Adolescent Stress Confers Resilience to Traumatic Stress Later in Life: Role of the Prefrontal Cortex.

Background: Adolescent brains are sensitive to stressors. However, under certain circumstances, developmental stress can promote an adaptive phenotype, allowing individuals to cope better with adverse situations in adulthood, thereby contributing to resilience. Methods: Sprague Dawley rats (50 males, 48 females) were subjected to adolescent chronic variable stress (adol CVS) for 2 weeks at postnatal day 45. At postnatal day 85, a group was subjected to single prolonged stress (SPS). After a week, animals were evaluated in an auditory-cued fear conditioning paradigm, and neuronal recruitment during reinstatement was assessed by Fos expression. Patch clamp electrophysiology (17-35 cells/group) was performed in male rats to examine physiological changes associated with resilience. Results: Adol CVS blocked fear potentiation evoked by SPS. We observed that SPS impaired extinction (males) and enhanced reinstatement (both sexes) of the conditioned freezing response. Prior adol CVS prevented both effects. SPS effects were associated with a reduction of infralimbic (IL) cortex neuronal recruitment after reinstatement in males and increased engagement of the central amygdala in females, both also prevented by adol CVS, suggesting different neurocircuits involved in generating resilience between sexes. We explored the mechanism behind reduced IL recruitment in males by studying the intrinsic excitability of IL pyramidal neurons. SPS reduced excitability of IL neurons, and prior adol CVS prevented this effect. Conclusions: Our data indicate that adolescent stress can impart resilience to the effects of traumatic stress on neuroplasticity and behavior. Our data provide a mechanistic link behind developmental stress-induced behavioral resilience and prefrontal (IL) cortical excitability in males.

Ventral striatum dopamine release encodes unique properties of visual stimuli in mice.

The mesolimbic dopamine system is an evolutionarily conserved set of brain circuits that play a role in attention, appetitive behavior, and reward processing. In this circuitry, ascending dopaminergic projections from the ventral midbrain innervate targets throughout the limbic forebrain, such as the ventral striatum/nucleus accumbens (NAc). Dopaminergic signaling in the NAc has been widely studied for its role in behavioral reinforcement, reward prediction error encoding, and motivational salience. Less well characterized is the role of dopaminergic neurotransmission in the response to surprising or alerting sensory events. To address this, we used the genetically encoded dopamine sensor dLight1 and fiber photometry to explore the ability of striatal dopamine release to encode the properties of salient sensory stimuli in mice, such as threatening looming discs. Here, we report that lateral NAc (LNAc) dopamine release encodes the rate and magnitude of environmental luminance changes rather than the visual stimulus threat level. This encoding is highly sensitive, as LNAc dopamine could be evoked by light intensities that were imperceptible to human experimenters. We also found that light-evoked dopamine responses are wavelength-dependent at low irradiances, independent of the circadian cycle, robust to previous exposure history, and involve multiple phototransduction pathways. Thus, we have further elaborated the mesolimbic dopamine system's ability to encode visual information in mice, which is likely relevant to a wide body of scientists employing light sources or optical methods in behavioral research involving rodents.

Prefrontal cortex PACAP signaling: organization and role in stress regulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuromodulatory peptide strongly implicated in nervous stress processing. Human polymorphism of the primary PACAP receptor (PAC1) is linked to psychiatric disorders, including posttraumatic stress disorder (PTSD). Prefrontal cortex PACAP signaling is associated with processing of traumatic stress and fear learning, suggesting a potential role in PTSD-related deficits. We used RNAscope to define the cellular location of PACAP and PAC1 in the infralimbic cortex (IL). Subsequent experiments used a pharmacological approach to assess the impact of IL PACAP infusion on behavioral and physiological stress response and fear memory. Adult male Sprague-Dawley rats were bilaterally microinjected with PACAP (1 ug) or vehicle into the IL, 30 minutes prior to forced swim test (FST). Blood was sampled at 15, 30, 60, and 120 minutes for analysis of hypothalamic pituitary adrenal (HPA) axis reactivity. Five days after, animals were tested in a 3-day passive avoidance paradigm with subsequent testing of fear retention two weeks later. We observed that PACAP is highly expressed in putative pyramidal neurons (identified by VGlut1 expression), while PAC1 is enriched in interneurons (identified by GAD). Pretreatment with PACAP increased active coping style in the FST, despite higher levels of ACTH and corticosterone. The treatment was also sufficient to cause an increase in anxiety-like behavior in a dark/light crossover test and enhanced retention of passive avoidance. Our data suggest that IL PACAP plays a role in driving stress responses and in processing of fear memories, likely mediated by inhibition of cortical drive.

