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Doctor of Philosophy (PhD) / GABAA receptors are implicated in the pathology of psychiatric disorders such as schizophrenia and depression. They are rapidly affected by stress in a sex-dependent fashion, suggesting that GABAA receptors may be relevant to understanding the association between stress and psychiatric disorders. Thus, this thesis examined how GABAA receptors are affected in both male and female mice exposed to stress in adulthood (Chapter 2), early-life (Chapter 3-5) and a combination of both early-life and adulthood stress (Chapter 6). 2. The effects of acute adulthood stress (3 minute warm swim stress) on GABAA receptor binding in the brains of male and female mice were examined using quantitative receptor autoradiography. The total number of GABAA receptor [3H]GABA binding sites was increased following swim stress in specific forebrain cortical regions of female mice swum individually or in a group, but decreased in male mice when swum in a group only. These findings confirm and extend previous studies, identifying the cortical regions involved in rapid stress-induced changes in GABAA receptors. 3. Post-natal handling models in rodents comparing control (brief handling sessions; EH) with no intervention stress conditions (NH), indicate that the NH condition results in an anxious adulthood phenotype and this was confirmed in the present thesis using the elevated plus-maze behavioural test. Using this model the effects of early-life stress on adulthood GABAA receptors were then examined. 4. Regional densities of GABAA receptor α1 and α2 subunit proteins were observed in the adult brain of male and female mice using immunoperoxidase histochemistry. NH males showed a loss of the α2 subunit from the thalamus and the lower layers (IV-VI) of the primary somatosensory cortex, whilst NH females showed a reduction of α2 but an increase in α1 protein in the lower layers of the primary somatosensory cortex only. These regionally specific alterations in the α1:α2 subunit ratio suggest that early-life stress disrupts the developmental α subunit switch, which occurs in a regionally-dependent fashion over the first two weeks of rodent life. 5. Double-labelling immunofluorescence and confocal microscopy were used to examine the effects of sex and early-life stress on GABAA receptor synaptic clustering. Regardless of sex, mice exposed to early-life stress (NH) showed reduced colocalisation of the GABAA receptor α2 subunit with the synaptic marker protein gephyrin relative to the control condition (EH). This suggests that early-life stress impairs adulthood inhibitory synaptic strength and is consistent with the increased anxiety of the stressed relative to control mice. 6. Finally, the effects of early-life stress on adulthood swim stress-induced changes in GABAA receptor binding were examined using quantitative receptor autoradiography in forebrain cortical regions. Findings showed that the effect of adulthood stress on the total number of GABAA receptor binding sites for [3H]GABA in forebrain cortical regions was altered by early-life stress in both male and female mice, suggesting that the rapid adulthood stress response of GABAA receptors is affected by early-life experience. 7. Together these results show that GABAA receptors are sensitive to subtle changes in the environment in both early-life and adulthood and that these neurochemical responses to stress in adulthood are sex-dependent. The short and long-term stress-sensitivity of the GABAergic system implicates GABAA receptors in the non-genetic aetiology of psychiatric illnesses in which sex and stress are important factors.

Doctor of Philosophy (PhD) / GABAA receptors are implicated in the pathology of psychiatric disorders such as schizophrenia and depression. They are rapidly affected by stress in a sex-dependent fashion, suggesting that GABAA receptors may be relevant to understanding the association between stress and psychiatric disorders. Thus, this thesis examined how GABAA receptors are affected in both male and female mice exposed to stress in adulthood (Chapter 2), early-life (Chapter 3-5) and a combination of both early-life and adulthood stress (Chapter 6). 2. The effects of acute adulthood stress (3 minute warm swim stress) on GABAA receptor binding in the brains of male and female mice were examined using quantitative receptor autoradiography. The total number of GABAA receptor [3H]GABA binding sites was increased following swim stress in specific forebrain cortical regions of female mice swum individually or in a group, but decreased in male mice when swum in a group only. These findings confirm and extend previous studies, identifying the cortical regions involved in rapid stress-induced changes in GABAA receptors. 3. Post-natal handling models in rodents comparing control (brief handling sessions; EH) with no intervention stress conditions (NH), indicate that the NH condition results in an anxious adulthood phenotype and this was confirmed in the present thesis using the elevated plus-maze behavioural test. Using this model the effects of early-life stress on adulthood GABAA receptors were then examined. 4. Regional densities of GABAA receptor α1 and α2 subunit proteins were observed in the adult brain of male and female mice using immunoperoxidase histochemistry. NH males showed a loss of the α2 subunit from the thalamus and the lower layers (IV-VI) of the primary somatosensory cortex, whilst NH females showed a reduction of α2 but an increase in α1 protein in the lower layers of the primary somatosensory cortex only. These regionally specific alterations in the α1:α2 subunit ratio suggest that early-life stress disrupts the developmental α subunit switch, which occurs in a regionally-dependent fashion over the first two weeks of rodent life. 5. Double-labelling immunofluorescence and confocal microscopy were used to examine the effects of sex and early-life stress on GABAA receptor synaptic clustering. Regardless of sex, mice exposed to early-life stress (NH) showed reduced colocalisation of the GABAA receptor α2 subunit with the synaptic marker protein gephyrin relative to the control condition (EH). This suggests that early-life stress impairs adulthood inhibitory synaptic strength and is consistent with the increased anxiety of the stressed relative to control mice. 6. Finally, the effects of early-life stress on adulthood swim stress-induced changes in GABAA receptor binding were examined using quantitative receptor autoradiography in forebrain cortical regions. Findings showed that the effect of adulthood stress on the total number of GABAA receptor binding sites for [3H]GABA in forebrain cortical regions was altered by early-life stress in both male and female mice, suggesting that the rapid adulthood stress response of GABAA receptors is affected by early-life experience. 7. Together these results show that GABAA receptors are sensitive to subtle changes in the environment in both early-life and adulthood and that these neurochemical responses to stress in adulthood are sex-dependent. The short and long-term stress-sensitivity of the GABAergic system implicates GABAA receptors in the non-genetic aetiology of psychiatric illnesses in which sex and stress are important factors.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AIIα subtype) or PD123177 (designated AIIβ subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AIIα and AIIβ subtypes.

