This thesis examined neurotoxic effects of nicotine exposure during a defined critical period of neonatal brain development in mice. In our environment there are numerous hazardous contaminants that an individual can be exposed to during its entire lifetime. In many mammalian species the neonatal period is characterised by a rapid development of the brain. The present studies have identified a defined critical period during the neonatal brain development in mice, where exposure to low doses of nicotine causes permanent disturbances in the cholinergic nicotinic receptors and altered behaviour response to nicotine at adult age. This adult reaction to nicotine, a hypoactive response, was the opposite of that observed in control animals and animals exposed to nicotine before or after this period. Animals showing a hypoactive response to nicotine lacked nicotinic low affinity binding sites in the cerebral cortex. Furthermore, neonatal exposure to nicotine affected learning and memory in adult animals, an effect that was time-dependent. This thesis also showed that neonatal exposure to nicotine increased adult susceptibility to a repeated exposure of nicotine, manifested as an even more pronounced effect in spontaneous behaviour after challenging doses of nicotine. In these animals the nicotinic receptors in the cerebral cortex, assayed by a-bungarotoxin, was decreased. Neonatal exposure to nicotine was also shown to increase adult susceptibility to the organophosphate paraoxon, a known cholinergic agent, and to the brominated flame retardant 2,2´,4,4´,5-pentabromodiphenyl ether, a novel environmental agent, at adult age. This was seen at doses that did not affect behaviour in control animals, and was manifested as deranged spontaneous behaviour and reduced habituation, aberrations that also worsened with age. The results indicate that differences in adult susceptibility to environmental pollutants are not necessarily an inherited condition. Rather they may well be acquired by low dose exposure to toxic agents during early life.

This thesis investigates the neurotoxic effects in mice neonatally co-exposed to different toxic environmental agents during a defined critical period of the brains's rapid growth and development. Environmental toxic agents are incorporated in our environment. The agents investigated in this thesis are ortho-substituted polychlorinated biphenyls (PCBs 52, and 153), co-planar PCB (PCB 126), polybrominated diphenyl ether (PBDE 99), methyl mercury (MeHg), and γ-radiation. Several epidemiological studies show that human exposure to environmental agents during early development can affect childhood cognitive development. The brain growth spurt (BGS) is defined by rapid growth and development of the immature brain. For rodents (rats and mice) the BGS is postnatal spanning the first 3-4 weeks after birth. For humans this period begins during the third trimester of pregnancy and continues throughout the first two years of life. Several studies have shown that the BGS period of the brain's development renders the brain vunerable and susceptible to insults caused by environmental agents. The combinations of environmental agents used in this thesis were: PCB 52 + PBDE 99, PCB 153 + MeHg, PCB 126 + MeHg, PBDE 99 + MeHg, and γ-radiation + MeHg. The studies presented in this thesis show that co-exposure to low doses of environmental agents lead to interaction effects. These effects of interaction include defective spontaneous behavior, diminished habituation capabilities and hyperactive condition, decreased learning and memory abilities, and reduction in the nicotinic cholinergic receptor densities. Traditionally environmental agents are evaluated one at a time to investigate their effects of toxicity. This thesis indicates that the effects of interaction caused by co-exposure were often seen at doses where exposure to the individual environmental agent alone did not cause any effect. The observed effect of co-exposure were often as pronounced as a dose up to ten times the individual environmental agent alone.

