Loss of Intercalated Cells (ITCs) in the Mouse Amygdala of Tshz1 Mutants Correlates with Fear, Depression, and Social Interaction Phenotypes.

The intercalated cells (ITCs) of the amygdala have been shown to be critical regulatory components of amygdalar circuits, which control appropriate fear responses. Despite this, the molecular processes guiding ITC development remain poorly understood. Here we establish the zinc finger transcription factor Tshz1 as a marker of ITCs during their migration from the dorsal lateral ganglionic eminence through maturity. Using germline and conditional knock-out (cKO) mouse models, we show that Tshz1 is required for the proper migration and differentiation of ITCs. In the absence of Tshz1, migrating ITC precursors fail to settle in their stereotypical locations encapsulating the lateral amygdala and BLA. Furthermore, they display reductions in the ITC marker Foxp2 and ectopic persistence of the dorsal lateral ganglionic eminence marker Sp8. Tshz1 mutant ITCs show increased cell death at postnatal time points, leading to a dramatic reduction by 3 weeks of age. In line with this, Foxp2-null mutants also show a loss of ITCs at postnatal time points, suggesting that Foxp2 may function downstream of Tshz1 in the maintenance of ITCs. Behavioral analysis of male Tshz1 cKOs revealed defects in fear extinction as well as an increase in floating during the forced swim test, indicative of a depression-like phenotype. Moreover, Tshz1 cKOs display significantly impaired social interaction (i.e., increased passivity) regardless of partner genetics. Together, these results suggest that Tshz1 plays a critical role in the development of ITCs and that fear, depression-like and social behavioral deficits arise in their absence.SIGNIFICANCE STATEMENT We show here that the zinc finger transcription factor Tshz1 is expressed during development of the intercalated cells (ITCs) within the mouse amygdala. These neurons have previously been shown to play a crucial role in fear extinction. Tshz1 mouse mutants exhibit severely reduced numbers of ITCs as a result of abnormal migration, differentiation, and survival of these neurons. Furthermore, the loss of ITCs in mouse Tshz1 mutants correlates well with defects in fear extinction as well as the appearance of depression-like and abnormal social interaction behaviors reminiscent of depressive disorders observed in human patients with distal 18q deletions, including the Tshz1 locus.

Genetic Approaches to Hypothalamic-Pituitary-Adrenal Axis Regulation.

The normal function of the hypothalamic-pituitary-adrenal (HPA) axis, and resultant glucocorticoid (GC) secretion, is essential for human health. Disruption of GC regulation is associated with pathologic, psychological, and physiological disease states such as depression, post-traumatic stress disorder, hypertension, diabetes, and osteopenia, among others. As such, understanding the mechanisms by which HPA output is tightly regulated in its responses to environmental stressors and circadian cues has been an active area of investigation for decades. Over the last 20 years, however, advances in gene targeting and genome modification in rodent models have allowed the detailed dissection of roles for key molecular mediators and brain regions responsible for this control in vivo to emerge. Here, we summarize work done to elucidate the function of critical neuropeptide systems, GC-signaling targets, and inflammation-associated pathways in HPA axis regulation and behavior, and highlight areas for future investigation.

Ontogeny of hypothalamic glucocorticoid receptor-mediated inhibition of the hypothalamic-pituitary-adrenal axis in mice.

Glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN) are important regulators of negative feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Previous evaluation of endogenous PVN GR function in adult mice demonstrated that mice with loss of GR exon 3 in the PVN (Sim1Cre-GRe3Δ) have a hyperactive HPA axis, growth impairment and metabolic disruptions. Here, we hypothesized that lack of negative feedback inhibition of the HPA axis through PVN GR, as demonstrated through loss of PVN GR early in life, will have developmental-stage-specific consequences. Immunofluorescence revealed that Sim1Cre-GRe3Δ mice display PVN GR loss as early as post-natal day 2 compared to control mice. Sim1Cre-GRe3Δ mice compared to controls also displayed increased corticotropin-releasing hormone (CRH) mRNA in the PVN at post-natal day 10, as shown by in situ hybridization. Corticosterone radioimmunoassay revealed that the disruptions in PVN GR and CRH expression led to elevated basal corticosterone secretion in male Sim1Cre-GRe3Δ mice by early adolescence and increased stress-induced (restraint) corticosterone secretion in late adolescence into adulthood. In comparison, female Sim1Cre-GRe3Δ mice did not display corticosterone disruption until adulthood. Circadian rhythmicity of corticosterone secretion was normal for male and female mice at all age groups regardless of genotype with one exception. In late adolescence, female Sim1Cre-GRe3Δ mice had disrupted circadian corticosterone secretion due to significantly elevated circulating levels at nadir. We conclude that PVN GR function matures at an earlier developmental time point in male than in female mice and thus leads to later differential stress responsiveness between sexes.

Dissection of glucocorticoid receptor-mediated inhibition of the hypothalamic-pituitary-adrenal axis by gene targeting in mice.

