The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Methamphetamine (MA) is a well-known, easily accessible and powerful psychostimulant, and its abuse has become a global problem. MA abuse affects millions of people worldwide and places an enormous burden on public healthcare resources. Documented consequences of MA abuse include cardiotoxic, neurotoxic and teratogenic effects, as well as long-term consequences of chronic abuse including affective disorders such as schizophrenia and major depressive disorder (MDD). MDD is a highly prevalent mood disorder in both adults and children, documented to contribute to approximately 850 000 suicides annually. This disorder is projected to become the 2nd leading disease of global burden by 2020, preceded only by ischemic heart disease. Depressive-like behaviour is documented as a symptom of chronic MA abuse and particularly during extensive MA withdrawal. Also, MA abuse during pregnancy is documented to cause neurodevelopmental changes that persist into later life. However, current understanding thereof is limited and warrants further investigation of the effects of early-life exposure to MA on outcome in adulthood, particularly in terms of mood disorders. The aim of the current study was to determine the effect of chronic exposure to MA on the depressive-like behaviour later in life in stress-sensitive (Flinders Sensitive Line) and control (Flinders Resistant Line) rats. Rats were exposed during one of the following natal day (ND) age groups: prenatal (ND-13 to ND+02), postnatal (ND+03 to ND+18), prepuberty (ND+19 to ND+34) or puberty (ND+35 to ND+50). These age groups represent different stages in neurodevelopment, as also seen in humans. For prenatal exposure, pregnant dams received 5 mg/kg daily subcutaneously (s.c.), and pups from postnatal, prepuberty and puberty age groups received an escalating dose regimen to simulate “binge-dosing” commonly seen in humans abusing MA. After MA exposure, rats were housed normally until behavioural testing on postnatal day 60 (ND+60), which included the novel object recognition test (NOR), open field test (OFT) and forced swim test (FST), measuring cognitive function, locomotor activity and depressive-like behaviour respectively. The FST data showed increased immobility behaviour of saline-treated FSL rats relative to that of FRL rats, in line with previous data validating FSL rats as a genetic rodent model of depression. Practically significant MA-induced increases in immobility behaviour were observed in all FSL and FRL treatment groups in the FST, reaching statistical significance in prenatally treated FRL rats, and in postnatally, prepuberty and puberty treated FSL rats. The data suggest that early-life MA exposure may alter neurodevelopment to predispose the rats to display depressive-like behaviour in early adulthood, and suggests that this detrimental effect of MA may be more expressed in stress-sensitive rats. Furthermore, all FSL groups plus prenatally and puberty treated FRL rats revealed practically and statistically significant decreases in swimming behaviour in the FST, whereas decreases in swimming behaviour in prepuberty treated FRL rats were practically significant but did not reach statistical significance. These data suggest that MA-induced depressive-like behaviour in FSL rats may be related to impaired serotonergic neurotransmission, and that this appears to be more robust in FSL rats. Climbing behaviour in the FST was generally not altered by early-life MA exposure, with a notable exception being a practically and statistically significant increase in puberty treated FRL rats. These data suggest that in general early-life MA exposure does not affect noradrenergic neurotransmission in early adulthood, except when normal rats were treated at puberty. The reason for the latter observation is not clear. The data from the NOR test revealed no discernible trends of MA-induced effects on memory and cognition, except for a small albeit practically significant increase in exploration time in prepuberty treated FRL rats and a practically and statistically significant decrease in exploration time in puberty-treated FRL rats. Lastly, locomotor activity in the OFT was mostly unaffected by MA treatments, except for practically significant decreases in locomotor activity in postnatally-and prepuberty-treated FRL rats and practically and statistically significant decreases in locomotor activity of prepuberty treated FSL rats. Altered locomotor activity is therefore not expected to explain any of the immobility results of the FST. In final conclusion, the study confirms that early-life MA exposure results in a depressogenic effect later in life in stress-sensitive (FSL) and control (FRL) rats, but appears to be more robust in stress-sensitive animals. Furthermore the data suggest that long-lasting MA-induced depressogenic effects may relate to impaired serotonergic neurotransmission. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2014

