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

This thesis examines developmental neurotoxic effects of polybrominated diphenyl ethers (PBDEs), PBDE 99, PBDE 153, and the fully brominated PBDE 209, after exposure during the newborn period in rodents. Our environment contains vast numbers of contaminants, including the flame retardants, PBDEs. The PBDEs are widely found in the environment and are increasing in human milk. Individuals can be exposed to PBDEs during their whole lifetime, and especially during the lactation period. The neonatal period, coinciding with the lactation period, is characterized in many mammalian species by rapid growth and development of the immature brain. It has been shown that numerous toxicants can induce permanent disorders in brain function when administered to the neonatal mouse during the brain growth spurt (BGS). In mice and rats this period is postnatal, spanning over the first 3-4 weeks of life, while in humans, BGS begins during the third trimester of pregnancy and continues throughout the first two years of life. The present studies identified a defined critical period during BGS in mice when the brain is vulnerable to insults of low doses of PBDEs and that it is the presence of PBDEs or their metabolites in the brain during this critical period that is crucial to evoking neurotoxic effects. The effects observed are permanent altered spontaneous behavior, reduced habituation, deficits in learning and memory, and disturbances in the cholinergic system. These effects worsen with age. The ability of PBDEs to induce neurotoxic effects does not appear to be gender-, strain- or species-specific, because the neurotoxic effects are induced in rats and male and female mice of different strains. The developmental neurotoxic effects of PBDEs are similar to those observed for polychlorinated biphenyls (PCBs) and possible interactive effects of PBDEs and other environmental contaminants are therefore of concern.

Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development. / February 2006

Thesis (M.S.)--State University of New York at Binghamton, Department of Psychology, 2009. / Includes bibliographical references.

The focus of this thesis was to investigate developmental neurotoxic effects of different persistent and non-persistent environmental pollutants, alone or in binary mixtures, when exposure occurs during a critical period of brain development, in mice. The compounds investigated included a perfluorinated compound, perfluorohexane sulphonate (PFHxS), and four different pesticides, endosulfan, cypermethrin, chlorpyrifos and carbaryl. Both persistent and non-persistent pollutants are detected in the environment and in humans, which shows that exposure to these compounds is occurring in real life. Humans can therefore be exposed to various pollutants during their whole lifetime, starting from the gestational period to adulthood. Furthermore, exposure to environmental pollutants is rarely exclusive to a single compound, but rather occurs through combinations of various pollutants present in the environment. Exposure to environmental pollutants during human brain development have been suggested to be a possible cause for neuropsychiatric disorders, such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Previous studies have shown that chemicals can induce irreversible disorders in brain function when exposure to these chemicals occurs during a critical defined period of the brain development known as the brain growth spurt (BGS). The BGS is characterized by a rapid growth and development of the immature brain. In humans, and mice, this period also overlaps the lactation period indicating that newborns and toddlers can be exposed via mothers’ milk as well. This thesis has shown that a single oral exposure to PFHxS, endosulfan, cypermethrin, chlorpyrifos or carbaryl can induce developmental neurotoxic effects in mice, when exposure occurs during a critical period of brain development. These effects are manifested as persistent altered adult spontaneous behavior in a novel home environment, modified habituation, altered susceptibility of the cholinergic system and changed levels of neuroproteins in the mouse brain. Furthermore, a single neonatal co-exposure to a binary mixture of carbaryl/chlorpyrifos or PFHxS/endosulfan can interact and exacerbate the adult behavioral effects. These effects were seen at dosages were the single compound did not elicit a response or induced a much weaker behavioral effect. This indicates that risk assessments conducted on single compounds might underestimate interaction effects of mixtures when co-exposed.

Not available

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.

