Although many signalling molecules appear relevant to the production of complex behaviours, those that are important to the physiological regulation of behaviour, and so those that characterise individual styles of behaviour, are unknown. Vasopressin is the strongest candidate regulator of social behaviour. Experiments were carried out in consideration that vasopressin may directly regulate aggressive behaviour in lactating rats. Patterns of immediate early gene expression during/subsequent to aggressive behaviour suggested specific neural circuits may have significant direct regulatory influence over particular behaviours, and that activation of the V1b vasopressin receptor, in these circuits, may contribute to this putative regulatory signalling. In situ hybridisation studies indicated that patterns of vasopressin release, rather than receptor expression, might be important for any peripartum changes in behaviour driven by vasopressin. Although their relative importance is unknown, central actions of vasopressin may exert a strong regulatory influence over a range of behaviours, across a range of species.

Maternal behaviour during the peri-partum period, albeit in differing forms, can be observed in all mammals, thus it must serve an important evolutionary purpose in enabling the successful raising of offspring. Maternal behaviour is comprised of a large suite of behaviours; in rodents these are generally defined as lactation, pup retrieval, maternal aggression and pup grooming. The maternal behaviour circuitry involves many brain regions including the hypothalamus and the limbic system which work together to regulate the motor, motivational and emotional demands of the lactation period. The main aim of this thesis is to understand how different neuromodulators, specifically oxytocin (OXT), vasopressin (AVP), allopregnanolone (AP) and GABA, influence the expression of maternal behaviour, especially maternal aggression, and where in the brain they act to control this. Maternal aggression in rats changes dramatically throughout pregnancy, parturition and lactation. This expression is highly influenced by pups and during early lactation, pup cues are essential in maintaining it. Towards the end of lactation pup cues appear to result in the down regulation of maternal aggression. The maternal aggression circuitry is highly complex and involves many of the brain regions highlighted to be involved in maternal behaviour. The neuropeptides, OXT and AVP, are observed to have significant changes in their systems that correlate with maternal aggression, specifically within the BnST and PVN. This leads to the proposal they work oppositely to control maternal aggression by regulating fear and anxiety in the lactating rat. There is also evidence the OXT system mediates the motor output of maternal aggression. AP and GABA are also important in maternal behaviour, especially in relation to fear; whether this in context with OXT to enable maternal aggression or if they are a back up mechanism for OXT secretion malfunctioning remains to be determined. By understanding the complex maternal behaviour neural circuitry and how neuromodulators work to control it, enables the development of potential therapies for disorders a woman may experience during the peri-partum period. Prevention of these disorders is not only beneficial to the mother and her immediate family but is also crucial for her offspring’s development in prevention of adulthood disorders stemming from their childhood experience which can impact their own paternal or maternal care ability.

The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.

No description available.

No description available.

Anxiety disorders are the most prevalent of all psychiatric conditions. However, current pharmacological treatments for anxiety disorders are characterized by one or more of the following deficiencies: 1) unwanted side effects, 2) partial efficacy, 3) addictive potential, and 4) delayed onset of therapeutic effects. These therapeutic liabilities motivate the search for better pharmacological treatments. This research effort has been concentrated in three broad, neuropharmacological domains: 1) Sub-unit specific GABAA receptor agonists, 2) Neurosteroids, and 3) Neuropeptides. The general purpose of this thesis was to advance our understanding of the putative anxiolytic potential of neurosteroids and neuropeptides, and their neural mechanisms of action, as revealed by intracerebral infusion studies in animal models of anxiety. Chapter 1 of this thesis will provide a systematic review of what is now known about the behavioral effects of intra-cerebrally infused agonists and antagonists of anxiolytic compounds in animal models of anxiety. A theoretical context in which to view the empirical work is also outlined. Chapter 2 will provide a brief introduction to neurosteroids and neuropeptides, and their potential as anxiolytic drugs as suggested by the current literature. In Chapter 3, the anxiolytic-like effects of the neurosteroid allopregnanolone were examined in the amygdala, the hippocampus or the medial prefrontal cortex. Allopregnanolone had site- and test-specific anxiolytic effects, causing anxiolysis following infusion into the amygdala and the medial prefrontal cortex. In Chapter 4, the anxiety-related effects of two receptor antagonists of the neuropeptide arginine vasopressin were investigated in the hippocampus. Anxiolytic effects were specific to both receptor sub-type and by infusion site. In chapter 5, the putative anxiolytic and antidepressant effects of the neuropeptide somatostatin were investigated. Intracerebroventricular microinfusion of somatostatin produced anxiolytic-like and antidepressant-like signatures in distinct domains. In chapter 6, selective agonists for each of the 5 G-protein coupled somatostatin receptors were administered to rats. Intracerebroventricular administration of an sst2 agonist produced anxiolytic-like effects, whereas an antidepressant-like effect was observed following the administration of both sst2 and sst3 agonists. In summary, the present thesis provides important clues to the neurochemical correlates of anxiety, and its potential treatment with alternative compounds such as neuropeptides.