Methamphetamine (MA) is a well-known, easily accessible and powerful psychostimulant, and its abuse has become a global problem. MA abuse affects millions of people worldwide and places an enormous burden on public healthcare resources. Documented consequences of MA abuse include cardiotoxic, neurotoxic and teratogenic effects, as well as long-term consequences of chronic abuse including affective disorders such as schizophrenia and major depressive disorder (MDD). MDD is a highly prevalent mood disorder in both adults and children, documented to contribute to approximately 850 000 suicides annually. This disorder is projected to become the 2nd leading disease of global burden by 2020, preceded only by ischemic heart disease. Depressive-like behaviour is documented as a symptom of chronic MA abuse and particularly during extensive MA withdrawal. Also, MA abuse during pregnancy is documented to cause neurodevelopmental changes that persist into later life. However, current understanding thereof is limited and warrants further investigation of the effects of early-life exposure to MA on outcome in adulthood, particularly in terms of mood disorders. The aim of the current study was to determine the effect of chronic exposure to MA on the depressive-like behaviour later in life in stress-sensitive (Flinders Sensitive Line) and control (Flinders Resistant Line) rats. Rats were exposed during one of the following natal day (ND) age groups: prenatal (ND-13 to ND+02), postnatal (ND+03 to ND+18), prepuberty (ND+19 to ND+34) or puberty (ND+35 to ND+50). These age groups represent different stages in neurodevelopment, as also seen in humans. For prenatal exposure, pregnant dams received 5 mg/kg daily subcutaneously (s.c.), and pups from postnatal, prepuberty and puberty age groups received an escalating dose regimen to simulate “binge-dosing” commonly seen in humans abusing MA. After MA exposure, rats were housed normally until behavioural testing on postnatal day 60 (ND+60), which included the novel object recognition test (NOR), open field test (OFT) and forced swim test (FST), measuring cognitive function, locomotor activity and depressive-like behaviour respectively. The FST data showed increased immobility behaviour of saline-treated FSL rats relative to that of FRL rats, in line with previous data validating FSL rats as a genetic rodent model of depression. Practically significant MA-induced increases in immobility behaviour were observed in all FSL and FRL treatment groups in the FST, reaching statistical significance in prenatally treated FRL rats, and in postnatally, prepuberty and puberty treated FSL rats. The data suggest that early-life MA exposure may alter neurodevelopment to predispose the rats to display depressive-like behaviour in early adulthood, and suggests that this detrimental effect of MA may be more expressed in stress-sensitive rats. Furthermore, all FSL groups plus prenatally and puberty treated FRL rats revealed practically and statistically significant decreases in swimming behaviour in the FST, whereas decreases in swimming behaviour in prepuberty treated FRL rats were practically significant but did not reach statistical significance. These data suggest that MA-induced depressive-like behaviour in FSL rats may be related to impaired serotonergic neurotransmission, and that this appears to be more robust in FSL rats. Climbing behaviour in the FST was generally not altered by early-life MA exposure, with a notable exception being a practically and statistically significant increase in puberty treated FRL rats. These data suggest that in general early-life MA exposure does not affect noradrenergic neurotransmission in early adulthood, except when normal rats were treated at puberty. The reason for the latter observation is not clear. The data from the NOR test revealed no discernible trends of MA-induced effects on memory and cognition, except for a small albeit practically significant increase in exploration time in prepuberty treated FRL rats and a practically and statistically significant decrease in exploration time in puberty-treated FRL rats. Lastly, locomotor activity in the OFT was mostly unaffected by MA treatments, except for practically significant decreases in locomotor activity in postnatally-and prepuberty-treated FRL rats and practically and statistically significant decreases in locomotor activity of prepuberty treated FSL rats. Altered locomotor activity is therefore not expected to explain any of the immobility results of the FST. In final conclusion, the study confirms that early-life MA exposure results in a depressogenic effect later in life in stress-sensitive (FSL) and control (FRL) rats, but appears to be more robust in stress-sensitive animals. Furthermore the data suggest that long-lasting MA-induced depressogenic effects may relate to impaired serotonergic neurotransmission. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2014

