Evan D. Paul

Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus.

The degree of behavioral control that an organism has over a stressor is a potent modulator of the stressors impact; uncontrollable stressors produce numerous outcomes that do not occur if the stressor is controllable. Research on controllability has focused on brainstem nuclei such as the dorsal raphe nucleus (DRN). Here we find that the infralimbic and prelimbic regions of the ventral medial prefrontal cortex (mPFCv) in rats detect whether a stressor is under the organisms control. When a stressor is controllable, stress-induced activation of the DRN is inhibited by the mPFCv, and the behavioral sequelae of uncontrollable stress are blocked. This suggests a new function for the mPFCv and implies that the presence of control inhibits stress-induced neural activity in brainstem nuclei, in contrast to the prevalent view that such activity is induced by a lack of control.

Previous experience with behavioral control over stress blocks the behavioral and dorsal raphe nucleus activating effects of later uncontrollable stress: role of the ventral medial prefrontal cortex.

Previous experience with stressors over which the subject has behavioral control blocks the typical behavioral consequences of subsequent exposure to stressors over which the organism has no behavioral control. The present experiments explored the involvement of the ventral medial prefrontal cortex (mPFCv) in mediating this “immunizing” or resilience producing effect of an initial experience with control. Behavioral immunization was blocked by inactivation of the mPFCv with muscimol at the time of the initial experience with control, as well as at the time of the later exposure to uncontrollable stress. Inhibition of protein synthesis within the mPFCv by anisomycin also blocked immunization when administered at the time of the initial controllable stress but had no effect when administered at the time of the later uncontrollable stress. Additional experiments found that the initial experience with control blocks the intense activation of serotonergic cells in the dorsal raphe nucleus that would normally be produced by uncontrollable stress, providing a mechanism for behavioral immunization. Furthermore, mPFCv activity during the initial controllable stressor was required for this effect to occur. These results suggest that the mPFCv is needed both to process information about the controllability of stressors and to utilize such information to regulate responses to subsequent stressors. Moreover, the mPFCv may be a site of storage or plasticity concerning controllability information. These results are consistent with recent research in other domains that explore the functions of the mPFCv.

5-Hydroxytryptamine 2C Receptors in the Basolateral Amygdala Are Involved in the Expression of Anxiety After Uncontrollable Traumatic Stress

BACKGROUND Exposure to uncontrollable stressors often increases anxiety-like behavior in both humans and rodents. In rat, this effect depends on stress-induced activity within the dorsal raphe nucleus (DRN). However, the role of serotonin in DRN projection regions is largely unknown. The goals of this study were to 1) assess the effect of uncontrollable stress on extracellular serotonin in the basolateral amygdala during the anxiety test, 2) determine whether DRN activity during a poststress anxiety test is involved in anxiety-like behavior, and 3) determine the role of the serotonin 2C receptor (5-HT(2C)) in uncontrollable stress-induced anxiety. METHOD Rats were exposed to tail shocks that were either controllable or uncontrollable. On the following day, anxiety-like behavior was assessed in a Juvenile Social Exploration (JSE) test. Basolateral amygdala (BLA) extracellular serotonin concentrations were assessed during JSE by in vivo microdialysis 24 hours after uncontrollable stress, controllable stress, or no stress. In separate experiments, drugs were administered before the JSE test to inhibit the DRN or to block 5-HT(2C) receptors. RESULTS Exposure to uncontrollable shock reduced later social exploration. Prior uncontrollable stress potentiated serotonin efflux in the BLA during social exploration, but controllable stress did not. Intra-DRN 8-OH-DPAT and systemic and intra-BLA 5-HT(2C) receptor antagonist SB 242,084 prevented the expression of potentiated anxiety in uncontrollably stressed rats. Intra-BLA injection of the 5-HT(2C) agonist CP 809,101 mimicked the effect of stress. CONCLUSIONS These results suggest that the anxiety-like behavior observed after uncontrollable stress is mediated by exaggerated 5-HT acting at BLA 5-HT(2C) receptors.

Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala.

