Despite their acknowledged efficacy, currently available antidepressants still demonstrate undesirable side effects, shortfalls in effectiveness and a delayed onset of action. All these agents act via monoaminergic mechanisms, although recent studies have begun to note the potential role of the cholinergic system as well as the amino acid pathways in affective isorders. It has been suggested that glutamate NMDA receptor activation may be involved in hippocampal degeneration seen in patients with depression, as well as contributing as a molecular target for the antidepressant action of known antidepressant drugs. Glutamate either separately or via the release of nitric oxide, regulates the release of various transmitters in the brain critical for affective state, e.g. monoamines (noradrenaline, dopamine), indoleamines (5HT), y-aminobutyric acid (GABA) and acetylcholine. The aim of this study was to investigate N-methyl-D-aspartate (I\IMDA) and muscarinic M1 receptor characteristics and also GABA and acetylcholine levels in a genetic animal model of depression, the Flinders Sensitive Line (FSL) rat, with respect to its control, viz. Flinders Resistant Line (FRL) rat, thereby establishing a possible role for the amino acid and cholinergic pathways in the hippocampus and frontal cortex, two brain areas implicated in depression. In addition, anxietylike behaviours were assessed using the open field and social interaction tests. A sensitive liquid chromatography tandem mass spectrometer (LC/MS/MS) method was used in the quantification of acetylcholine as well as high performance liquid chromatography with electrochemical detection (HPLG-EGD) for the quantification of GABA in the above-mentioned brain areas of FSL and FRL rats. NMDA and muscarinic M1 receptor characteristics were expressed in terms of receptor denSity (Bmax) and affinity (Kd) values and were performed using [3H]-MK801 (27.5 Gi/mmol) and quinuclidinyl benzilate (52.0 Gilmmol) for NMDA and M1 receptors, respectively. In addition, to provide evidence for face validity, behavioural assessments were routinely performed using the open field test and social interaction test. Significantly elevated levels of acetylcholine were found in the frontal cortex but with significantly reduced levels in the hippocampus of FSL rats. Cortical and hippocampal muscarinic receptor binding characteristics remained unchanged, while no differences with regard to GABA levels and NMDA receptor binding characteristics were noted in these brain areas. In concordance with studies from the literature, aversive and locomotor behaviour as measured in the open field test, provided evidence of anxiogenic behaviour in the FSL rat, evinced by significantly less social interaction than their FRL counterparts. In addition, evidence for a lack in general activity of the FSL rat in the open field was also noted. Our data therefore suggest the presence of a cholinergic dysfunction in both the frontal cortex and hippocampus of the FSL rat, although this is not accompanied by simultaneous changes in muscarinic M1 receptor binding in key limbic brain regions. Although increased cholinergic drive is a recognised characteristic of FSL rats and is representative of the model's' construct validity, we suggest that the depressive phenotype of these animals is not related to altered cholinergic activity in a single brain region, but instead involves various limbic brain regions, possibly being more dependent on opposing cholinergic deficits in the cortex and hippocampus. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Despite their acknowledged efficacy, currently available antidepressants still demonstrate undesirable side effects, shortfalls in effectiveness and a delayed onset of action. All these agents act via monoaminergic mechanisms, although recent studies have begun to note the potential role of the cholinergic system as well as the amino acid pathways in affective isorders. It has been suggested that glutamate NMDA receptor activation may be involved in hippocampal degeneration seen in patients with depression, as well as contributing as a molecular target for the antidepressant action of known antidepressant drugs. Glutamate either separately or via the release of nitric oxide, regulates the release of various transmitters in the brain critical for affective state, e.g. monoamines (noradrenaline, dopamine), indoleamines (5HT), y-aminobutyric acid (GABA) and acetylcholine. The aim of this study was to investigate N-methyl-D-aspartate (I\IMDA) and muscarinic M1 receptor characteristics and also GABA and acetylcholine levels in a genetic animal model of depression, the Flinders Sensitive Line (FSL) rat, with respect to its control, viz. Flinders Resistant Line (FRL) rat, thereby establishing a possible role for the amino acid and cholinergic pathways in the hippocampus and frontal cortex, two brain areas implicated in depression. In addition, anxietylike behaviours were assessed using the open field and social interaction tests. A sensitive liquid chromatography tandem mass spectrometer (LC/MS/MS) method was used in the quantification of acetylcholine as well as high performance liquid chromatography with electrochemical detection (HPLG-EGD) for the quantification of GABA in the above-mentioned brain areas of FSL and FRL rats. NMDA and muscarinic M1 receptor characteristics were expressed in terms of receptor denSity (Bmax) and affinity (Kd) values and were performed using [3H]-MK801 (27.5 Gi/mmol) and quinuclidinyl benzilate (52.0 Gilmmol) for NMDA and M1 receptors, respectively. In addition, to provide evidence for face validity, behavioural assessments were routinely performed using the open field test and social interaction test. Significantly elevated levels of acetylcholine were found in the frontal cortex but with significantly reduced