The rising number of the population that present with major depressive disorder has intensified the need to identify and elucidate new biological markers for the diagnosis and treatment of depression. Depression presents with evidence of changes in the nitric oxide (NO) pathway. In this study, levels of various endogenous markers of the NO cascade, viz. nitrite (NO2–), asymmetrical dimethylarginine (ADMA) and arginase II activity, were investigated in the Flinders Sensitive Line (FSL) rat, a genetic animal model of depression. The aim of the current study was to determine if there are differences between these markers in the plasma of the FSL rat compared to its healthy control, the (Flinders Resistant Line) FRL rat, with the possibility of considering their use as biomarkers of depression. Nitrite was chosen as metabolite over nitrate (NO3–) because the dietary intake of nitrite and/or nitrate does not significantly affect nitrite (NO2–) levels in plasma. Although this is of no significance if applied to rats, it is an important factor to be considered when doing clinical studies. For neurochemical determination of nitrite a sensitive fluorometric reversed phase high–performance liquid chromatographic (HPLC) assay was developed to analyze nitrite in human and rat plasma. Derivatization of sample nitrite was performed with 2,3–diaminonaphthalene (DAN) followed by the quantification of the stable and highly fluorescent product, 2,3–naphthotriazole (NAT). Determination of arginase II activity was performed by measuring L–arginine and L–ornithine concentrations in the plasma, while ADMA was measured simultaneously with L–arginine and L–ornithine using liquid chromatography/tandem mass spectrometry, or LC/MS/MS. Plasma nitrite levels of FSL rats were significantly decreased compared to plasma nitrite levels in the FRL rat, but neither the levels of ADMA nor arginase II activity showed a significant difference between the FSL and FRL rat groups. From these results it is concluded that in accordance with previous studies, the NO pathway plays an important role in the pathophysiology of depression, as depicted in the differences found between plasma nitrite levels in the FSL rat compared to its healthy control. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.

The rising number of the population that present with major depressive disorder has intensified the need to identify and elucidate new biological markers for the diagnosis and treatment of depression. Depression presents with evidence of changes in the nitric oxide (NO) pathway. In this study, levels of various endogenous markers of the NO cascade, viz. nitrite (NO2–), asymmetrical dimethylarginine (ADMA) and arginase II activity, were investigated in the Flinders Sensitive Line (FSL) rat, a genetic animal model of depression. The aim of the current study was to determine if there are differences between these markers in the plasma of the FSL rat compared to its healthy control, the (Flinders Resistant Line) FRL rat, with the possibility of considering their use as biomarkers of depression. Nitrite was chosen as metabolite over nitrate (NO3–) because the dietary intake of nitrite and/or nitrate does not significantly affect nitrite (NO2–) levels in plasma. Although this is of no significance if applied to rats, it is an important factor to be considered when doing clinical studies. For neurochemical determination of nitrite a sensitive fluorometric reversed phase high–performance liquid chromatographic (HPLC) assay was developed to analyze nitrite in human and rat plasma. Derivatization of sample nitrite was performed with 2,3–diaminonaphthalene (DAN) followed by the quantification of the stable and highly fluorescent product, 2,3–naphthotriazole (NAT). Determination of arginase II activity was performed by measuring L–arginine and L–ornithine concentrations in the plasma, while ADMA was measured simultaneously with L–arginine and L–ornithine using liquid chromatography/tandem mass spectrometry, or LC/MS/MS. Plasma nitrite levels of FSL rats were significantly decreased compared to plasma nitrite levels in the FRL rat, but neither the levels of ADMA nor arginase II activity showed a significant difference between the FSL and FRL rat groups. From these results it is concluded that in accordance with previous studies, the NO pathway plays an important role in the pathophysiology of depression, as depicted in the differences found between plasma nitrite levels in the FSL rat compared to its healthy control. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.