The work reported in this thesis is an investigation of certain aspects of both the blood pressure (BP) elevating properties and BP lowering properties of arginine vasopressin (AVP). The hypothesis that endothelin (ET) contributes to the exaggerated pressor responsiveness of the spontaneously hypertensive rat (SHR) to AVP was tested by comparing the changes of BP, cardiac output (CO), and total peripheral conductance (TPC) to AVP in SHR to those in Wistar-Kyoto rats (WKY) both in the presence and absence of bosentan, a non-selective ET antagonist. Bosentan antagonized the BP responses to exogenous ET-1 in a competitive fashion. The pressor effects of AVP and Ang II were exaggerated in the SHR compared to WKY. Except for the highest dose of AVP, pre-treatment with bosentan blunted the increases in BP and the decreases in total peripheral conductance (TPC) evoked by AVP in the SHR, but not in the WKY. In contrast to AVP, bosentan blunted the increases in BP evoked by lower doses of Ang II in both strains, although the effect was more pronounced in the SHR. These results suggest that ET contributes to the hemodynamic effects of AVP in the SHR and to the effects of Ang II in both strains. The findings support the hypothesis that ET contributes to the exaggerated pressor responsiveness of SHR to AVP. Cessation of a 3 hour infusion of AVP (20 ng/kg/min) results in a dramatic and prolonged decrease in BP below pre-infusion basal levels in hypertensive rats, but not in normotensive control rats. This phenomenon has been termed the "withdrawal-induced antihypertensive phenomenon" (WAP). In order to determine the time course of the WAP, and the role of CO and TPC in the WAP, BP was recorded by radiotelemetry and CO was recorded from aonic flowprobes in conscious unrestrained rats before, during, and after a 3 hr i.v. infusion of 20 ng/kg/min of AVP. Baseline mean arterial BP values were lower, and the magnitude of the WAP was less in SHR when BP was recorded with radiotelemetric implants than in another group in which BP was recorded with conventional externalized femoral arterial catheters. Strikingly, absolute BP values recorded both during and after the AVP infusion were similar in the two groups. BP remained decreased for several days in SHR infused with AVP with complete recovery requiring 6-7 days. In rats instrumented with aortic flow probes, the fall in pressure following cessation of the AVP infusion was associated with a large decrease in CO below control levels in the SHR. The time-course of the CO responses approximated the time-course of the pressure responses. These results lead to the following conclusions: firstly, telemetry is a superior method for recording BP in hypertensive animals, and the lower magnitude of the WAP was probably related to the lower basal BPs recorded by this method; secondly, the mechanism accounting for the WAP must be of a long duration; thirdly, the WAP is mediated by a fall in CO and not by an increase in TPC. In conclusion, the results of the thesis support the hypothesis that ET contributes to the BP elevating properties of AVP and, consequently, the exaggerated pressor responsiveness of SHR to the peptide, and that the BP lowering properties of AVP are mediated by a fall in CO.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The hormone relaxin regulates the release of the magnocellular hormones, oxytocin and vasopressin, from the central nervous system. Studies have yet to determine whether relaxin regulates magnocellular hormone release through the circumventricular organs alone, or whether relaxin can act on the brain regions containing the magnocellular neurons as well. The paraventricular nucleus of the hypothalamus was isolated from other brain regions and maintained in vitro, in order evaluate the effects of the relaxin and relaxin-3 on the somatodendritic release of oxytocin and vasopressin. At 50 nM concentrations, relaxin induced oxytocin release, while relaxin-3 inhibited oxytocin release. Neither relaxin nor relaxin-3 had an effect on the vasopressin release, however the RXFP3 specific agonist, R3/I5, induced vasopressin release. The effect of the relaxin peptides on the electrical activity of neurons in the paraventricular nucleus was also evaluated. Relaxin depolarized magnocellular neurons while relaxin-3 hyperpolarized the neurons. Relaxin and relaxin-3 appear to have differential effects on the magnocellular neurons of the paraventricular nucleus.

No description available.