Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 105-134).

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains xiv, 223 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 187-220).

Indiana University-Purdue University Indianapolis (IUPUI) / Anxiety disorders are estimated to impact 1 in 4 individuals within their lifetime. For some individuals, repeated episodes of the stress response leads to pathological anxiety and depression. The stress response is linked to increased levels of corticotropin-releasing factor (CRF) in the basolateral nucleus of the amygdala (BLA), a putative site for regulating anxiety and associative processes related to aversive emotional memories, and activation of CRF receptors in the BLA of rats produces anxiety-like behavior. Mimicking repeated episodes of the stress response, sub-anxiogenic doses of urocortin 1 (Ucn1), a CRF receptor agonist, are microinjected into the BLA of rats for five consecutive days, a procedure called priming. This results in 1) behavioral sensitization, such that a previously non-efficacious dose of Ucn1 will elicit anxiety-like response after the 3rd injection and 2) the development of a persistent anxiety-like phenotype that lasts at least five weeks after the last injection without any further treatment. Therefore, the purpose of this thesis was to identify mechanisms involved in the Ucn1-priming-induced anxiogenesis. The first a set of experiments revealed that the anxiety-like behavior was not due to aversive conditioning to the context or partner cues of the testing environment. Next, Ucn1-priming-induced gene expression changes in the BLA were identified: mRNA expression for Sst2, Sst4, Chrna4, Chrma4, and Gabrr1 was significantly reduced in Ucn1-primed compared to Vehicle-primed rats. Of these, Sst2 emerged as the primary receptor of interest. Subsequent studies found that antagonizing the Sstr2 resulted in anxiety-like behavior and activation of Sstr2 blocked acute Ucn1-induced anxiety-like responses. Furthermore, pretreatment with a Sstr2 agonist delayed the behavioral sensitization observed in Ucn1-induced priming but did not stop the development of persistent anxiety-like behavior or the Ucn1-priming-induced decrease in the Sstr2 mRNA. These results suggest that the decrease in Sstr2 mRNA is associated with the expression of persistent anxiety-like behavior but dissociated from the mechanisms causing the behavioral sensitization. Pharmacological studies confirmed that a reduced Sstr2 mediated effect in the BLA is likely to play a role in persistent anxiety and should be investigated further.

Lors d’un stress, l’activation de l’axe hypothalamo-hypophyso-surrénalien (HHS) conduit à une augmentation de la production de glucocorticoïdes (tel que la corticostérone) par les glandes surrénales. Le rôle de la corticotropin-releasing hormone (CRH), à l’origine de l’activation de l’axe HHS, est encore méconnu. En effet, les récepteurs à la CRH sont présents aussi bien au niveau du système nerveux central (SNC), notamment au niveau du cervelet, qu’au niveau du système immunitaire (SI). Cela suggère donc une action directe possible de cette hormone sur ces deux systèmes. Au cours de ce projet, nous avons étudié les régulations des SNC et SI lors d’un stress, et plus particulièrement le rôle de la CRH et de ses récepteurs dans ces régulations. Suite à des injections chroniques de corticostérone, mimant un stress, nous avons observé une altération des fonctions locomotrices qui semble être reversée lorsque le CRH-R1 est inhibé avec un antagoniste. Ces premiers résultats permettent de mettre en avant un éventuel rôle de la CRH dans la régulation des fonctions motrices au niveau du cervelet en conditions de stress. En parallèle, d’autres études in vitro réalisées sur des splénocytes murins stimulés avec de la CRH ont montré une diminution de la viabilité des lymphocytes B (LB). Suite à ces résultats, nous avons caractérisé pour la première fois la présence de récepteurs à la CRH sur cette population de LB murins. Ces résultats montrent l’importance de la CRH dans les régulations des SNC et SI en condition de stress et le rôle de cette hormone dans les interactions entre les deux systèmes / In stress conditions, the Hypothalamo-Pituitary-Adrenal (HPA) axis activation leads to an overproduction of glucocorticoïds (such as corticosterone in rodent) by adrenal glands and this activation is well characterized. However, various questions remain about the precise role of corticotropin-releasing hormone (CRH), which is at the beginning of the HPA activation. Indeed, CRH receptors are presents both in central nervous system (CNS), especially in cerebellum, and in immune system (IS). This suggest a possible direct action of this hormone on both system. In this project, we studied the regulations on CNS and IS in stress conditions and more particularly the CRH role and these receptors in these regulations. After chronic corticsterone injections, to mimic a stress, we observed a locomotor alteration which seems to be inverted when CRH-R1 were inhibited with an antagonist. These first results show an possible CRH role in locomotor regulation in cerebellum under stress condition. In parallel, others in vitro studies performed on murine splenocytes stimulated with CRH showed a B lymphocyte (LB) viability decrease. Furthermore, we are the first to characterise the CRH receptors on murine LB. This work show the CRH importance in CNS and IS regulations under stress conditions and its role in interactions between the two systems

