The brainstem localization of acetylcholinesterase (AChE)-containing neurons projecting to the anteroventral thalamic nucleus (AVN) was studied in rats. The AVN is one of several forebrain regions innervated by the AChE-containing dorsal tegmental pathway described by Shute and Lewis. In the present study, horseradish peroxidase (HRP) was injected into the region of the AVN to determine the brainstem origin of afferent projections. Alternate sections of tissue were stained for HRP or AChE. HRP-labeled neurons were found in the laterodorsal tegmental nucleus (LTN) and the locus coeruleus. Examination of adjacent sections revealed AChE-containing neurons in both of these nuclear regions. Combined HRP/AChE histochemistry demonstrated that transported HRP and AChE were in the same cells. In further experiments, unilateral lesions of the LTN were found to cause a decrease in AChE staining of the ipsilateral AVN. Destruction of the locus coeruleus had no effect. In combination with available evidence, the present findings suggest that cholinergic neurons in the LTN innervate the AVN. © 1980, All rights reserved.

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.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, is registered for the treatment of male erectile dysfunction (Viagra®) and pulmonary hypertension (Revatio®) in the United States. PDE5 is found in the endothelium of blood vessels in the penile corpus cavernosum, pulmonary vessels and also brain and other peripheral tissue. Sildenafil crosses the blood brain barrier, leading to side-effects such as headache and dizziness, as well as behavioural manifestations including depression, anxiety and aggression (Milman & Arnold, 2002). According to the Food and Drug Administration (2001), 12378 adverse events were reported after the use of sildenafil and 274 of these reports implicated sildenafil in neurologic, emotional, or psychological disturbances between January 1998 and 21 February 2001. In addition, in vivo studies in rats indicate that sildenafil has anxiogenic and stressogenic actions (Harvey et al., 2005; Volke et al., 2003). This is a clear indication that sildenafil influences neurological processes in the brain and may influence various signalling systems, which play major roles in the neural circuitry of the above-mentioned disturbances. Recent in vitro studies in our laboratory suggest that sildenafil may potentiate cholinergic muscarinic receptor signalling (Eager, 2004). These results suggest potential depressogenic actions, since an increase in acetylcholine is associated with depression-like symptoms (El- Yousef et al., 1973). It was therefore postulated that sildenafil may in fact possess antidepressant activity that is masked by a cholinergic-driven depressogenic activity. In a study conducted by Muller and Benkert in 2000, patients reported a decrease in depression-like symptoms when treated with sildenafil for erectile dysfunction. This implied that sildenafil not only had a direct effect on erectile function in about 50-80% of men with erectile dysfunction (Langtry and Markham, 1999; Padma-Nathan, 1999) but might also improve anhedonia and depression. The substantial correlation between the International Index of Erectile Function and Epidemiologic Studies-Depression Scale scores supported this assumption (Muller & Benkert, 2000). In addition, Raffaele et al. (2002) reported an indirect improvement in depressive-like symptoms in patients treated for erectile dysfunction with idiopathic Parkinson's disease. Aims: The current study investigated the behavioural and neuroreceptor properties of sildenafil in a rat model of depression. We also investigated a hypothesis that sildenafil displays antidepressant-like properties, but which are masked by its potentiation of the cholinergic system. Methods: The experimental layout was divided into three pilot studies. Pilot Study 1 validated the FST under our laboratory conditions, Sprague-Dawley rats received saline intraperitoneally (i.p.) for 7 days, whereafter half of the rats were pre-exposed to a 15 minute swim trial, while the remaining rats were not pre-exposed. All rats were then evaluated 24 hours later in the 5 minute scored swim trial. In Pilot Study 2 Sprague-Dawley rats were treated for 3, 7 or 11 days with vehicle (control) or 20 mg/kg fluoxetine to establish the time-dependency of the onset of antidepressant-like effects in a rat model of depression. We measured immobility in the rat forced swim test (FST), as well as changes in P-adrenergic receptor (P-AR) concentration in rat frontal cortex. In pilot study 3, rats were treated for 7 days with vehicle (control), 20 mg/kg fluoxetine, 10 mg/kg sildenafil, 1 mg/kg atropine or various combinations of these drugs. Again we employed the FST and measured cortical p-AR concentration. Results: In the FST pre-exposure to a 15 minute swim trial 24 hours before the scored swim trial significantly increased immobility. Fluoxetine inhibited this development of increased immobility in FST and decreased P-AR concentration after 7 and 11 days of treatment with fluoxetine, but not after 3 days. Seven days of treatment with atropine and sildenafil alone did not exert any changes in immobility in the FST or changes in p-AR concentration. However, a combination of atropine and sildenafil exerted a significant antidepressant-like behavioural effect, comparable with fluoxetine. Moreover, the combination of atropine and fluoxetine as well as the a triple combination of fluoxetine, sildenafil and atropine was superior to fluoxetine alone. Conclusion: Muscarinic cholinergic mechanisms mask the antidepressant-like properties of sildenafil in a rat model of depression. The antidepressant properties of the combination of sildenafil and atropine are comparable to that of fluoxetine in an animal model of depression. The combination of fluoxetine with atropine, and atropine and sildenafil enhances the antidepressant-like properties of fluoxetine. