Jodi L. Lukkes

Adult rats exposed to early-life social isolation exhibit increased anxiety and conditioned fear behavior, and altered hormonal stress responses.

Social isolation of rodents during development is thought to be a relevant model of early-life chronic stress. We investigated the effects of early-life social isolation on later adult fear and anxiety behavior, and on corticosterone stress responses, in male rats. On postnatal day 21, male rats were either housed in isolation or in groups of 3 for a 3 week period, after which, all rats were group-reared for an additional 2 weeks. After the 5-week treatment, adult rats were examined for conditioned fear, open field anxiety-like behavior, social interaction behavior and corticosterone responses to restraint stress. Isolates exhibited increased anxiety-like behaviors in a brightly-lit open field during the first 10 min of the test period compared to group-reared rats. Isolation-reared rats also showed increased fear behavior and reduced social contact in a social interaction test, and a transient increase in fear behavior to a conditioned stimulus that predicted foot-shock. Isolation-reared rats showed similar restraint-induced increases in plasma corticosterone as group-reared controls, but plasma corticosterone levels 2 h after restraint were significantly lower than pre-stress levels in isolates. Overall, this study shows that isolation restricted to an early part of development increases anxiety-like and fear behaviors in adulthood, and also results in depressed levels of plasma corticosterone following restraint stress.

Consequences of post-weaning social isolation on anxiety behavior and related neural circuits in rodents

Exposure to adverse experiences in early-life is implicated in the later vulnerability to development of psychiatric disorders, including anxiety and affective disorders in humans. Adverse early-life experiences likely impart their long-term consequences on mental health by disrupting the normal development of neural systems involved in stress responses, emotional behavior and emotional states. Neural systems utilizing the neurotransmitters serotonin, dopamine and the neuropeptide corticotropin-releasing factor (CRF) are implicated in mediating emotive behaviors, and dysfunction of these neurochemical systems is associated with mood/anxiety disorders. These neural systems continue maturing until early or mid-adolescence in humans, thus alterations to their development are likely to contribute to the long-term consequences of adverse early-life experiences. A large body of literature suggests that post-weaning isolation rearing of rodents models the behavioral consequences of adverse early-life experiences in humans. Overall, the majority findings suggest that post-weaning social isolation that encompasses pre-adolescence produces long-lasting alterations to anxiety behavior, while measures of monoaminergic activity in various limbic regions during social isolation suggest alterations to dopamine and serotonin systems. The goal of this review is to evaluate and integrate findings from post-weaning social isolation studies specifically related to altered fear and anxiety behaviors and associated changes in neuroendocrine function and the activity of monoaminergic systems.

Does Serotonin Influence Aggression? Comparing Regional Activity before and during Social Interaction*

Serotonin is widely believed to exert inhibitory control over aggressive behavior and intent. In addition, a number of studies of fish, reptiles, and mammals, including the lizard Anolis carolinensis, have demonstrated that serotonergic activity is stimulated by aggressive social interaction in both dominant and subordinate males. As serotonergic activity does not appear to inhibit agonistic behavior during combative social interaction, we investigated the possibility that the negative correlation between serotonergic activity and aggression exists before aggressive behavior begins. To do this, putatively dominant and more aggressive males were determined by their speed overcoming stress (latency to feeding after capture) and their celerity to court females. Serotonergic activities before aggression are differentiated by social rank in a region‐specific manner. Among aggressive males baseline serotonergic activity is lower in the septum, nucleus accumbens, striatum, medial amygdala, anterior hypothalamus, raphe, and locus ceruleus but not in the hippocampus, lateral amygdala, preoptic area, substantia nigra, or ventral tegmental area. However, in regions such as the nucleus accumbens, where low serotonergic activity may help promote aggression, agonistic behavior also stimulates the greatest rise in serotonergic activity among the most aggressive males, most likely as a result of the stress associated with social interaction.