Anxiety is an affective state defined by heightened arousal and unease in the absence of a clear and present fear-inducing stimulus. Chronic and inappropriate anxiety leads to anxiety disorders, the most common class of human mental disorder. Recent work suggests projections to the ventral tegmental area (VTA), are critical for anxiety behavior expression. However, the relationship between efferent VTA projections and anxiety is unclear. This thesis resolves anxiety circuitry connecting the dopaminergic (DAergic) VTA to the interpeduncular nucleus (IPN), coined the mesointerpeduncular circuit. I hypothesize the mesointerpeduncular circuit affects anxiety through the release of anxiogenic corticotropin releasing factor (CRF) during nicotine withdrawal and anxiolytic dopamine (DA) during drug naïve behavior. Electrophysiological and pharmacological data suggest CRF release from the DAergic VTA during nicotine withdrawal activates CRF receptor 1 (CRFR1) potentiating the glutamatergic activation of “Type 2” neurons and anxiety-like behavior in mice. However, in nicotine naïve conditions CRF production is negligible. Instead, in vivo DA release is anticorrelated with anxiety-like behaviors. Optogenetic stimulation and inhibition drives decreased and increased anxiety-like behaviors, respectively. Electrophysiological experiments reveal a complex interpeduncular microcircuit where D1-like DA receptor expressing “Type C” neurons in the caudal IPN (cIPN) regulate glutamatergic release in the ventral IPN (vIPN) through presynaptic GABA receptors. The result is propagation of the signal to excite “Type A” and inhibit “Type B” vIPN neurons. Finally, pharmacological activation or inhibition of interpeduncular D1-like DA receptors is sufficient to decrease and increase anxiety-like behaviors respectively. Thus, this circuit is important for modulating anxiety-like behavior.

Leung Tse Ngong. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 169-197). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

The stress response is an evolutionarily conserved mechanism critical for survival that requires orchestration of different systems in the body. Corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) represent the final common pathway leading to HPA axis activation in response to stress. Noradrenergic inputs to CRH neurons in the PVN provide a powerful drive to activate the HPA axis. Previous anatomical studies have shown that noradrenergic afferents synapse directly on CRH neurons, but electrophysiological analyses indicate that the noradrenergic activation of CRH neurons is mediated primarily by the stimulation of presynaptic glutamatergic neurons. Here, using whole cell patch clamp recordings in identified CRH neurons, I demonstrate that norepinephrine (NE) stimulates excitatory synaptic inputs by activating postsynaptic α1 adrenergic receptors in CRH neurons and inducing the release of the retrograde messenger nitric oxide, which drives upstream glutamate neurons to elicit spike-dependent synaptic glutamate release onto the CRH neurons. Notably, the NE effect is dependent on ATP transmission and astrocytic function, suggesting that astrocytes serve as an intermediary in the retrograde activation of glutamateregic synaptic inputs to the CRH neurons. In addition, I also show that the NE-induced excitation of CRH neurons is stress-status sensitive and corticosterone dependent, in that stress-induced corticosterone causes internalization of membrane α1 adrenergic receptors to desensitize the CRH neurons to NE. Taken together, my findings provide evidence that NE excites CRH neurons in a stress state-dependent manner by a retrograde NO stimulation of local glutamate circuits that is dependent on glial activation. This retrograde trans-neuronal-glial regulation of excitatory synaptic inputs to CRH neurons by NE provides a mechanism for the NE activation of the HPA axis in the early stage of stress response. The stress-/corticosterone-induced desensitization of CRH neurons to NE modulation by the internalization of α1 adrenergic receptors confers a stress state-dependent resistance of the CRH neurons to repeated noradrenergic activation, which provides a mechanism for the negative feedback regulation of the CRH neurons and the HPA axis by stress and glucocorticoids, and a means to restore neuroendocrine homeostasis after stress exposure. / acase@tulane.edu