In vitro receptor autoradiography was performed on rat brain and kidney sections stored frozen at -20°C for extended time periods (17, 40, 64, 121, 183, 251, and 333 days). The results indicate that prolonged tissue storage has a differential effect upon125I sar1ile8 angiotensin II binding to AT1 and AT2 receptor sites. Binding at AT1 receptor rich tissues studied (renal medulla, renal cortex, anterior pituitary, ventral hippocampus, spinal trigeminal nucleus, and nucleus of the solitary tract) shows a first order exponential decay pattern. The logarithmic linear regression slope (log(e) specific binding versus time), is significantly different from zero (p<0.05) in all AT1 rich tissues except for nucleus of the solitary tract (p=0.086). There is no detected loss of 125I sar1ile8 angiotensin II binding at the AT2 prominent regions in the superior colliculus, medial geniculate nucleus, and the inferior olivary nucleus. The half lives of AT1 receptors are highly variable, ranging from 36 days in the anterior pituitary to 442 days in the nucleus of the solitary tract, and this might be related to variable stability of AT(1A) and AT(1B) receptors. These observations should be taken into account when assessing and comparing AT1 and AT2 receptor subtype densities.

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1, Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 > 10-4 M). DuP 753 sensitive binding sites (designated AIIα subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AIIβ) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.

Previous studies have used new angiotensin II (AII) receptor subtype selective compounds to localize AII receptor subtypes within discrete rat brain nuclei. The purpose of this autoradiographic study was to extend these preliminary findings and provide a comprehensive analysis of AII binding sites in 22 rat brain nuclei and the anterior pituitary, to include estimates of the binding affinity for 125I sar1 ile8 AII (125I SIAII) at each nucleus, and determine the fractional distribution of each subtype at each nucleus. Estimates of K(D), in separate experiments revealed that AT1 nuclei had a consistently higher affinity for 125I SIAII than AT2 nuclei (0.66 vs. 2.55 nM). Displacement of subsaturating concentrations of 125I SIAII by 10-8-10-4 M DuP753 (selective for the AT1 subtype) or PD123177 (selective for the AT2 subtype) indicated that approximately half of the brain regions surveyed contained predominantly AT1 sites and half contained predominantly AT2 sites. Binding was partially displaced by both compounds in several regions and two site analyses were performed to estimate the distribution of subtypes within each nucleus. The data were then corrected for differential occupancy by 125I SIAII. Brain nuclei associated with cardiovascular or dipsogenic actions of AII, e.g., subfornical organ, organum vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and area postrema, contained pure, or almost pure, populations of AT1 receptors. The functions of AII in brain regions containing predominantly AT2 binding sites, e.g., thalamus, colliculi, inferior olive and locus ceruleus, remain undefined. Thus, AII binding sites in the rat brain have been differentiated into two subtypes with similar characteristics to those reported in peripheral tissues. However, the unexpected finding that they can be differentiated on the basis of their affinity for 125I SIAII raises questions concerning their coidentity with peripheral receptor subtypes.

Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1, isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1, isoleucine8 angiotensin II binding was strongly inhibited by 30 mM β-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AIIα) 125I-sarcosine1, isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AIIβ) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AIIα) receptor subtype.