Corticotropin-Releasing Factor Receptor Antagonism within the Dorsal Raphe Nucleus Reduces Social Anxiety-Like Behavior after Early-Life Social Isolation

Social isolation of rats during the early part of development increases social anxiety-like behavior in adulthood. Furthermore, early-life social isolation increases the levels of corticotropin-releasing factor (CRF) receptors in the serotonergic dorsal raphe nucleus (dRN) of adult rats. Interactions between serotonin and CRF systems are thought to mediate anxiety behavior. Therefore, we investigated the effects of CRF receptor antagonism within the dRN on social anxiety-like behavior after early-life social isolation. Male rats were reared in isolation or in groups from weaning until midadolescence, and rehoused in groups and allowed to develop into adulthood. Adult rats underwent surgery to implant a drug cannula into the dRN. After recovery from surgery and acclimation to the testing arena, rats were infused with vehicle or the CRF receptor antagonist d-Phe-CRF(12-41) (50 or 500 ng) into the dRN before a social interaction test. Isolation-reared rats pretreated with vehicle exhibited increased social anxiety-like behavior compared with rats reared in groups. Pretreatment of the dRN with d-Phe-CRF(12-41) significantly reduced social anxiety-like behaviors exhibited by isolation-reared rats. Overall, this study shows that early-life social stress results in heightened social anxiety-like behavior, which is reversed by CRF antagonism within the dRN. These data suggest that CRF receptor antagonists could provide a potential treatment of stress-related social anxiety.

Repeated social defeat increases reactive emotional coping behavior and alters functional responses in serotonergic neurons in the rat dorsal raphe nucleus

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.

Early life social isolation alters corticotropin-releasing factor responses in adult rats

Stress induced by early life social isolation leads to long-lasting alterations in stress responses and serotonergic activity. Corticotropin-releasing factor (CRF) is a neurotransmitter that mediates stress responses and alters serotonergic activity. We tested the hypothesis that the stress of early life isolation enhances responses to CRF in adulthood by determining the effect of CRF infusions into the dorsal raphe nucleus (dRN) on 5-HT release in the nucleus accumbens (NAc) of adult rats using in vivo microdialysis. Juvenile male rats were either isolated or housed in groups of three for a 3-week period beginning on postnatal day 21 after which, all rats were group-reared for an additional 2 weeks. Following the isolation/re-socialization procedure, infusion of 100 ng CRF into the dRN decreased 5-HT release in the NAc of group-reared rats. This treatment did not significantly affect 5-HT release in the NAc of isolation-reared animals. In contrast, infusion of 500 ng CRF into the dRN transiently increased 5-HT release in the NAc of both group-reared and isolated animals with isolated animals showing a more prolonged serotonergic response. Western blot and immunofluorescent staining for CRF receptors in the dRN showed that CRF(2) receptor levels were increased in the dRN of isolation-reared animals when compared with group-reared rats. Taken together, the results suggest that isolation during the early part of development causes alterations in both CRF receptor levels and CRF-mediated serotonergic activity. These effects may underlie the increased sensitivity to stress observed in isolates.

Investigation of a central nucleus of the amygdala/dorsal raphe nucleus serotonergic circuit implicated in fear-potentiated startle

Serotonergic systems are thought to play an important role in control of motor activity and emotional states. We used a fear-potentiated startle paradigm to investigate the effects of a motor-eliciting stimulus in the presence or absence of induction of an acute fear state on serotonergic neurons in the dorsal raphe nucleus (DR) and cells in subdivisions of the central amygdaloid nucleus (CE), a structure that plays an important role in fear responses, using induction of the protein product of the immediate-early gene, c-Fos. In Experiment 1 we investigated the effects of fear conditioning training, by training rats to associate a light cue (conditioned stimulus, CS; 1000 lx, 2 s) with foot shock (0.5 s, 0.5 mA) in a single session. In Experiment 2 rats were given two training sessions identical to Experiment 1 on days 1 and 2, then tested in one of four conditions on day 3: (1) placement in the training context without exposure to either the CS or acoustic startle (AS), (2) exposure to 10 trials of the 2 s CS, (3) exposure to 40 110 dB AS trials, or (4) exposure to 40 110 dB AS trials with 10 of the trials preceded by and co-terminating with the CS. All treatments were conducted during a 20 min session. Fear conditioning training, by itself, increased c-Fos expression in multiple subdivisions of the CE and throughout the DR. In contrast, fear-potentiated startle selectively increased c-Fos expression in the medial subdivision of the CE and in serotonergic neurons in the dorsal part of the dorsal raphe nucleus (DRD). These data are consistent with previous studies demonstrating that fear-related stimuli selectively activate DRD serotonergic neurons. Further studies of this mesolimbocortical serotonergic system could have important implications for understanding mechanisms underlying vulnerability to stress-related psychiatric disorders, including anxiety and affective disorders.

