction, but not in control conditions. Human interaction also reduced behavioral signs of excitation/social solicitation (vocalizing) and fear (panting). Finally, pretest cortisol levels were found to predict levels of panting and another fear-related behavior (tongue protrusions) that dogs exhibited during subsequent testing. The findings suggest practical means of reducing stress, and potentially of improving the welfare, of dogs in shelters.

Persistent sensitization of depressive-like behavior and thermogenic response during maternal separation in pre- and post-weaning guinea pigs.

Early attachment disruption is thought to promote later onset of depressive illness through a process involving sensitization. Maternal separation in guinea pig pups produces depressive-like behavior and core body temperature fluctuations that appear to be mediated by proinflammatory activity. In pups near the age of weaning (~20 days of age), these responses are increased during repeated separations occurring over several days. Here, enhanced depressive-like behavior and core body temperature responses were observed during repeated separations in guinea pigs from ~10 to 30 days of age. The sensitization lasted for more than a week, with the greatest temperature response occurring during the final separation. These results demonstrate persisting sensitization of behavioral and thermogenic responses to maternal separation over the age range in which these responses are known to occur. The findings are consistent with the hypothesis that proinflammatory activity contributes to the sensitization response and provide further suggestion that the impact of early attachment disruption on susceptibility to depression may involve proinflammatory processes.

Sensitization of depressive-like behavior during repeated maternal separation is associated with more-rapid increase in core body temperature and reduced plasma cortisol levels.

Infant guinea pigs exhibit a 2-stage response to maternal separation: an initial active stage, characterized by vocalizing, and a second passive stage marked by depressive-like behavior (hunched posture, prolonged eye-closure, extensive piloerection) that appears to be mediated by proinflammatory activity. Recently we found that pups showed an enhanced (i.e., sensitized) depressive-like behavioral response during repeated separation. Further, core body temperature was higher during the beginning of a second separation compared to the first, suggesting a more-rapid stress-induced febrile response to separation the second day, though the possibility that temperature was already elevated prior to the second separation could not be ruled out. Therefore, the present study examined temperature prior to, and during, 2 daily separations. We also examined the temperature response to a third separation conducted 3 days after the second, and assessed the effect of repeated separation on plasma cortisol levels. Core temperature did not differ just prior to the separations, but showed a more-rapid increase and then decline during both a second and third separation than during a first. Temperature responses were not associated with changes in motor activity. Depressive-like behavior was greater during the second and third separations. Pups separated a first time showed a larger plasma cortisol response at the conclusion of separation than did animals of the same age separated a third time. In all, the results indicate that the sensitization of depressive-like behavior during repeated separations over several days is accompanied by a more-rapid febrile response that may be related to a reduction of glucocorticoid suppression.

Proinflammatory activity and the sensitization of depressive-like behavior during maternal separation.

When guinea pig pups are isolated for a few hours in a novel environment, they exhibit a distinctive passive behavioral response that appears to be mediated by proinflammatory activity. Recently, we observed that pups separated on two consecutive days show an enhanced (sensitized) passive response on the second day. In Experiment 1, pups receiving intracerebroventricular infusion of 50 ng of the anti-inflammatory cytokine interleukin-10 prior to a first separation failed to show a sensitized behavioral response to separation the next day. In Experiment 2, pups separated on Days 1 and 2, or just 2, showed an increase in passive responding during separation on Day 5. Pups injected with the bacterial antigen lipopolysacchride (LPS; 75 µg/kg body weight, intraperitoneal) prior to separation on Day 1 showed an increase in passive behavior several days later not shown by pups injected with saline prior to Day 1 separation. However, injection of LPS without separation on the first day did not enhance responding during an initial separation on the second day. These results suggest that immune activation is necessary, but not sufficient, to account for the sensitization of passive behavior of isolated guinea pig pups the following day, that boosting proinflammatory activity during an initial separation may promote sensitization several days later, and that the sensitized response persists for at least several days.