The present research project was designed to analyse some of the mechanisms implicated in the autoregulation of serotonin (5-HT) function in rats and mice. It focuses on (i) the study of 5-HT reuptake activity following the long-term administration of the selective 5-HT reuptake inhibitor (SSRI) paroxetine and the tricyclic drug tianeptine, and (ii) autoreceptor-mediated control of 5-HT release at the cell body level and its modifications following long-term antidepressant treatments. / The adaptative properties of the 5-HT transporter were assessed using a combined methodological approach of in vivo binding studies as well as $ rm lbrack sp3H rbrack$5-HT uptake assays. Such an approach indicated that long-term administration of an SSRI: (a) reduced the efficacy of paroxetine to prolong the time for recovery of firing activity of CA$ sb3$ pyramidal neurons following the suppression induced on this parameter by microiontophoretic application of 5-HT, (b) reduced the amount of $ rm lbrack sp3H rbrack$5-HT captured by hippocampal and midbrain raphe slices, and (iii) reduced the number of 5-HT transporters in hippocampus and cortex. From these results, it was concluded that prolonged administration of paroxetine down-regulated 5-HT transporters. On the other hand, the sustained administration of the tricyclic drug tianeptine, which enhances 5-HT uptake activity, did not induce any long-lasting changes either in hippocampal 5-HT uptake activity, or in the efficacy of 5-HT synaptic transmission in this terminal projection field. / A second series of studies was then undertaken to assess how is extracellular availability of somatodendritic 5-HT regulated in in vivo electrophysiological and voltammetric experiments. The non-selective 5-HT agonist TFMPP inhibited 5-HT release in the dorsal raphe nucleus, independently of the firing activity of 5-HT neurons. In vitro superfusion experiments, using midbrain raphe slices from rats and mice (wild type and 5-HT$ rm sb{1B}$ knock-out) were then used to assess pharmacological and functional properties of this non-5-HT$ rm sb{1A}$ receptor. It was concluded that G$ rm sb{i/o}$-coupled 5-HT$ rm sb{1D}$ autoreceptors negatively regulate 5-HT release in midbrain raphe nuclei of rats and mice, and that these receptors desensitise following prolonged administration of SSRI's and monoamine oxidase inhibitors (MAOI's).

Application of potassium chloride (KCl) to a brain surface, a procedure which consistently elicits spreading depression, resulted in a marked induction of the proto-oncogene c-fos in the treated cerebral cortex in a rapid and transient manner. Most of the cerebral cortex throughout the treated hemisphere showed an increase in c-fos-like immunoreactivity (IR). The only area spared was that immediately below the exposed surface. On the other hand, ubiquitin-like IR appeared to increase in pyramidal-shaped cells in the damaged area as early as 90 minutes and persisted up to 2 days after treatment. Mechanical cortical brain injury was accompanied by a similar widespread expression of c-fos protein(s) throughout the wounded cortex even far from the lesion site. Rats with a mechanical cortical injury, which were sacrificed 1.5 hours later, showed an increase in c-fos immunoreactive nuclei in the piriform cortex ipsilateral to the lesion at post-natal day (PD) 22, but not PD 10 or 15. This pattern was maintained up to at least PD 90. Similarly, the presence of c-fos IR cells was observed in the ipsilateral cingulate cortex since PD 22. The pattern of c-fos expression after mechanical wound was compared with topical potassium application to a cortical surface. Though both models generated c-fos expression far from the lesion site, K+ application resulted in a higher number of c-fos immunoreactive cells, particularly in the cingulate cortex. / High potassium concentrations are generally used to induce neurotransmitter release. In order to establish a possible link between c-fos expression and stimulating conditions for neurotransmitter release in microdialysis procedures, we administered KCl (100 mM) into the hippocampus. C-fos-like IR was up regulated in the dentate gyrus and pyramidal cells of the hippocampus. The expression of c-fos induced by KCl was not altered in the animals with fimbria-fornix (FF) lesions despite the marked decrease of ACh releaae in the hippocampus. Glutamate concentrations measured in the same superfusates showed that a significantly greater glutamate release occurs in denervated hippocampi. Administration of pentobarbital (1ml/kg i.p.) in order to abolish any seizure-like activity induced by KCl, did not alter expression of c-fos IR in the K$ sp+$-stimulated hippocampi. / The topical application of high K$ sp+$ concentrations to a brain surface (parietal cortex), which induced c-fos expression, preceded an increase in both NGF mRNA and NGF-like protein(s). A maximal increase in c-fos was detected within 3 hours, whereas NGF mRNA levels peaked at 12 hours and NGF-like protein(s) reached its maximum at 24 hours after KCl application. The most prominent increase in NGF mRNA was measured in the entorhinal cortex (50 fold) but it was also moderately increased in other cortical areas (2-3 fold). / Application of K+ to the cortex induced the expression of glial fibrillary acidic protein (GFAP) in astrocytes, as assessed by immunohistochemical techniques, throughout the cortex ipsilateral to K+ exposure. Administration of the non- competitive NMDA antagonist MX-801 (4mg/kg i.p.) prior to the injury prevented the rise in GFAP IR at 2 but not 7 days after the treatment. GFAP IR was also studied after disruption of a restricted area of the pia-arachnoid which compromises vascular irrigation of the underlying cortex. GFAP+ cells were present in the ipsilateral remaining cortex, distant from the wound, between days 4 and 15. / These findings suggest selective changes in gene expression following different types of cortical brain injury. The interpretation of these observations is given in the discussion of this thesis.