levels in the hippocampus of FSL rats. Cortical and hippocampal muscarinic receptor binding characteristics remained unchanged, while no differences with regard to GABA levels and NMDA receptor binding characteristics were noted in these brain areas. In concordance with studies from the literature, aversive and locomotor behaviour as measured in the open field test, provided evidence of anxiogenic behaviour in the FSL rat, evinced by significantly less social interaction than their FRL counterparts. In addition, evidence for a lack in general activity of the FSL rat in the open field was also noted. Our data therefore suggest the presence of a cholinergic dysfunction in both the frontal cortex and hippocampus of the FSL rat, although this is not accompanied by simultaneous changes in muscarinic M1 receptor binding in key limbic brain regions. Although increased cholinergic drive is a recognised characteristic of FSL rats and is representative of the model's' construct validity, we suggest that the depressive phenotype of these animals is not related to altered cholinergic activity in a single brain region, but instead involves various limbic brain regions, possibly being more dependent on opposing cholinergic deficits in the cortex and hippocampus. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

This master thesis report describes signal processing parameters of electroencephalography (EEG) signals with a significant difference between the signals from the animal model of clinical depression and the non-depressed animal model. The signal from the depressed model had a weaker power in gamma (30 - 80 Hz) than the non-depressed model during awake and it had a stronger power in delta (1.5 - 4 Hz) during sleep. The report describes the process of using visualisation to understand the shape of the signal which helps with interpreting results and helps with the development of parameters. A generic tool for time-frequency analysis was improved to cope with the size of the weeklong EEG dataset. A method for evaluating the quality of how well the EEG parameters are able to separate the strains with as short recordings as possible was developed. This project shows that it is possible to separate an animal model of depression from an animal model of non-depression based on its EEG and that EEG-classifiers may work as indicative classifiers for depression. Not a lot of data is needed. Further studies are needed to verify that the results are not overly sensitive to recording setup and to study to what extent the results are translational. It might be some of the EEG parameters with significant differences described here are limited to describe the difference between the two strains FSL and SD. But the classifiers have reasonable biological explanations that makes them good candidates for being translational EEG-based classifiers for clinical depression.

Methamphetamine (METH) abuse has become a fast growing drug problem that has developed into a global epidemic. In fact, METH is one of the most commonly abused substances with an estimated 35 million abusers worldwide and is said to be the second most popular illicit drug. The Western Province of South Africa has seen a dramatic increase in drug abuse in recent years where METH is the primary or secondary drug of abuse. Interestingly, more than 50% of these individuals are under the age of 20 years. The longer duration of euphoric effects of METH has attracted many users away from cocaine in favour of METH. In addition to the rapid euphoric effect of METH, the direct short-term effects include arousal, reduced fatigue, an increase in blood pressure, reduced appetite as well as sustained attention. Chronic METH abuse may result in debilitating and long-lasting effects that includes mood disorders such as depression. Studies suggest a strong relationship between exposure to adverse environmental factors early in life and the later development of a neuropsychiatric disorder, such as depression. However, these severe consequences do not seem to invoke cessation of the drug. The euphoric and addictive properties of METH causes users to abuse the drug with an increase in frequency and dose, even though it might not have been their original intention. The primary objective of this study was to investigate the effect of early-life administration of METH to stress-sensitive (Flinders Sensitive Line - FSL) and control (Flinders Resistant Line - FRL) rats on depressive-like behaviour and regional brain monoamine levels later in life. The study implemented a sixteen-day period for administration of METH or a vehicle control from postnatal day 19 (PnD19) to postnatal day 34 (PnD34). The latter developmental stage corresponds to pre-adolescence in the rat when neurological development are similar to that seen in human adolescents, and represents the stage when drug abuse is most common in humans. Chronic dosing of METH and saline was performed twice daily at 09:00 and at 15:00. The animals received a sub-cutaneous (SC) escalating dose regimen of METH during the 16 day period (mimicking binging behaviour in humans), with every dose escalating in increments of 0.2 mg/kg from 0.2 mg/kg to 6.0 mg/kg. The study then investigated whether early-life administration of METH would cause depressive-like behaviours directly after the injection period (immediate drug effects before withdrawal on PnD35) or later in life (after the withdrawal period in early adulthood on PnD60). The behavioural effects were assessed in a battery of tests and thereafter the rats were sacrificed and the frontal cortex removed and snap frozen for later analyses of altered neurochemistry. The study demonstrated that chronic METH treatment during pre-adolescence induces significant behavioural changes related to depression in humans directly after the injection period (PnD35) and later in life (PnD60). The animals displayed antidepressant-like behaviour in the forced swim test (FST) before withdrawal, yet a depressogenic effect was observed 25 days post-withdrawal. This effect also seems to be additive to the congenital depressive-like phenotype of FSL rats, suggesting a role for genetic susceptibility. This observation would be in line with the two-hit hypothesis of depression, suggesting that the manifestation of depression will result when a genetic predisposition is followed by an environmental stressor (i.e. METH) later in life. The data suggests a working hypothesis that individuals that already have a predisposition to depression may be more susceptible to developing depression when abusing METH. The fact that the FSL control rats were more immobile than FRL control rats also confirmed the face validity of the FSL genetic rat model of depression. Locomotor activity assessment indicated that METH treatment decreased locomotor activity in FSL and FRL rats compared to their vehicle controls on PnD35 but not on PnD60. It is important to note that the effects observed in locomotor activity could not have contributed to the immobility observed in the FST, confirming that the immobility in the FST indeed reflects psychomotor and not locomotor effects. The study also demonstrated that METH significantly lowers social interaction behaviour in both FRL and FSL rats, both immediately following drug treatment (PnD35) and after withdrawal (PnD60). It is therefore clear that this effect of METH is long-lasting, putatively related to neurodevelopmental effects. In addition, the rats investigated the familiar object for a greater amount of time in the novel object recognition test (nORT) on PnD35 and PnD60 and may be the result of loss of recognition memory for the familiar object. This data confirms that METH results in cognitive memory deficits probably due to sustained adverse neurodevelopmental effects. Neurochemical analyses of the frontal cortex indicated decreased serotonin (5-HT) and norepinephrine (NE) levels on PnD35. METH is widely recognised for its pro-inflammatory effects, while the reduced 5-HT levels observed may have been the result of an increase in circulating pro-inflammatory cytokines. Neurochemical analyses provided thought-provoking data concerning the role of the permissive hypotheses of depression, indicating that dopamine (DA) is most likely not responsible for the behavioural effects observed, at least under the current study conditions, whereas 5-HT is decidedly more involved than expected. The data also suggest that depletion in NE plays a role in the development of depressive-like behaviours following METH exposure. Based on these findings, we propose that disturbances in 5-HT and NE are a crucial mechanism in how METH abuse may precipitate or worsen depressive-like symptoms in individuals who abuse METH. It should be noted that this study does not discard the role of DA in the development of depression after METH exposure, although under the current study conditions it appears that DA does not play a central role. The current study demonstrated that pre-adolescent exposure to METH can reproduce most of the behavioural changes seen in depressed individuals, and that these behavioural data can be used to identify causal neurochemical factors. Environmental stressors such as METH abuse should be regarded as an additional diagnostic criterion and is relevant to an accumulative risk factor hypothesis. Furthermore, although further study is required, the data suggests that early-life exposure to METH may predispose an individual to mood disorders and behavioural abnormalities later in life. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

Methamphetamine (METH) abuse has become a fast growing drug problem that has developed into a global epidemic. In fact, METH is one of the most commonly abused substances with an estimated 35 million abusers worldwide and is said to be the second most popular illicit drug. The Western Province of South Africa has seen a dramatic increase in drug abuse in recent years where METH is the primary or secondary drug of abuse. Interestingly, more than 50% of these individuals are under the age of 20 years. The longer duration of euphoric effects of METH has attracted many users away from cocaine in favour of METH. In addition to the rapid euphoric effect of METH, the direct short-term effects include arousal, reduced fatigue, an increase in blood pressure, reduced appetite as well as sustained attention. Chronic METH abuse may result in debilitating and long-lasting effects that includes mood disorders such as depression. Studies suggest a strong relationship between exposure to adverse environmental factors early in life and the later development of a neuropsychiatric disorder, such as depression. However, these severe consequences do not seem to invoke cessation of the drug. The euphoric and addictive properties of METH causes users to abuse the drug with an increase in frequency and dose, even though it might not have been their original intention. The primary objective of this study was to investigate the effect of early-life administration of METH to stress-sensitive (Flinders Sensitive Line - FSL) and control (Flinders Resistant Line - FRL) rats on depressive-like behaviour and regional brain monoamine levels later in life. The study implemented a sixteen-day period for administration of METH or a vehicle control from postnatal day 19 (PnD19) to postnatal day 34 (PnD34). The latter developmental stage corresponds to pre-adolescence in the rat when neurological development are similar to that seen in human adolescents, and represents the stage when drug abuse is most common in humans. Chronic dosing of METH and saline was performed twice daily at 09:00 and at 15:00. The animals