Negative feedback regulation of glucocorticoid (GC) synthesis and secretion occurs through the function of glucocorticoid receptor (GR) at sites in the hypothalamic-pituitary-adrenal (HPA) axis, as well as in brain regions such as the hippocampus, prefrontal cortex, and sympathetic nervous system. This function of GRs in negative feedback coordinates basal glucocorticoid secretion and stress-induced increases in secretion that integrate GC production with the magnitude and duration of the stressor. This review describes the effects of GR loss along major sites of negative feedback including the entire brain, the paraventricular nucleus of the hypothalamus (PVN), and the pituitary. In genetic mouse models, we evaluate circadian regulation of the HPA axis, stress-stimulated neuroendocrine response and behavioral activity, as well as the integrated response of organism metabolism. Our analysis provides information on contributions of region-specific GR-mediated negative feedback to provide insight in understanding HPA axis dysregulation and the pathogenesis of psychiatric and metabolic disorders.

Site-specific modulation of brain glucocorticoid receptor and corticotropin-releasing hormone expression using lentiviral vectors.

The glucocorticoid receptor (GR) and corticotropin-releasing hormone (CRH) are important molecular regulators of an individual's ability to respond to stressful stimuli in an adaptive manner. Impaired signaling of both GR and CRH often leads to dysfunction of the hypothalamic-pituitary-adrenal axis, which underlies the etiology of many affective disorders such as anxiety and depression. Studies focusing on how GR and CRH influence the stress response are limited as they generalize to broad brain regions, thus hindering identification of how specific CNS nuclei contribute to maladaptive stress responses. Our objective is to distinguish the site-specific involvement of GR and CRH in limbic regions involved in the stress response. With that intent, we use lentiviral (LV) vectors in combination with transgenic mouse lines, enabling us to modify expression of GR or CRH in a very localized manner. This paper describes the generation of several distinct LV vectors and transgenic mice models that will help further elucidate the site-specific actions of GR and CRH.

Behavioral Studies and Genetic Alterations in Corticotropin-Releasing Hormone (CRH) Neurocircuitry: Insights into Human Psychiatric Disorders.

To maintain well-being, all organisms require the ability to re-establish homeostasis in the presence of adverse physiological or psychological experiences. The regulation of the hypothalamic-pituitary adrenal (HPA) axis during stress is important in preventing maladaptive responses that may increase susceptibility to affective disorders. Corticotropin-releasing hormone (CRH) is a central stress hormone in the HPA axis pathway and has been implicated in stress-induced psychiatric disorders, reproductive and cardiac function, as well as energy metabolism. In the context of psychiatric disorders, CRH dysfunction is associated with the occurrence of post-traumatic stress disorder, major depression, anorexia nervosa, and anxiety disorders. Here, we review the synthesis, molecular signaling and regulation, as well as synaptic activity of CRH. We go on to summarize studies of altered CRH signaling in mutant animal models. This assembled data demonstrate an important role for CRH in neuroendocrine, autonomic, and behavioral correlates of adaptation and maladaptation. Next, we present findings regarding human genetic polymorphisms in CRH pathway genes that are associated with stress and psychiatric disorders. Finally, we discuss a role for regulators of CRH activity as potential sites for therapeutic intervention aimed at treating maladaptive behaviors associated with stress.

Behavioral insights from mouse models of forebrain--and amygdala-specific glucocorticoid receptor genetic disruption.

Genetic modulation of glucocorticoid receptor (GR) function in the brain using transgenic and gene knockout mice has yielded important insights into many aspects of GR effects on behavior and neuroendocrine responses, but significant limitations regarding interpretation of region-specific and temporal requirements remain. Here, we summarize the behavioral phenotype associated with two knockout mouse models to define the role of GRs specifically within the forebrain and amygdala. We report that forebrain-specific GR knockout mice exhibit impaired negative feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis and increased despair- and anxiety-like behaviors. In addition, mice with a disruption of GR specifically within the central nucleus of the amygdala (CeA) are deficient in conditioned fear behavior. Overall, these models serve as beneficial tools to better understand the biology of GR signaling in the normal stress response and in mood disorders.

Pro-NGF, sortilin, and p75NTR: potential mediators of injury-induced apoptosis in the mouse dorsal root ganglion.

The nerve growth factor precursor (pro-NGF) may function as a death-inducing ligand that mediates its apoptotic effects via p75NTR. Pro-NGF-induced apoptosis is postulated to be dependent upon membrane expression of the sortilin receptor, which interacts with p75NTR to promote a high-affinity binding site for pro-NGF. Here, we explore the expression of pro-NGF, sortilin and p75NTR in the mouse lumbar dorsal root ganglion (DRG) to understand the potential for this trimeric signaling complex to function in injury-induced neuronal death of DRG neurons. Our results reveal the expression of all 3 components within the DRG and that a subpopulation of neurons coexpresses sortilin and p75NTR. Following sciatic nerve transection, the expression of these proteins appears insensitive to injury; however, the majority of small p75NTR-sortilin coexpressing neurons are lost 25 days after sciatic nerve transection. These results propose pro-NGF-induced, p75NTR-sortilin-mediated neuronal death as a critical aspect of nerve injury-induced death in the DRG.

Apoptosis: understanding the new molecular pathway.

Advances in science have increased the knowledge of how cells die in the body (apoptosis). A basic understanding of this process can improve nurses' ability to review new scientific literature and enable them to provide safer bedside care.