This research project deals with the electroretinogram (ERG) and the role played by the oscillatory potentials (OPs) in its generation. Investigators are still debating over the cellular origin of these fast components but the current view is that the OPs are produced by structures different from those involved in the genesis of the b-wave (positive wave of the ERG). Another claim would be that the OPs should be considered the primary electrical events produced by the retina and their summation would yield the b-wave. A pharmacological approach was chosen to investigate the OP-b-wave relationship. 2-amino-4-phosphonobutyric acid (APB) and glycine were injected intravitreally in rabbits. Our results indicate that the b-wave and OPs demonstrate identical chemical sensitivity. These findings could support the claim that the b-wave results from the summation of the OPs.

Excitotoxic neonatal lesion of the ventral hippocampus (NVH) in rats has been proposed as a putative neurodevelopmental animal model of schizophrenia, with characteristic post-pubertal behavioral and neurochemical abnormalities comparable to those observed in this disease including alterations in dopaminergic (DA) neurotransmission and prefrontal cortical functions. Dysfunctions in the mesocorticolimbic DA system are believed to contribute to some of the behavioural manifestations of schizophrenia. Accumulating evidence also suggests that alterations in the activity of cortical cholinergic neurotransmission may be involved in the cognitive abnormalities seen in major neuropsychiatric disorders. These two neurotransmitter systems are functionally interlinked as the mesocorticolimbic dopaminergic system stimulates acetylcholine (ACh) release in the prefrontal cortex (PFC) mainly through the activation of D 1-like receptors. Given the involvement of the PFC and DA abnormalities in NVH lesioned animals and evidence of cholinergic abnormalities in schizophrenia, the purpose of the present thesis was to investigate the status of cholinergic system in pre and post-pubertal NVH lesioned animals, and its regulation by DA. / In vivo microdialysis studies revealed that PFC ACh release was increased to a greater extent in response to the systemic injection of the D1-like agonist SKF 81297 in post-pubertal NVH lesioned rats compared to sham control rats. Furthermore, intra-cortical administration of SKF 81297 increased PFC ACh release in post-pubertal NVH lesioned rats but not in sham animals. Interestingly, tail pinch stress, which activates mesocorticolimbic DA neurotransmission, also increased PFC ACh release to a greater extent in post-pubertal NVH lesioned rats. This increase was normalised by D1 or D2-like antagonists. Quantitative receptor autoradiographic studies revealed increases in M1 and M2-like muscarinic receptor levels in some limbic and PFC regions in post-pubertal NVH lesioned rats. These increases were associated with enhanced physiological responses to the muscarinic receptor agonist, oxotremorine. Finally, the muscarinic receptor antagonist biperiden normalized deficits in prepulse inhibition of the acoustic startle in post-pubertal NVH lesioned rats. Taken together, these data reveal the developmental alterations of cholinergic neurotransmission and its regulation by DA with functional changes seen only in post-pubertal NVH lesioned animals. While being relevant to the behavioural characteristics of the NVH lesioned animal model, these data also support the possible role of cholinergic neurotransmission in schizophrenia.