Brain mechanisms of HPA axis regulation: neurocircuitry and feedback in context Richard Kvetnansky lecture.

Regulation of stress reactivity is a fundamental priority of all organisms. Stress responses are critical for survival, yet can also cause physical and psychological damage. This review provides a synopsis of brain mechanisms designed to control physiological responses to stress, focusing primarily on glucocorticoid secretion via the hypothalamo-pituitary-adrenocortical (HPA) axis. The literature provides strong support for multi-faceted control of HPA axis responses, involving both direct and indirect actions at paraventricular nucleus (PVN) corticotropin releasing hormone neurons driving the secretory cascade. The PVN is directly excited by afferents from brainstem and hypothalamic circuits, likely relaying information on homeostatic challenge. Amygdala subnuclei drive HPA axis responses indirectly via disinhibition, mediated by GABAergic relays onto PVN-projecting neurons in the hypothalamus and bed nucleus of the stria terminalis (BST). Inhibition of stressor-evoked HPA axis responses is mediated by an elaborate network of glucocorticoid receptor (GR)-containing circuits, providing a distributed negative feedback signal that inhibits PVN neurons. Prefrontal and hippocampal neurons play a major role in HPA axis inhibition, again mediated by hypothalamic and BST GABAergic relays to the PVN. The complexity of the regulatory process suggests that information on stressors is integrated across functional disparate brain circuits prior to accessing the PVN, with regions such as the BST in prime position to relay contextual information provided by these sources into appropriate HPA activation. Dysregulation of the HPA in disease is likely a product of inappropriate checks and balances between excitatory and inhibitory inputs ultimately impacting PVN output.

Chemogenetic Inhibition of Infralimbic Prefrontal Cortex GABAergic Parvalbumin Interneurons Attenuates the Impact of Chronic Stress in Male Mice.

Hypofunction of the prefrontal cortex (PFC) contributes to stress-related neuropsychiatric illnesses. Mechanisms leading to prefrontal hypoactivity remain to be determined. Prior evidence suggests that chronic stress leads to an increase in activity of parvalbumin (PV) expressing GABAergic interneurons (INs) in the PFC. The purpose of the study was to determine whether reducing PV IN activity in the Infralimbic (IL) PFC would prevent stress-related phenotypes. We used a chemogenetic approach to inhibit IL PFC PV INs during stress. Mice were first tested in the tail suspension test (TST) to determine the impact of PV IN inhibition on behavioral responses to acute stress. The long-term impact of PV IN inhibition during a modified chronic variable stress (CVS) was tested in the forced swim test (FST). Acute PV IN inhibition reduced active (struggling) and increased passive coping behaviors (immobility) in the TST. In contrast, inhibition of PV INs during CVS increased active and reduced passive coping behaviors in the FST. Moreover, chronic inhibition of PV INs attenuated CVS-induced changes in Fos expression in the prelimbic cortex (PrL), basolateral amygdala (BLA), and ventrolateral periaqueductal gray (vlPAG) and also attenuated adrenal hypertrophy and body weight loss associated with chronic stress. Our results suggest differential roles of PV INs in acute versus chronic stress, indicative of distinct biological mechanisms underlying acute versus chronic stress responses. Our results also indicate a role for PV INs in driving chronic stress adaptation and support literature evidence suggesting cortical GABAergic INs as a therapeutic target in stress-related illnesses.

Lasting Impact of Chronic Adolescent Stress and Glucocorticoid Receptor Selective Modulation in Male and Female Rats.