The intra dorsal raphe nucleus (DRN) administration of corticotropin releasing hormone (CRF) inhibits serotonergic (5-HT) activity in this structure, an effect blocked by antagonists selective for the type 1 CRF receptor (CRF1). The DRN has a high density of the type 2 receptor (CRF2), and so the present experiments explored the impact of CRF2 activation within the DRN on 5-HT function. The intra-DRN administration of the selective CRF2 agonist urocortin 2 (Ucn 2) dose dependently increased 5-HT efflux in the basolateral amygdala, a projection region of the DRN. Intra-DRN Ucn 2 also increased c-fos expression in labeled 5-HT neurons. Both of these effects of Ucn 2 were completely blocked by intra-DRN antisauvagine-30 (ASV-30), a relatively selective CRF2 antagonist. These data suggest that CRF1 and CRF2 activation within the DRN affect 5-HT neurons in opponent fashion. Implications of these results for understanding the behavioral effects of CRF and other CRF-like ligands are discussed.

The role of prior stressor controllability and the dorsal raphé nucleus in sucrose preference and social exploration

Research investigating how control over stressors affects behavior often utilizes freezing and shuttle escape learning as the behavioral endpoints. These endpoints have been argued to reflect anxious or depressed states, but these descriptions are problematic. The present study sought to determine the impact of stressor controllability and the dorsal raphé nucleus (DRN) on sucrose preference and juvenile social exploration, putative measures of anhedonia and anxiety that are commonly used in studies of stress per se. In Experiment 1 rats were exposed to escapable stress (ES) or yoked-inescapable stress (IS) tailshocks. In Experiment 2 ES or IS was given 7 days before all rats received IS. In Experiment 3 the DRN was inactivated during IS by microinjection of 8-OH-DPAT. Sucrose preference and social exploration were tested for several days after stress. A fourth experiment confirmed that juvenile social exploration is sensitive to traditional beta-carboline and benzodiazepine manipulations. Both ES and IS reduced sucrose preference, but only IS reduced social exploration. Prior treatment with ES prevented the effect of IS on social exploration but did not prevent the effect of IS on sucrose preference and inactivation of the DRN prevented the effect of IS on social exploration but did not change sucrose preference. The present results indicate that social exploration but not sucrose preference is sensitive to prior stressor controllability, and that DRN activation mediates the effect of IS on social exploration. We argue that DRN-5-HT activation mediates a state of generalized anxiety produced by uncontrollable stress and that juvenile social exploration is a useful behavioral endpoint in stressor controllability studies.

Functional topography of serotonergic systems supports the Deakin/Graeff hypothesis of anxiety and affective disorders

Over 20 years ago, Deakin and Graeff hypothesized about the role of different serotonergic pathways in controlling the behavioral and physiologic responses to aversive stimuli, and how compromise of these pathways could lead to specific symptoms of anxiety and affective disorders. A growing body of evidence suggests these serotonergic pathways arise from topographically organized subpopulations of serotonergic neurons located in the dorsal and median raphe nuclei. We argue that serotonergic neurons in the dorsal/caudal parts of the dorsal raphe nucleus project to forebrain limbic regions involved in stress/conflict anxiety-related processes, which may be relevant for anxiety and affective disorders. Serotonergic neurons in the “lateral wings” of the dorsal raphe nucleus provide inhibitory control over structures controlling fight-or-flight responses. Dysfunction of this pathway could be relevant for panic disorder. Finally, serotonergic neurons in the median raphe nucleus, and the developmentally and functionally-related interfascicular part of the dorsal raphe nucleus, give rise to forebrain limbic projections that are involved in tolerance and coping with aversive stimuli, which could be important for affective disorders like depression. Elucidating the mechanisms through which stress activates these topographically and functionally distinct serotonergic pathways, and how dysfunction of these pathways leads to symptoms of neuropsychiatric disorders, may lead to the development of novel approaches to both the prevention and treatment of anxiety and affective disorders.