Purpose. This work investigates the effect of anti-migraine serotonin (5-HT) receptor agonists (the triptans) on in vivo 5-HT synthesis rates in the brain. In rats, rates are examined after acute and chronic drug administration. In humans, rates are measured in different migrainous conditions, namely, during a headache, after sumatriptan, and between attacks. Methods. Synthesis rates are determined using the alpha-[14C or 11C]methyl- L-tryptophan tracer kinetic model, combined in rats with autoradiography and in humans with positron emission tomography. Results & conclusions . Acute triptan administration reduces 5-HT synthesis rates in rat brain while chronic delivery increases rates at serotonergic projection areas. Preliminary results in human migraine sufferers suggest a trend, though not significant, towards an acute decrease in central rates after sumatriptan injection and higher rates during migraine pain than between attacks. Such central effects might or might not help alleviate pain but would have repercussions for chronic triptan users.

Our group studies the interaction between antibiotic peptides and phospholipids. These peptides are known to form channels in the lipid membrane. With X-ray diffraction, oriented circular dichroism and in-plane neutron scattering measurements, and evidences from experiments done by others, we have established channel models for alamethicin and magainin, and measured the channel sizes. In this thesis, neutron off-plane scattering of aligned peptide-lipid samples is discussed. Compared to in-plane scattering, this new technique provides a much richer structural information of the sample. We therefore are able to easily distinguish different structures based on the scattering pattern, and, ideally, reconstruct the channel scattering length density in real space. Inter-membrane correlation was observed in the magainin samples.

<p> High-dose pharmacologic ascorbate has promise as an adjuvant to traditional therapies for cancer. It is hypothesized that the peak plasma concentration is a key determinant in treatment efficacy. From the Phase I clinical trails on the use of pharmacological ascorbate as an adjuvant to Gemcitabine in the treatment of stage IV pancreatic cancer at the University of Iowa Hospitals and Clinics, we found that monitoring plasma ascorbate concentration [<i> AscH</i>^&ndash;]<i>_pl</i> with each infusion is both very time consuming and expensive for large scale implementation. A method to determine the amount and protocol to infuse ascorbate to achieve a desired patient [<i>AscH</i>^&ndash;]<i>_pl</i> would be of great benefit. Current models lack flexibility for various infusion protocols. Additionally, constructing a model of ascorbate pharmacokinetics would allow investigation of an optimal dosing regime to maintain constant plasma ascorbate levels.</p><p> A mechanistic model and an empirical model were developed and validated. The mechanistic model suitably replicated the results obtained in the clinical trial but contained too many variables to be useful in a clinical setting. The empirical model showed good results in replicating the trial results and requires only a few easily measured variables to generate predictions.</p><p> High dose ascorbate has been shown to produce hydrogen peroxide. In furthering the studies of how ascorbate affects tumor cells, the action of glucose-6-phosphate dehydrogenase (G6PD) is considered because it supplies NADPH to several peroxide removal pathways. To this end, the kinetics of G6PD were studied using kinetic simulations. G6PD exhibits a reserve capacity, which is the difference between the activity when all intracellular NADP is oxidized to the rate at which is operates when intracellular NADP is at the physiologic 90 % reduced to 10 % oxidized ratio.</p><p> These simulations yielded an interesting pattern which is also seen by evolutionary biologists. G6PD exhibits a response capacity, which is the difference between the maximum G6PD activity exhibited when there is no demand for NADPH greater than normal cell functions and the activity exhibited when all cellular NADP is oxidized.</p>