Corticotropin-releasing factor (CRF) is the primary hormone of the hypothalamo-pituitary adrenal axis (HPA-axis). In addition to its endocrine function, it has been proposed that CRF acts as a neurotransmitter. The widespread distribution of CRF immunoreactivity and CRF receptors in the rat central nervous system (CNS) supports this theory. Immunohistochemical studies have demonstrated high levels of CRF immunoreactivity the rat hypothalamus, a brain region involved in the regulation and integration of a variety of endocrine and autonomic homeostatic mechanisms. CRF has been shown to be involved in a number of these activities such as blood pressure control, food and water intake, behaviour and emotional integration. Many of these activities demonstrate progressive dysfunction as ageing proceeds. The aim of this thesis was to investigate the regulation of CRF in the rat CNS, particularly over the period of maturation and ageing. Tissue extraction and peptide radioimmunoassay (RIA) techniques were developed in order to measure regional CRF concentrations as a function of age in the rat CNS. Seven brain regions were examined including the hypothalamus, pituitary, medulla oblongata, pons, cerebral cortex, cerebellum and midbrain. Three age ranges were investigated: 3 – 4 weeks, 4 – 5 months and 14 – 18 months, representing young, mature and old age groups. Data for the tissues of individual rats from each age group were analysed using one-way analysis of variance (ANOVA) with post-hoc Scheffé tests (SPSS Release 6 for Windows, 1989 – 1993). CRF were detected in measurable quantities in all brain regions examined. Different age-related patterns of change were observed in each brain region. CRF concentrations (ng/g tissue) were highest in the pituitaries of young rats and were significantly reduced over the period of maturation (P< 0.05). However, the high CRF concentration of the young rat pituitary was likely to be a factor of the smaller tissue mass. Although the absolute CRF content (ng/tissue) of this tissue appeared to decline with maturation and ageing, the reduction was not significant (P>0.05). Therefore the pituitary of the young rat was relatively enriched with CRF per gram tissue. The highest CRF concentration in mature and aged rats was measured in the hypothalamus, in accordance with previous immunohistochemical studies. Hypothalamic CRF concentrations (ng/g tissue) demonstrated no significant alterations with maturation and ageing. The absolute CRF content (ng/tissue) of the hypothalamus was significantly less in the young rat compared to mature and aged animals, however this was accompanied by a smaller tissue mass (P<0.05). The CRF concentrations (ng/g tissue) of the rat cerebral cortex and medulla oblongata demonstrated significant reduction with advancing age (P<0.05), however in both cases this appeared to be due to significant increases in mean tissue mass. The absolute CRF content of these tissues (ng/tissue) were not significantly different over the period of maturation and ageing (P>0.05). CRF concentration (ng/g tissue) and absolute content (ng/tissue) of the pons demonstrated a trend to increase with advanced age in the rat, however this was not significant in both cases (P>0.05). Of interest were the significant increases observed in the CRF concentrations of the cerebellum and midbrain (ng/g tissue with advanced ageing (P<0.05). Significant increases were also observed in the mean tissue mass and absolute CRF content (ng/tissue) of these regions in aged rats (P<0.05). These findings perhaps indicate increased CRF synthesis and or decreased CRF turnover in these tissues with advancing age. The second stage of these studies examined age-related alterations in basal and potassium-stimulated hypothalamic CRF and overflow over the period of maturation and ageing in the rat, and required the preliminary development of an in vitro tissue superfusion system. The concomitant release of the co-modulatory compound, neuropeptide Y (NPY) was also measured. NPY has been shown to positively regulate CRF release and gene expression in the hypothalamus. In addition, NPY has been demonstrated to be involved in a number of hypothalamic activities, including blood pressure control and food intake regulation. Hypothalamic superfusion data were analysed using one factor repeated measures ANOVA (SPSS Release 6 for Windows, 1989-1993) followed by least significant difference tests ( Snedecor and Cochran, 1967) to enable both time and age comparisons. Basal hypothalamic CRF overflow was unaltered with maturation and ageing in the rat. Potassium stimulation (56 mM) elicted a significant 2 – 3 fold increase in hypothalamic CRF overflow across age groups (P<0.05). Stimulated hypothalamic CRF overflow was significantly greater in the young rat compared to the mature and aged animals (P<0.05). The enhanced response to depolarizing stimulus was observed at an age when the absolute CRF content of the hypothalamus was significantly less that of other age groups. It is possible that the enhanced responsiveness of the young rat may be of survival advantage in life threatening situations. Basal hypothalamic NPY overflow was much less than that of CRF, and potassium stimulation resulted in a very different age-related profile. The hypothalamic NPY response to depolarization was significantly reduced in the young rat and declined significantly with advanced ageing (P<0.05). The contrasting profiles of stimulated CRF and NPY overflow may indicate the activity of alternative regulatory factors present in the hypothalamus, whose activity may also be affected in an age-related manner. The final stage of these studies examined the nature of NPY modulation of hypothalamic CRF overflow in the mature rat. The facilitatory effect of NPY on hypothalamic CRF overflow was confirmed. The application of NPY (0.1 µM) significantly increased CRF overflow approximately 4 fold of basal (P<0.05). In addition, the role of the NPY-Y1 receptor was investigated by the prior application of Y1 receptor antagonists, GW1229 (0.05 µM). At this concentration GW1229 significantly reduced hypothalamic CRF overflow induced by perfusion with NPY (0.1 µm), P<0.05. It was concluded the Y1 receptor does have a role in the regulation of hypothalamic CRF overflow by NPY.

