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

Ethanol causes neurotoxicity via several mechanisms at different points in the cycle of dependence, including neuroinflammation and oxidative stress during ethanol exposure as well as excitotoxicity during ethanol withdrawal. The primary therapeutic implication is that ethanol-induced neurotoxicity requires multifunctional pharmacotherapies which reduce all mechanisms. Using an innovative pharmacological high throughput screening method on a large plant extract library we discovered flavonoids with alpha7 nicotinic acetylcholine receptor (nAChR) activity. In addition to their well-known anti-inflammatory and antioxidant properties, this novel activity means they can potentially reduce excitotoxicity and therefore makes them ideal for inhibition of ethanol-induced neurotoxicity. Rhamnetin, the candidate compound, was first found to inhibit lipopolysaccharide induced inflammation in immortalized BV2 microglia, in part, via alpha7 nAChRs. We then established an in vitro model of ethanol induced-neurotoxicity using organotypic hippocampal slice cultures which incorporated both neuroinflammatory and excitotoxic components. Neuroinflammation enhanced excitotoxicity under control conditions but the reverse was observed during ethanol withdrawal. Both mechanisms are important but their interaction is not simple. Finally, rhamnetin was evaluated in this model and found to reduce neuroinflammation and excitotoxicity associated with ethanol withdrawal. In conclusion, the studies herein provide strong evidence for alpha7 nAChRs selective flavonoids as potential pharmacotherapies for the treatment of ethanol-induced neurotoxicity and further implicate neuroinflammation, excitotoxicity, and their interaction as critical mechanisms in this pathology.

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.

Pentameric ligand-gated ion channels (pLGICs) are membrane bound receptors found in the nervous system. They are responsible for detecting neurotransmitters released from neurons and subsequently mediating responses of the cells on which they are found. Thus, pLGICs play an invaluable role in communication between cells of the nervous system and understanding their function is pivotal to understanding how the nervous system works in general. One factor which is known to mediate pLGIC function is lipids found in the membrane environment in which pLGICs are embedded. This dissertation explores the various ways in which lipids interact with and modulate the function of pLGIC. Potential mechanisms and biological consequences of this modulation will be presented and discussed within the context of our current state of knowledge of pLGIC and nervous system function.

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

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.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-128). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of South Florida, 2004. / Includes vita. Includes bibliographical references (leaves 144-164).

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 166-195).