Adolescent animals are vulnerable to the effects of stress on brain development. We hypothesized that long-term effects of adolescent chronic stress are mediated by glucocorticoid receptor (GR) signaling. We used a specific GR modulator (CORT108297) to pharmacologically disrupt GR signaling in adolescent rats during exposure to chronic variable stress (CVS). Male and female rats received 30 mg/kg of drug during a 2-week CVS protocol starting at PND46. Emotional reactivity (open field) and coping behaviors (forced swim test (FST)) were then tested in adulthood, 5 weeks after the end of the CVS protocol. Blood samples were collected two days before FST and serial samples after the onset of the swim test to determine baseline and stress response levels of HPA hormones respectively. Our results support differential behavioral, physiological and stress circuit reactivity to adolescent chronic stress exposure in males and females, with variable involvement of GR signaling. In response to adolescent stress, males had heightened reactivity to novelty and exhibited marked reduction in neuronal excitation following swim stress in adulthood, whereas females developed a passive coping strategy in the FST and enhanced HPA axis stress reactivity. Only the latter effect was attenuated by treatment with the GR modulator C108297. In summary, our data suggest that adolescent stress differentially affects emotional behavior and circuit development in males and females, and that GR manipulation during stress can reverse at least some of these effects.

Conditional deletion of glucocorticoid receptors in rat brain results in sex-specific deficits in fear and coping behaviors.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

Long-term impact of chronic variable stress in adolescence versus adulthood.

Adolescence is a period of active development of stress regulatory neurocircuitry. As a consequence, mechanisms that control the responses to stress are not fully matured during this developmental period, which may result in vulnerability to chronic stress. We hypothesized that adolescent chronic stress would have negative consequences on stress adaptation later in life. Male Wistar rats (PND40) were subjected to chronic variable stress (CVS) for 2 weeks, with 2 daily stressors randomly presented and overnight social stressors twice a week. After five weeks, animals were evaluated during adulthood, using the elevated plus maze (EPM) and the forced swim test (FST). The hypothalamic-pituitary adrenal (HPA) axis response to a 30-min restraint was also assessed. Results are compared to those of adult rats tested 5 weeks following CVS cessation. Our results demonstrate that the long-term effects of CVS are specific to the age of application of the stress regime. We show how behavior and HPA axis response as well as hypothalamic paraventricular nucleus activation can differ with age, resulting in differential behavioral adaptations for animals stressed in adolescence and dysregulation of the HPA axis in the animals stressed in adulthood, These data underscore the importance of the adolescent period in determining resilience of the HPA axis and programming behavioral responses later in life.

A double-hit model of stress dysregulation in rats: implications for limbic corticosteroid receptors and anxious behavior under amitriptyline treatment.

Adversity during early life can lead to diverging endocrine and behavioral responses to stress in adulthood. In our laboratory, we evaluated the long-term effects of early life adversity and its interaction with chronic stress during adulthood. We propose this as a model of vulnerability to dysregulation of the stress response. We hypothesized that rats subjected to both protocols would show differential expression of corticosteroid receptors measured as number of neurons immunoreactive for glucocorticoid receptors (GR) or mineralocorticoid receptors (MR), in limbic areas related to the control of anxiety-like behavior. We also evaluated the effect of amitriptyline expecting to prevent the outcomes of the model. Male Wistar rats were separated from the mother (MS) for 4.5 h every day for the first 3 weeks of life. From postnatal day 50, rats were subjected to chronic variable stress (CVS) during 24 d (five types of stressor at different times of day). During the stress protocol, the rats were administered amitriptyline (10 mg/kg i.p.) daily. MS evoked lower MR expression in the central amygdaloid nucleus and this was reversed by amitriptyline. Furthermore, CVS increased MR immunoreactivity in the hippocampal area CA2 and increased anxious behavior; both effects were prevented by the antidepressant. When MS was combined with CVS during adulthood, there was a reduction of locomotor activity, with no corrective effect of amitriptyline. The differential effects among groups could mean that MS would promote an alternative phenotype that is expressed when facing CVS (a double hit) later in life.

Amitriptyline treatment under chronic stress conditions: effect on circulating catecholamines and anxiety in early maternally separated rats.

The aim of this work was to determine the effect of amitriptyline (AMI) on peripheral outcomes such as plasma epinephrine (E) and norepinephrine (NE) concentration and anxiety-like behavior displayed in the plus maze test in adult male Wistar rats under variable chronic stress and daily oral administration of AMI (5 mg/kg). Animals were previously isolated from the mother for 4.5 hr every day for the first 3 weeks of life. Administration of the antidepressant AMI reduced anxiety-like behavior in animals submitted only to chronic stress but not in early maternally separated (MS) subjects or in animals subjected to the two types of stresses.