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

Fluoxetine and escitalopram are the only antidepressants approved by the Food and Drug Administration of the United States of America (FDA) for treatment of major depression in children and adolescents. Both drugs are selective serotonin reuptake inhibitors (SSRIs). In recent years there has been a growing concern over the long-term developmental effects of early-life exposure to SSRIs. The current study employed male Flinders Sensitive Line (FSL) rats, a well described and validated translational model of depression, to investigate the long term effects of pre-pubertal fluoxetine exposure. First we examined the effect of such early-life exposure on the development of depressive-like behaviour, locomotor activity and anxiety-like behaviour as manifested in early adulthood. Next, the current study investigated the effect of pre-pubertal fluoxetine exposure on the acute monoaminergic stress response, as displayed later in life. Animals received either saline (vehicle control), or 10 mg/kg/day fluoxetine from postnatal day (ND+) 21 to ND+34 (pre-puberty). The treatment period was chosen to coincide with a developmental phase where the serotonergic system’s neurodevelopment had been completed, yet the noradrenergic and dopaminergic systems had not, a scenario comparable to neurodevelopment in human adolescents. Both behavioural and in vivo intra-cerebral microdialysis experiments were conducted after ND+60 (early adulthood). On ND+60 rats allocated to behavioural experiments were evaluated for depressive-like behaviour in the forced swim test (FST), locomotor activity in the open field test (OFT), and anxiety-like behaviour in the OFT. Corticosterone concentrations were shown to be significantly higher in male FSL rats exposed to a 10 minute forced swim stress when compared to male FSL rats not exposed to a forced swim stress on ND+60. In the microdialysis experiments the rats were exposed to an acute 10 minute forced swim stress and the concentrations of the monoamines and their metabolites were measured before, during, and after the acute stressor. Relative to saline-treated (control) rats, fluoxetine-treated FSL rats did not show long-term changes in immobility in the FST (i.e. no anti-depressant-like activity) on ND+60. Like-wise anxiety-like behaviour in the OFT did not change. However, a significant decrease in locomotor activity was observed in fluoxetine-treated FSL rats compared to saline-treated (control) rats. These data suggest that a long-lasting anti-depressant-like effect of fluoxetine may be masked by the effect on locomotor activity. With measurements from the microdialysis experiments a significant attenuation of the noradrenergic stress response was observed in fluoxetine-treated rats compared to saline controls. A similar picture was observed for 5-hydroxyindole-3-acetic acid (5-HIAA), a metabolite of serotonin (5-HT), although the latter was not statistically significant. At baseline, before the stressor, significant increase in dopamine (DA) levels were observed in fluoxetine treated rats when compared to saline controls, suggesting that enhanced dopamine neurotransmission may comprise a long-term effect of pre-pubertal fluoxetine treatment. There were no discernible differences in homovanilllic acid (HVA) concentrations between fluoxetine-treated rats and saline controls. In conclusion significant developmental effects of pre-pubertal fluoxetine exposure were observed later in life and these findings warrant further investigation. / MPharm (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

Fluoxetine and escitalopram are the only antidepressants approved by the Food and Drug Administration of the United States of America (FDA) for treatment of major depression in children and adolescents. Both drugs are selective serotonin reuptake inhibitors (SSRIs). In recent years there has been a growing concern over the long-term developmental effects of early-life exposure to SSRIs. The current study employed male Flinders Sensitive Line (FSL) rats, a well described and validated translational model of depression, to investigate the long term effects of pre-pubertal fluoxetine exposure. First we examined the effect of such early-life exposure on the development of depressive-like behaviour, locomotor activity and anxiety-like behaviour as manifested in early adulthood. Next, the current study investigated the effect of pre-pubertal fluoxetine exposure on the acute monoaminergic stress response, as displayed later in life. Animals received either saline (vehicle control), or 10 mg/kg/day fluoxetine from postnatal day (ND+) 21 to ND+34 (pre-puberty). The treatment period was chosen to coincide with a developmental phase where the serotonergic system’s neurodevelopment had been completed, yet the noradrenergic and dopaminergic systems had not, a scenario comparable to neurodevelopment in human adolescents. Both behavioural and in vivo intra-cerebral microdialysis experiments were conducted after ND+60 (early adulthood). On ND+60 rats allocated to behavioural experiments were evaluated for depressive-like behaviour in the forced swim test (FST), locomotor activity in the open field test (OFT), and anxiety-like behaviour in the OFT. Corticosterone concentrations were shown to be significantly higher in male FSL rats exposed to a 10 minute forced swim stress when compared to male FSL rats not exposed to a forced swim stress on ND+60. In the microdialysis experiments the rats were exposed to an acute 10 minute forced swim stress and the concentrations of the monoamines and their metabolites were measured before, during, and after the acute stressor. Relative to saline-treated (control) rats, fluoxetine-treated FSL rats did not show long-term changes in immobility in the FST (i.e. no anti-depressant-like activity) on ND+60. Like-wise anxiety-like behaviour in the OFT did not change. However, a significant decrease in locomotor activity was observed in fluoxetine-treated FSL rats compared to saline-treated (control) rats. These data suggest that a long-lasting anti-depressant-like effect of fluoxetine may be masked by the effect on locomotor activity. With measurements from the microdialysis experiments a significant attenuation of the noradrenergic stress response was observed in fluoxetine-treated rats compared to saline controls. A similar picture was observed for 5-hydroxyindole-3-acetic acid (5-HIAA), a metabolite of serotonin (5-HT), although the latter was not statistically significant. At baseline, before the stressor, significant increase in dopamine (DA) levels were observed in fluoxetine treated rats when compared to saline controls, suggesting that enhanced dopamine neurotransmission may comprise a long-term effect of pre-pubertal fluoxetine treatment. There were no discernible differences in homovanilllic acid (HVA) concentrations between fluoxetine-treated rats and saline controls. In conclusion significant developmental effects of pre-pubertal fluoxetine exposure were observed later in life and these findings warrant further investigation. / MPharm (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