received a sub-cutaneous (SC) escalating dose regimen of METH during the 16 day period (mimicking binging behaviour in humans), with every dose escalating in increments of 0.2 mg/kg from 0.2 mg/kg to 6.0 mg/kg. The study then investigated whether early-life administration of METH would cause depressive-like behaviours directly after the injection period (immediate drug effects before withdrawal on PnD35) or later in life (after the withdrawal period in early adulthood on PnD60). The behavioural effects were assessed in a battery of tests and thereafter the rats were sacrificed and the frontal cortex removed and snap frozen for later analyses of altered neurochemistry. The study demonstrated that chronic METH treatment during pre-adolescence induces significant behavioural changes related to depression in humans directly after the injection period (PnD35) and later in life (PnD60). The animals displayed antidepressant-like behaviour in the forced swim test (FST) before withdrawal, yet a depressogenic effect was observed 25 days post-withdrawal. This effect also seems to be additive to the congenital depressive-like phenotype of FSL rats, suggesting a role for genetic susceptibility. This observation would be in line with the two-hit hypothesis of depression, suggesting that the manifestation of depression will result when a genetic predisposition is followed by an environmental stressor (i.e. METH) later in life. The data suggests a working hypothesis that individuals that already have a predisposition to depression may be more susceptible to developing depression when abusing METH. The fact that the FSL control rats were more immobile than FRL control rats also confirmed the face validity of the FSL genetic rat model of depression. Locomotor activity assessment indicated that METH treatment decreased locomotor activity in FSL and FRL rats compared to their vehicle controls on PnD35 but not on PnD60. It is important to note that the effects observed in locomotor activity could not have contributed to the immobility observed in the FST, confirming that the immobility in the FST indeed reflects psychomotor and not locomotor effects. The study also demonstrated that METH significantly lowers social interaction behaviour in both FRL and FSL rats, both immediately following drug treatment (PnD35) and after withdrawal (PnD60). It is therefore clear that this effect of METH is long-lasting, putatively related to neurodevelopmental effects. In addition, the rats investigated the familiar object for a greater amount of time in the novel object recognition test (nORT) on PnD35 and PnD60 and may be the result of loss of recognition memory for the familiar object. This data confirms that METH results in cognitive memory deficits probably due to sustained adverse neurodevelopmental effects. Neurochemical analyses of the frontal cortex indicated decreased serotonin (5-HT) and norepinephrine (NE) levels on PnD35. METH is widely recognised for its pro-inflammatory effects, while the reduced 5-HT levels observed may have been the result of an increase in circulating pro-inflammatory cytokines. Neurochemical analyses provided thought-provoking data concerning the role of the permissive hypotheses of depression, indicating that dopamine (DA) is most likely not responsible for the behavioural effects observed, at least under the current study conditions, whereas 5-HT is decidedly more involved than expected. The data also suggest that depletion in NE plays a role in the development of depressive-like behaviours following METH exposure. Based on these findings, we propose that disturbances in 5-HT and NE are a crucial mechanism in how METH abuse may precipitate or worsen depressive-like symptoms in individuals who abuse METH. It should be noted that this study does not discard the role of DA in the development of depression after METH exposure, although under the current study conditions it appears that DA does not play a central role. The current study demonstrated that pre-adolescent exposure to METH can reproduce most of the behavioural changes seen in depressed individuals, and that these behavioural data can be used to identify causal neurochemical factors. Environmental stressors such as METH abuse should be regarded as an additional diagnostic criterion and is relevant to an accumulative risk factor hypothesis. Furthermore, although further study is required, the data suggests that early-life exposure to METH may predispose an individual to mood disorders and behavioural abnormalities later in life. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

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.

No / The Flinders Sensitive Line (FSL) rat is a genetic animal model of depression. Following recent findings that the brain fatty acid composition of FSL is characterised by increased arachidonic acid (AA), we used electrospray tandem mass spectrometry and 1H-NMR to examine lipid species in different brain areas. Cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, arachidonic acid-containing phosphatidylcholine species (AA-PC) were elevated with PC16:0/20:4, PC18:1/20:4 and PC18:0/20:4 (p<0.003) increased in the hypothalamus and striatum. In contrast, there was a decrease in some docosahexaenoic acid (DHA)-containing species, specifically PC18:1/22:6 (p<0.003) in the striatum and PE18:1/22:6 (p<0.004) in the prefrontal cortex. Since no significant differences were observed in the erythrocyte fatty acid concentrations, dietary or environmental causes for these observations are unlikely. The increase in AA-PC species which in this animal model may be associated with altered neuropathy target esterase activity, an enzyme involved in membrane PC homeostasis, may contribute to the depressive phenotype of the FSL rats.

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