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, is registered for the treatment of male erectile dysfunction (Viagra®) and pulmonary hypertension (Revatio®) in the United States. PDE5 is found in the endothelium of blood vessels in the penile corpus cavernosum, pulmonary vessels and also brain and other peripheral tissue. Sildenafil crosses the blood brain barrier, leading to side-effects such as headache and dizziness, as well as behavioural manifestations including depression, anxiety and aggression (Milman & Arnold, 2002). According to the Food and Drug Administration (2001), 12378 adverse events were reported after the use of sildenafil and 274 of these reports implicated sildenafil in neurologic, emotional, or psychological disturbances between January 1998 and 21 February 2001. In addition, in vivo studies in rats indicate that sildenafil has anxiogenic and stressogenic actions (Harvey et al., 2005; Volke et al., 2003). This is a clear indication that sildenafil influences neurological processes in the brain and may influence various signalling systems, which play major roles in the neural circuitry of the above-mentioned disturbances. Recent in vitro studies in our laboratory suggest that sildenafil may potentiate cholinergic muscarinic receptor signalling (Eager, 2004). These results suggest potential depressogenic actions, since an increase in acetylcholine is associated with depression-like symptoms (El- Yousef et al., 1973). It was therefore postulated that sildenafil may in fact possess antidepressant activity that is masked by a cholinergic-driven depressogenic activity. In a study conducted by Muller and Benkert in 2000, patients reported a decrease in depression-like symptoms when treated with sildenafil for erectile dysfunction. This implied that sildenafil not only had a direct effect on erectile function in about 50-80% of men with erectile dysfunction (Langtry and Markham, 1999; Padma-Nathan, 1999) but might also improve anhedonia and depression. The substantial correlation between the International Index of Erectile Function and Epidemiologic Studies-Depression Scale scores supported this assumption (Muller & Benkert, 2000). In addition, Raffaele et al. (2002) reported an indirect improvement in depressive-like symptoms in patients treated for erectile dysfunction with idiopathic Parkinson's disease. Aims: The current study investigated the behavioural and neuroreceptor properties of sildenafil in a rat model of depression. We also investigated a hypothesis that sildenafil displays antidepressant-like properties, but which are masked by its potentiation of the cholinergic system. Methods: The experimental layout was divided into three pilot studies. Pilot Study 1 validated the FST under our laboratory conditions, Sprague-Dawley rats received saline intraperitoneally (i.p.) for 7 days, whereafter half of the rats were pre-exposed to a 15 minute swim trial, while the remaining rats were not pre-exposed. All rats were then evaluated 24 hours later in the 5 minute scored swim trial. In Pilot Study 2 Sprague-Dawley rats were treated for 3, 7 or 11 days with vehicle (control) or 20 mg/kg fluoxetine to establish the time-dependency of the onset of antidepressant-like effects in a rat model of depression. We measured immobility in the rat forced swim test (FST), as well as changes in P-adrenergic receptor (P-AR) concentration in rat frontal cortex. In pilot study 3, rats were treated for 7 days with vehicle (control), 20 mg/kg fluoxetine, 10 mg/kg sildenafil, 1 mg/kg atropine or various combinations of these drugs. Again we employed the FST and measured cortical p-AR concentration. Results: In the FST pre-exposure to a 15 minute swim trial 24 hours before the scored swim trial significantly increased immobility. Fluoxetine inhibited this development of increased immobility in FST and decreased P-AR concentration after 7 and 11 days of treatment with fluoxetine, but not after 3 days. Seven days of treatment with atropine and sildenafil alone did not exert any changes in immobility in the FST or changes in p-AR concentration. However, a combination of atropine and sildenafil exerted a significant antidepressant-like behavioural effect, comparable with fluoxetine. Moreover, the combination of atropine and fluoxetine as well as the a triple combination of fluoxetine, sildenafil and atropine was superior to fluoxetine alone. Conclusion: Muscarinic cholinergic mechanisms mask the antidepressant-like properties of sildenafil in a rat model of depression. The antidepressant properties of the combination of sildenafil and atropine are comparable to that of fluoxetine in an animal model of depression. The combination of fluoxetine with atropine, and atropine and sildenafil enhances the antidepressant-like properties of fluoxetine. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.