The sensory insular cortex mediates the stress-buffering effects of safety signals but not behavioral control

Safety signals are learned cues that predict stress-free periods whereas behavioral control is the ability to modify a stressor by behavioral actions. Both serve to attenuate the effects of stressors such as uncontrollable shocks. Internal and external cues produced by a controlling behavior are followed by a stressor-free interval, and so it is possible that safety learning is fundamental to the effect of control. If this is the case then behavioral control and safety should recruit the same neural machinery. Interestingly, safety signals that prevented a behavioral outcome of stressor exposure that is also blocked by control (reduced social exploration) failed to inhibit activity in the dorsal raphé nucleus or use the ventromedial prefrontal cortex, the mechanisms by which behavioral control operates. However, bilateral lesions to a region of posterior insular cortex, termed the “sensory insula,” prevented the effect of safety but not of behavioral control, providing a double-dissociation. These results indicate that stressor-modulators can recruit distinct neural circuitry and imply a critical role of the sensory insula in safety learning.

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.

MicroRNA-19b Associates with Ago2 in the Amygdala Following Chronic Stress and Regulates the Adrenergic Receptor Beta 1

Activation of the stress response in the presence of diverse challenges requires numerous adaptive molecular and cellular changes. To identify specific microRNA molecules that are altered following chronic stress, mice were subjected to the chronic social defeat procedure. The amygdala from these mice was collected and a screen for microRNAs that were recruited to the RNA-induced silencing complex and differentially expressed between the stressed and unstressed mice was conducted. One of the microRNAs that were significantly altered was microRNA-19b (miR-19b). Bioinformatics analysis revealed the adrenergic receptor β-1 (Adrb1) as a potential target for this microRNA with multiple conserved seed sites. Consistent with its putative regulation by miR-19b, Adrb1 levels were reduced in the basolateral amygdala (BLA) following chronic stress. In vitro studies using luciferase assays showed a direct effect of miR-19b on Adrb1 levels, which were not evident when miR-19b seed sequences at the Adrb1 transcript were mutated. To assess the role of miR-19b in memory stabilization, previously attributed to BLA-Adrb1, we constructed lentiviruses designed to overexpress or knockdown miR-19b. Interestingly, adult mice injected bilaterally with miR-19b into the BLA showed lower freezing time relative to control in the cue fear conditioning test, and deregulation of noradrenergic circuits, consistent with downregulation of Adrb1 levels. Knockdown of endogenous BLA-miR-19b levels resulted in opposite behavioral and noradrenergic profile with higher freezing time and increase 3-methoxy-4-hydroxyphenylglycol/noradrenaline ratio. These findings suggest a key role for miR-19b in modulating behavioral responses to chronic stress and Adrb1 as an important target of miR-19b in stress-linked brain regions.

Chronic Activation of Corticotropin-Releasing Factor Type 2 Receptors Reveals a Key Role for 5-HT1A Receptor Responsiveness in Mediating Behavioral and Serotonergic Responses to Stressful Challenge

Background The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation. Methods Transgenic mice overexpressing the highly specific CRFR2 ligand urocortin 3 (UCN3OE) were analyzed for stress-related behaviors and hypothalamic-pituitary-adrenal axis responses. Responses to 5-HT receptor agonist challenge were assessed by local cerebral glucose utilization, while 5-HT and 5-hydroxyindoleacetic acid content were quantified in limbic brain regions. Results Mice overexpressing urocortin 3 exhibited increased stress-related behaviors under basal conditions and impaired retention of spatial memory compared with control mice. Following acute stress, unlike control mice, they exhibited no further increase in these stress-related behaviors and showed an attenuated adrenocorticotropic hormone response. 5-HT and 5-hydroxyindoleacetic acid content of limbic nuclei were differentially regulated by stress in UCN3OE mice as compared with control mice. Responses to 5-HT type 1A receptor challenge were significantly and specifically reduced in UCN3OE mice. The distribution pattern of local cerebral glucose utilization and 5-HT type 1A receptor messenger RNA expression levels suggested this effect was mediated in the raphé nuclei. Conclusions Chronic activation of CRFR2 promotes an anxiety-like state, yet with attenuated behavioral and hypothalamic-pituitary-adrenal axis responses to stress. This is reminiscent of stress-related atypical psychiatric syndromes such as posttraumatic stress disorder, chronic fatigue, and chronic pain states. This new understanding indicates CRFR2 antagonism as a potential novel therapeutic target for such disorders.