Early Life Adversity Alters the Developmental Profiles of Addiction-Related Prefrontal Cortex Circuitry

Early adverse experience is a well-known risk factor for addictive behaviors later in life. Drug addiction typically manifests during adolescence in parallel with the later-developing prefrontal cortex (PFC). While it has been shown that dopaminergic modulation within the PFC is involved in addiction-like behaviors, little is known about how early adversity modulates its development. Here, we report that maternal separation stress (4 h per day between postnatal days 2–20) alters the development of the prelimbic PFC. Immunofluorescence and confocal microscopy revealed differences between maternally-separated and control rats in dopamine D1 and D2 receptor expression during adolescence, and specifically the expression of these receptors on projection neurons. In control animals, D1 and D2 receptors were transiently increased on all glutamatergic projection neurons, as well as specifically on PFC→nucleus accumbens projection neurons (identified with retrograde tracer). Maternal separation exacerbated the adolescent peak in D1 expression and blunted the adolescent peak in D2 expression on projection neurons overall. However, neurons retrogradely traced from the accumbens expressed lower levels of D1 during adolescence after maternal separation, compared to controls. Our findings reveal microcircuitry-specific changes caused by early life adversity that could help explain heightened vulnerability to drug addiction during adolescence.

Topographical distribution of corticotropin-releasing factor type 2 receptor-like immunoreactivity in the rat dorsal raphe nucleus: co-localization with tryptophan hydroxylase

Corticotropin-releasing factor (CRF) and CRF-related neuropeptides are involved in the regulation of stress-related physiology and behavior. Members of the CRF family of neuropeptides bind to two known receptors, the CRF type 1 (CRF₁) receptor, and the CRF type 2 (CRF₂) receptor. Although the distribution of CRF₂ receptor mRNA expression has been extensively studied, the distribution of CRF₂ receptor protein has not been characterized. An area of the brain known to contain high levels of CRF₂ receptor mRNA expression and CRF₂ receptor binding is the dorsal raphe nucleus (DR). In the present study we investigated in detail the distribution of CRF₂ receptor immunoreactivity throughout the rostrocaudal extent of the DR. CRF₂ receptor-immunoreactive perikarya were observed throughout the DR, with the highest number and density in the mid-rostrocaudal DR. Dual immunofluorescence revealed that CRF₂ receptor immunoreactivity was frequently co-localized with tryptophan hydroxylase, a marker of serotonergic neurons. This study provides evidence that CRF₂ receptor protein is expressed in the DR, and that CRF₂ receptors are expressed in topographically organized subpopulations of cells in the DR, including serotonergic neurons. Furthermore, these data are consistent with the hypothesis that CRF₂ receptors play an important role in the regulation of stress-related physiology and behavior through actions on serotonergic and non-serotonergic neurons within the DR.

Amphetamine treatment increases corticotropin-releasing factor receptors in the dorsal raphe nucleus.

Psychostimulant use increases anxious behavior, likely through interactions between central corticotropin-releasing factor (CRF) and serotonergic systems. The current study examined whether chronic amphetamine treatment (2.5mg/kg, 14 days) or withdrawal altered CRF receptor densities in the serotonergic dorsal raphe nucleus (dRN). Amphetamine treatment increased CRF(2) receptor densities in most subregions of the dRN, and CRF(2) receptors were still elevated following 6 weeks of withdrawal. No changes in CRF(1) receptor densities were observed following amphetamine treatment or during withdrawal. Selective increases in dRN CRF(2) receptors may be related to increased anxiety-like behaviors following psychostimulant use.