Excitatory amino acids (EAAs) are known to play an important role in the regulation of dopaminergic neurotransmission. This thesis examined effects of EAAs and modulators of EAA receptor activity on the release of ($ sp3$H) dopamine ( ($ sp3$H) DA) from dissociated cell cultures of ventral mesencephalon. The results provide evidence that multiple EAA receptor subtypes are present on DA-releasing neurons and participate in regulating this release. NMDA, quisqualate and kainate, preferred agonists for respectively named EAA receptor subtypes, stimulated Ca$ sp{2+}$-dependent, tetrodotoxin (TTX)-insensitive ($ sp3$H) DA release, with 3 distinct profiles of sensitivity to antagonists and of ontogenic development in culture. Low concentrations of the endogenous EAAs, glutamate, homocysteate, homocysteine sulfinate, cysteate and cysteine sulfinate evoked Ca$ sp{2+}$-dependent ($ sp3$H) DA release through activation of NMDA receptors, although non-NMDA receptors mediated effects of these agonists at higher concentrations ($ geq$100 $ mu$M, depending upon the EAA tested). Aspartate induced ($ sp3$H) DA release that was mediated solely by the NMDA receptor. / Glycine potentiated NMDA-evoked ($ sp3$H) DA release when the test was made under nominally Mg$ sp{2+}$-free conditions, but caused strychnine-sensitive inhibition of the NMDA response in the presence of 1.2 mM Mg$ sp{2+}$. It is suggested that these results demonstrate how voltage-dependent blockade of the NMDA receptor by Mg$ sp{2+}$ may regulate sensitivity of NMDA responses to convergent inputs. / Micromolar concentrations of the psychotomimetic agent, phencyclidine (PCP) blocked NMDA-evoked ($ sp3$H) DA release. At higher concentrations ($ geq$100 $ mu$M), PCP-related compounds stimulated TTX-insensitive ($ sp3$H) DA release. This stimulatory effect was not due to actions of these compounds at the PCP/NMDA receptor, sigma binding site, dopamine uptake site, or cation channels for Ca$ sp{2+}$ or Na$ sp{+}$, but may have been caused by K$ sp{+}$ channel blockade.

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism. Severe enzymatic deficiency results in metabolic disease with occasional early demise. Mild deficiency is due to a common polymorphism (A222V) which increases risk for several common disorders. Pharmacogenetic studies of the antifolate methotrexate on Mthfr heterozygous knockout mice (Mthfr +/-), a model of the mild human deficiency, revealed significantly higher apoptosis rates in several tissues compared to wildtype mice (Mthfr +/+). These results suggest that mild MTHFR deficiency is a risk factor for methotrexate toxicity in mice and possibly in humans. A new medication, metafolin, was administered to Mthfr +/- mothers to assess survival of their Mthfr -/- pups. Survival rate of pups at 5 weeks of age from treated mothers was significantly higher than that from untreated mothers (54% versus 20%). The results of this study suggest that metafolin could be useful for treatment of severe MTHFR deficiency in humans.

The aim of the studies underlying this thesis was to characterize the effect of catecholamines on developing rat brain oligodendrocytes by characterizing the alpha1-adrenoceptor (alpha1-AR) expressed in oligodendrocytes, determining the signaling pathway downstream from the second messenger molecules, and demonstrating a cytotoxic response to catecholamines. / Norepinephrine (NE) caused a time- and concentration-dependent increase in total inositol phosphate formation (IPt). Using various selective antagonists, we identified that, similar to progenitors, the alpha 1A-AR subtype in mature oligodendrocytes is mediating NE-induced IP t formation. / In examining the signaling transduction pathway, we found that in oligodendrocyte progenitors NE only in the presence of propranolol, a beta-AR antagonist, increased mitogen-activated protein kinase (MAPK) activity. / We further revealed that catecholamines exposure results in cytotoxicity to oligodendrocyte cultures, which is dependent on the dose of dopamine or norepinephrine used, and on the developmental stage of the cultures, with oligodendrocyte progenitors being more vulnerable. Catecholamines caused an increase in oxidative stress as elucidated from reduced intracellular glutathione levels, and increased accumulation in reactive oxygen species and in stress-induced protein, heme oxygenase-1. / In conclusion, our findings showed that developing oligodendrocytes express all three alpha1-AR subtypes but that only the alpha1A -AR was involved in NE-mediated IPt, formation. We also demonstrated that NE activated MAPK and c-fos expression in oligodendrocyte progenitors, suggesting trophic response. Lastly, we demonstrate that catecholamine could induce cytotoxicity in oligodendrocyte cultures, which is developmentally regulated, mediated by oxidative stress and have characteristics of apoptosis in progenitor cells. (Abstract shortened by UMI.)