Cholinergic input to, and cholinergic mechanisms within the lower lumbar (L6) and upper sacral (S1) spinal cord of rat may influence neuronal excitability and afferent transmission (Thor et al, 2000) and may provide the environment necessary for appropriate central nervous system control of bladder and bowel function. It is unclear, however, if cholinergic terminals and receptors are present in the L6 & S1 spinal segments of rat and when this may develop. Cholinergic mechanisms have been shown to alter sensory afferent transmission, enhance motoneuron excitability, induce plateau potentials via non-linear membrane properties in motoneurons and reveal oscillations in locomotor-related interneurons. The enhanced activity of sphincter motoneurons was attributed to non-linear properties during the continence phase of distention-evoked voiding in the decerebrate cat (Paroschy & Shefchyk, 2000). Candidate neurotransmitters inducing non-linear properties in cat sphincter motoneurons are 5-HT (Paroschy & Shefchyk, 2000) and acetylcholine via motoneuron axon collaterals (Sasaki, 1994) and other spinal sources. We have established using the antibody to the vesicular acetylcholine transporter (VAChT) that cholinergic terminals are present on ventrolateral Onuf (VLO), dorsomedial Onuf (DMO) motoneurons and parasympathetic preganglionic motoneurons (PGN) in the L6 and S1 rat spinal cord segments. Muscarinic receptor (M2), nicotinic-α4 and α7 receptor subunit immunoreactivity was also present on Onuf motoneurons and in regions dorsal to the PGN. One source of the cholinergic puncta on Onuf motoneurons may be from motoneuron axon collaterals which we observed on a postnatal day 15 VLO motoneuron. Cholinergic terminals were observed on vasoactive intestinal polypeptide-immunoreactive (VIP) afferents, interneurons in the intermediolateral (IML) region and perhaps on other afferents in the lateral and medial collateral pathway of L6 and S1 spinal segments. In the ventral horn, the cholinergic puncta and receptors appear to have a mature distribution around two weeks postnatal and the cholinergic terminals appeared to have a mature distribution in the IML region by three weeks postnatal. Using whole cell patch clamp recording techniques and thick slices of the L6 and S1 rat spinal cord, we observed excitatory responses of ventral horn neurons and motoneurons to carbachol (10-50 μM), a non-specific cholinergic agonist. Ventral horn neurons (postnatal day 8- 16) exhibited prolonged firing and prolonged depolarizations (plateau potentials) beyond the duration of the applied excitatory input from cholinergic (n=6/33) and other (n= 4/37) neurotransmitter systems. In a selection of the neurons with plateau potentials, the L-type calcium current played a role in the plateau production (n=5/5) and low frequency oscillations (n=2/2) as revealed by nifedipine. Postnatally, the voiding reflex changes from a perineal-evoked reflex, to the adult bladder-bladder reflex. Cholinergic input may be responsible in part for the bursting activity of the external urethral sphincter and the activation of the bladder, which is required for complete voiding reflexes in the adult rat. Plateau potentials and enhanced excitability due to cholinergic mechanisms could render inessential a constant excitatory drive that is required in the perineal-evoked voiding reflex in the neonatal rat and may underlie changes in the voiding reflexes that occur during postnatal development.

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.

Stroke is the leading cause of long-term disability in the developed world and can have devastating effects on the health and everyday functioning of individuals. In most cases stroke is ischemic and is caused by the obstruction of blood flow due to a clot in the brain blood vessels. This initiates a cascade of events that result in tissue death and loss of behavioural function associated with the damaged region. The peri-infarct cortex is a region surrounding the infarct core that survives the ischemic event and is most susceptible to pharmacological treatments and rehabilitation. α4ß2 nicotinic acetylcholine receptor (nAChR) signalling has been implicated as a mechanism that affects cell survival and cell death in the acute response after stroke. Nicotinic receptor signalling is also involved in modulating brain excitability, which can affect neural plasticity and restoration of cortical circuits and lead to recovery of lost function after stroke. In order to elucidate the role of α4ß2 nAChRs on acute and chronic recovery after stroke, we tested two hypotheses: (1) blocking α4ß2 nAChRs triggers acute neuroprotection and (2) α4ß2 nAChRs play a role in regulating plasticity and long-term functional recovery. In the first set of experiments a new model of targeted photothrombotic stroke was induced in a distal branch of the middle cerebral artery (MCA) in awake and anaesthetized mice. Mice treated with the α4ß2 nAChR antagonist dihydro-ß-erythroidine (DHßE) showed smaller lesion sizes relative to vehicle controls and this effect was greater in mice that were awake during stroke induction. To determine the mechanism of α4ß2 nAChRmediated neuroprotection, changes in collateral flow were measured using Evans bluestained surface angiograms and laser Doppler flowmetry. Contrary to what was expected, DHßE did not appear to induce neuroprotection by altering collateral flow. In the second set of experiments, we first used confocal imaging to quantify and characterize the expression of α4ß2 nAChRs after stroke. Next, mice were induced with a targeted photothrombotic stroke in the forelimb somatosensory cortex. Mice were then chronically treated with DHßE to determine if α4ß2 nAChR antagonism could improve recovery of function. Behavioural tests showed that blocking α4ß2 nAChRs chronically had no effect on forelimb function after stroke. Voltage-sensitive dye imaging was used to measure forelimb-evoked responses in the somatosensory cortex and revealed no differences in cortical responsiveness between treated and non-treated groups. Altogether, these results show that changes in α4ß2 nAChR signalling that occur after stroke mediate ischemic cell death but do not have an effect on long-term recovery and plasticity. Moreover, they present a novel pathway for investigating stroke pathophysiology and the development of acute neuroprotective treatments. / Graduate / 0317 / aseto@uvic.ca