The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing hormone (CRH) are involved in the hypothalamic-pituitary-interrenal (HPI) axis. During stress, the HPI axis is activated and cortisol is released into the blood. In addition to their role in the HPI axis, AVT and CRH also have behavioural effects. The roles of AVT and CRH in stress responses and agonistic behaviour were studied in this thesis, using two different models. In the first model, two strains of rainbow trout (Onchorhynchus mykiss) divergent in stress-induced release of cortisol were investigated. This was done by observing behaviour and stress responses under different conditions. These strains were found to have divergent stress coping strategies based on the observed behaviour and levels of plasma cortisol. This divergence in behaviour could be associated with the CRH system, since the mRNA levels of CRH differed between the strains during stress. However, no differences between strains were observed in AVT or its receptor expressions. In the second model, non-selected rainbow trout were paired and the effect of intracerebroventricular (icv) injections of an active substance (AVT, CRH or the CRH related peptide Urotensin-I (UI)) on fights for dominance was investigated. One fish of the pair received the active substance icv and the other received saline icv. Fish receiving AVT became subordinate in accordance with the suggestion that AVT attenuates aggression in territorial vertebrates. Fish receiving CRH became subordinate whereas UI showed no effect on fights for dominance. Further, both CRH and UI induced an anxiety-related behaviour similar to non-ambulatory motor activity in rats. In addition, CRH appeared to affect the dopaminergic and serotonergic systems. In this thesis, it is suggested that CRH is involved in the behavioural modulation of the stress coping strategies in teleost fish. Further, AVT and CRH seem to act inhibitory on aggressive behaviour.

The neuropeptide, corticotropin-releasing factor (CRF), plays a critical role in the central regulation of various stress-related behaviours, including those unique to subjects with prior cocaine experience. The three series of experiments presented in this dissertation explored the role of two neurochemical systems, the teneurin C-terminal associated peptides (TCAP) and the endocannabinoids (eCBs), in several cocaine- and anxiety-related behaviours induced or mediated by CRF. The first series of experiments examined the effects of TCAP-1 on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization. Repeated (5-day), but not acute, TCAP-1 treatment blocked the reinstatement of cocaine seeking induced by central injections of CRF. TCAP-1 was, however, without effect on footshock- or cocaine-induced reinstatement. Repeated TCAP-1 further interfered with the expression of behavioural sensitization to a CRF, but not a cocaine, challenge. These findings suggest that TCAP-1 normalizes CRF signaling dysregulated by cocaine exposure to interfere in the subsequent effects of CRF on cocaine-related behaviours. A parallel series of experiments investigated the role of eCB signaling at CB1 receptors in the reinstatement of cocaine seeking and cocaine-sensitized locomotion. Pretreatment with the CB1 receptor antagonist, AM251, selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 further blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine. These findings reveal a mediating role for CB1 receptor transmission in the effects of CRF on cocaine-related behaviours. A final series of experiments examined the role of CB1 receptor transmission in the behavioural anxiety induced by central injections of CRF, and by withdrawal from chronic cocaine exposure. AM251, although itself anxiogenic, reversed anxiety induced by CRF and cocaine withdrawal. Furthermore, AM251 elevated plasma corticosterone levels, indicative of increased HPA axis activity, irrespective of CRF treatment or cocaine withdrawal. These findings suggest that CRF- and cocaine withdrawal-induced anxiety are mediated, at least in part, by CB1 receptor transmission, independent of HPA axis regulation. The collective findings are discussed within a framework of CRF-TCAP-eCB interactions, wherein TCAP-1 and AM251 are proposed to act in parallel to modulate amygdalar CRF transmission, and thus regulate the expression of cocaine- and anxiety-related behaviours.