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease. Neuropeptides, especially vasoactive intestinal peptide (VIP) and substance P (SP), have long been considered to play key roles in UC. Among other effects, these neuropeptides have trophic and growth-modulating as well as wound-healing effects. Furthermore, whilst VIP has anti-inflammatory properties, SP has pro-inflammatory effects. It is generally assumed that the main source of SP and VIP in the intestine is the tissue innervation. It is not known whether or not they are produced in the epithelial layer. The details concerning the expressions of their receptors in UC are also, to a great extent, unclear. Apart from the occurrence of peptidergic systems in the intestine, there are also neuronal as well as non-neuronal cholinergic systems. The pattern concerning the latter is unknown with respect to UC. The studies in this thesis aimed to investigate the expression of SP and VIP and their major receptors (NK-1R and VPAC1) in UC colon, compared to non-UC colon. The main emphasis was devoted to the epithelium. A second aim was to examine for levels of these neuropeptides in blood plasma in UC. Another aim was to examine for the non-neuronal cholinergic system in UC, thus, to investigate whether there is acetylcholine production outside nerves in the UC colon. Methods used in the thesis were immunohistochemistry, in situ hybridization, enzyme immunosorbent assay, and in vitro receptor autoradiography. For the first time, mRNA for VIP and SP has here been found in the colonic epithelium. That was especially noted in UC mucosa showing a rather normal morphology, and in non-UC mucosa. Marked derangement of the mucosa was found to lead to a distinct decrease in VIP binding, and also a decrease in the expression level of VIP receptor VPAC1 in the epithelium. In general, there was an upregulation of the SP receptor NK-1R in the epithelium when the mucosa was deranged. The plasma levels of SP and VIP were higher for UC patients compared to healthy controls. There were marked correlations between the levels of the peptides in plasma, their levels in the mucosa and the degree of mucosal derangement/inflammation. A pronounced nonneuronal cholinergic system was found in both UC and non-UC colon. Certain changes occurred in this system in response to inflammation/derangement in UC. The present study shows unexpectedly that expressions for VIP and SP are not only related to the nerve structures and the inflammatory cells. The downregulation of VPAC1 expression, and the tendencies of upregulation of NK-1R expression levels when there is marked tissue derangement, may be a drawback for the intestinal function. The study also shows that there is a marked release of neuropeptides to the bloodstream in parallel with a marked derangement of the mucosa in UC. The cholinergic effects in the UC colon appear not only to be associated with nerverelated effects, but also effects of acetylcholine produced in local non-neuronal cells. The thesis shows that local productions for not only acetylcholine, but also SP and VIP, occur to a larger extent than previously considered.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 150-191).

Heart rate is tonically regulated by the two branches of the autonomic nervous system (ANS); parasympathetic activation decreases heart rate while sympathetic activation increases it. Previous studies on the ANS in chickens have focused mainly on embryonic development, but it is largely unknown what happens in the weeks following hatching. The present study focused on the development of the autonomic nervous system in 2 and 5 week old broiler and Red Junglefowl (RJF). Since broilers are less fearful, less stressed and less active than their wild ancestor RJF, a possible domestication effect on the sympathetic nervous system was investigated by evaluating both physiological and behavioural responses during stress. I found that the heart is mainly under control from the sympathetic nervous system in 2 and 5 week old broiler and RJF as propranolol significantly decreased heart rate during baseline and stress conditions while injection of atropine had little or no effect on baseline heart rates. When the adrenergic tone was blocked, heart rate still increased during stress, more so in 5 week old birds than in 2 week old birds. This suggests that some other physiological regulatory mechanism with fast recruitment is involved in the stress response and it matures in the weeks following hatch. No differences in behaviour between broiler and RJF were observed when the sympathetic nervous system was blocked. As both breeds show similar responses, a domestication effect on the ANS from these results cannot be confirmed.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

The ascending cholinergic and serotonergic projections are central to cortical activation and normal behavior. The objective of this thesis was to determine whether unilaterally damaging both of these systems would disrupt the production of skilled movements on the contralateral side of the body. Rats received unilateral damage to either the ascending cholinergic, or serotonergic, or both projections. The respective lesions reduced neocortical leveles of acetylcholine and serotonin as assessed by acetylcholinesterase reactivity and immunohistochemical staining for serotonin. Subjects were assessed on a battery of sensorimotor tasks sensitive to neocortical integrity. The cholinergic lesion produced mild deficits on some taks but damage to both together did not abolish skilled movement. The impairments are decreased in relation to the severe effects of bilateral lesions. The results show that the sensorimotor cortex remains functional following deafferentation of both cholinergic and serotonergic afferents. / vii, 166 leaves : ill. ; 28 cm.