No Brasil, populações ribeirinhas da Amazônia estão expostas ao metilmercúrio (MeHg) e ao chumbo (Pb) oriundos, respectivamente, de peixes e farinha de mandioca contaminados. Embora a toxicidade destes elementos químicos seja explorada há tempo, pouco se sabe sobre os efeitos decorrentes da exposição crônica a baixas doses destes toxicantes e, menos ainda, acerca da exposição simultânea a estes dois metais. Neste sentido, este trabalho foi desenvolvido objetivando avaliar a ocorrência de efeitos bioquímicos, genotóxicos e relacionados ao estresse oxidativo, decorrentes da exposição crônica de ratos a baixas doses de MeHg e Pb, associados ou não, bem como a distribuição tecidual destes metais. Adicionalmente, foram investigados os efeitos da administração da vitamina antioxidante niacina (NA) diante destas exposições. Para isto, ratos machos Wistar foram divididos em 8 grupos (n = 6): Grupo controle; Grupo MeHg, tratado com cloreto de MeHg (140 ?g/Kg/dia) por gavagem; Grupo Pb, tratado com acetato de Pb (648 ?g/Kg/dia) por gavagem; Grupo MeHg + Pb, tratado com MeHg (140 ?g/Kg/dia) e Pb (648 ?g/Kg/dia) por gavagem. Grupos paralelos (NA; NA + MeHg; NA + Pb e NA + MeHg + Pb) receberam o mesmo tratamento associado à suplementação de niacina (50 mg/Kg/dia) adicionada na água. O tratamento teve duração de 92 dias. Foram avaliados os parâmetros bioquímicos colesterol total e frações, glicose, atividade das enzimas hepáticas aspartato aminotransferase (AST) e alanina aminotransferase (ALT), e hemoglobina. Os marcadores relacionados ao estresse oxidativo determinados foram tióis totais (GSH); lipoperoxidação, avaliada pela concentração de malondialdeído (MDA) e espécies reativas ao ácido barbitúrico (TBARS); além da atividade das enzimas antioxidantes superóxido dismutase (SOD), glutationa peroxidase (GSH-Px) e catalase (CAT). Ainda, foi avaliada a concentração de óxido nítrico (NO) plasmático e a genotoxicidade por meio do Ensaio Cometa. As concentrações de mercúrio (Hg) e Pb foram determinadas em sangue total e tecidos dos animais por ICP-MS. A exposição a baixas doses de MeHg causou genotoxicidade, redução na atividade da CAT no cérebro e nas concentrações de GSH no sangue e fígado dos animais, além de peroxidação lipídica, evidenciada pelo aumento nas concentrações de MDA e TBARS no plasma e cérebro dos ratos. Os animais expostos exclusivamente ao MeHg apresentaram ainda atividade de ALT aumentada e concentração plasmática de NO reduzida. A distribuição do Hg no organismo foi maior no rim, seguido por sangue e, posteriormente, cérebro. A exposição exclusivamente ao Pb promoveu redução na concentração de GSH e na atividade de CAT, além de induzir peroxidação lipídica no cérebro dos ratos. A exposição ao Pb também resultou em genotoxicidade, além de redução na concentração de hemoglobina e NO. As maiores concentrações de Pb foram observadas no osso (fêmur) > rim > cérebro > sangue. A exposição simultânea ao MeHg e Pb não resultou em sinergismo de efeitos tóxicos em comparação ao tratamento com os metais individualmente. Considerando parâmetros como concentração plasmática de NO e GSH no sangue, fígado e cérebro, a exposição ii conjunta aos metais antagonizou os efeitos desencadeados por cada metal individualmente. Entretanto, a coexposição MeHg/Pb também promoveu genotoxicidade e lipoperoxidação. Já a administração de niacina apresentou efeitos protetores frente às alterações desencadeadas pela exposição aos metais, sem alterar a concentração e distribuição destes nos órgãos/tecidos. Em resumo, nossos resultados mostram que a exposição ao MeHg e Pb, até mesmo em doses baixas, induz toxicidade em roedores. Visto que a niacina apresentou efeitos antioxidantes e antigenotóxicos relevantes, a suplementação com esta vitamina pode ser uma alternativa para amenizar os efeitos tóxicos decorrentes da exposição ao MeHg e/ou Pb. / In Brazil, riverside populations of the Amazon are exposed to methylmercury (MeHg) and lead (Pb) coming respectively from contaminated fish and cassava flour. Although the toxicity of these elements has been explored for some time, little is known about the effects of chronic exposure at low doses and even less about the simultaneous exposure. Thus, this work aimed to evaluate the occurrence of biochemical, genotoxic and oxidative stress related effects, resulting from chronic exposure of rats at low doses of MeHg and Pb, associated or not, as well as the tissue distribution of these elements. Additionally, the effects of co-administration of the antioxidant vitamin niacin (NA) on the toxic effects were investigated. For this, male Wistar rats were divided into 8 groups (n = 6): Control group; MeHg group, received MeHg chloride (140 ?g/Kg/day) by gavage; Pb group, received Pb acetate (648 ?g/Kg/day) by gavage; MeHg + Pb group, received MeHg (140 ?g/Kg/day) and Pb (648 ?g/Kg/day) by gavage. Parallel groups (NA; NA + MeHg; NA + Pb and NA + MeHg + Pb) received the same treatment associated with niacin supplementation (50 mg/kg/day) added to the water. The treatment lasted 92 days. Biochemical parameters such as total and fractions cholesterol, glucose, hepatic enzyme activity such as aspartate transaminase (AST) and alanine transaminase (ALT), and hemoglobin were determined. Oxidative stress markers such as total thiols (GSH); lipid peroxidation, measured by malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS) concentrations; besides the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) antioxidant enzymes were evaluated in treated and non-treated animals. Also, the concentration of plasmatic nitric oxide (NO) and genotoxicity by the Comet Assay was assessed. Levels of mercury (Hg) and Pb were determined in whole blood and tissues of animals by ICP-MS. Low doses of exposure to MeHg caused reduction of CAT activity in the brain and reduction of GSH concentrations in the blood and liver of animals, besides genotoxicity and lipid peroxidation, as evidenced by the increase levels of MDA and TBARS in plasma and brain of rats. Animals exposed to MeHg still had increased ALT activity and decreased plasmatic NO levels. Levels of Hg were found higher in kidney, followed by blood and brain. Pb exposure alone promoted reduction of GSH concentration and CAT activity, and induced lipid peroxidation in the brain of rats. Moreover, genotoxicity, and reduction of hemoglobin and plasmatic NO levels were also observed in the group of animals treated only with Pb. The highest Pb levels were observed in bone (femur) > kidney > brain > blood. Simultaneous exposure to MeHg and Pb did not result in synergistic toxic effects. Considering parameters such as plasmatic NO and GSH levels in blood, liver and brain, the joint exposure to metals antagonized the effects produced by each metal individually. However, the MeHg/Pb co-exposure also promoted genotoxicity and lipid peroxidation. On the other hand, administration of niacin exhibited protective effects under the changes triggered by exposure to metals without altering the concentration and distribution of these metals in the organs/tissues of animals. Taken together, our results demonstrated that exposure to MeHg and Pb, even at low doses, are toxic to iv rodents. Moreover, since niacin presented relevant antioxidant and antigenotoxic effects, supplementation with this vitamin can be an alternative to mitigate the toxic effects resulting from exposure to MeHg and/or Pb.