Agrin and neuregulin are neurotrophic molecules well known for their roles at synapses in the peripheral and central nervous systems. The expression of these two molecules is not restricted to these sites however, as they are broadly expressed across multiple organ systems. What roles do agrin and neuregulin play within these alternate systems; what is the function of these molecules outside the nervous system? Here I investigate potential roles for agrin and neuregulin in inflammation. Inflammation is an immediate response by innate immune cells, primarily macrophages, to infection and is characterized by the synthesis of pro-inflammatory mediators. The innate immune system possesses multiple redundant mechanisms to locally control inflammation. The neuro-immune axis is one means of control. Often called the cholinergic anti-inflammatory pathway, it acts to regulate local inflammation via nerve-secreted acetylcholine signaling through the homopentameric α7 nicotinic acetylcholine receptors (α7nAChR) present on macrophages. Both agrin and neuregulin have been independently described to share an intricate relationship with acetylcholine receptors (AChR) in the nervous system. Agrin is best known for its role in AChR aggregation at the neuromuscular junction while neuregulin has related roles in AChR transcription, cell survival, communication and differentiation. Based on the common characteristics of synapses in the nervous and immune systems we were curious to see if agrin and neuregulin played analogous roles on macrophages. Here we show that agrin and its receptor dystroglycan are expressed on RAW264.7 macrophages. In addition, agrin treated macrophages demonstrate increased endogenous agrin and α7nAChR expression. By blocking α-dystroglycan (α-DG), a receptor for agrin, with an anti-α-DG antibody we further saw a reduction in agrin expression. We also show that agrin is able to aggregate surface α7nAChRs and transmembrane agrin co-localizes with α7nAChRs therein. Agrin appears to induce approximately a 15-fold increase in anti-inflammatory cytokine IL-10 in macrophages but does not increase pro-inflammatory cytokine TNF-α or IL-6 synthesis. Agrin-treated macrophages challenged with LPS, a potent activator of inflammation, exhibit a 57% decrease in IL-6. Macrophages treated with agrin also exhibit a 4-fold increase in STAT3, a regulator of anti-inflammatory action. The potential anti-inflammatory effects of agrin in the periphery parallel previous work describing the effects of neuregulin in the brain. Previous work completed by our lab suggests a role for neuregulin in augmenting the expression of α7nAChRs on microglia, the macrophages of the brain, but not in peripheral macrophages. Here we show that treatment of LPS challenged microglia with neuregulin produces an 88% decrease in IL-6 and a 33% decrease in TNF-α. These results indicate both agrin and neuregulin are able to induce an increase in α7nAChRs and augment the synthesis of pro- and anti-inflammatory cytokines in their respective systems. These results also further the support the evidence of neuro-immune crosstalk in the immune system. Taken together these results present two novel players in inflammatory regulation by macrophages in the periphery and CNS.

Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the treatment of male erectile dysfunction (MED) in the United States early 1998, 274 adverse event reports were filed by the Food and Drug Administration (FDA) between 4 Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various neurological disturbances, including amnesia and aggressive behaviour (Milman and Arnold, 2002). These and other research findings have prompted investigations into the possible central effects of sildenafil. The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004). GPCRs signal through the phosphatidylinositol signal transduction pathway known to activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal transduction pathway is also known to involve the activation of PKs (via cyclic guanosine monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the action of antidepressants by elevating cGMP levels. It is generally assumed that excitotoxic delayed cell death is pathologically linked to an increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate antagonists, especially those that block the define NMDA-receptors, are neuroprotective, showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels. Aims: The aims of the study were to investigate any neuroprotective properties of sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function. Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug (control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX - ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ - 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used to determine mAChR function by constructing dose-response curves of methacholine or to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for cell viability. Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of methacholine in neuronal cells but did not show a significant increase in epithelial cells The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay could also not be used, due to severe cell loss. Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a neurodegenerative, and not a protective property, in neuronal cells. This is not likely to be associated with its PDE5 inhibitory action, but may possibly be linked to an increase in cGMP levels via the NO-cGMP pathway. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

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.