Attention-deficit/hyperactivity disorder (ADHD) is one of the most common disorders of childhood. It is theorized to be caused by catecholamine dysfunction in the striatum (Str) and frontal cortex (FC). The spontaneously hypertensive rat (SHR) has been used as a model for ADHD because of its attention deficits, impulsiveness, and hyperactivity. Prior studies of dopamine (DA) in the Str and FC have revealed conflicting results in the SHR compared to control, indicative of a need for a better understanding of DA dynamics in this model. In addition to the DA hypothesis, studies have begun implicating glutamate in the etiology of ADHD. Previous evaluations of the SHR model of ADHD found that the SHR have increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity and elevated calcium levels in the FC, suggesting that altered glutamatergic neurotransmission exists in the SHR. The first set of studies presented here suggest that increased surface expression of DA transporters may exist in the SHR model of ADHD, lowering basal DA levels. Second, we discovered that the glutamate system in the FC of the SHR model of ADHD is hyperfunctional, thus raising the possibility that targeting glutamate dysfunction in the FC could lead to the development of novel therapeutics for the treatment of ADHD. The third and fourth set of studies explored glutamate signaling in the awake rodent to fully understand glutamate neurotransmission as well as the effects of methylphenidate (MPH) on glutamate signaling in the prelimbic cortex, a region heavily implicated in ADHD. The SHR displayed similar phasic glutamate signaling compared to control; however, in the SHR but not the WKY control, chronic treatment with MPH lowered phasic glutamate amplitude. Additionally, intermediate treatment with MPH increased tonic glutamate in the SHR only, whereas chronic MPH treatment increased tonic levels in both the SHR and WKY compared to saline. Taken together, this body of work characterizes DA and glutamate signaling in the anesthetized SHR model of ADHD. Additionally, glutamate dynamics and the effects of the stimulant medication MPH were explored in the awake animal, providing evidence that glutamate is a likely target for future neuropharmacology for the treatment of ADHD.

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--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.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.

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.

Glutamate is the essential neurotransmitters in pain pathways. The discovery of the vesicular glutamate transporters (VGLUT1-3) has been a fundamental step on the way to describe glutamate-dependent pain pathways. We used the Cre-lox system to construct conditional knockouts with deficient Vglut2 transmission in specific neuronal populations. We generated a Vglut2f/f;Ht-Pa-Cre line to selectively delete Vglut2 from the peripheral nervous system. These Vglut2 deficient mice showed decreased acute nociceptive responses and were less prone to develop an inflammatory state. They did not develop cold allodynia, or heat hyperalgesia and were less hypersensitive to mechanical stimuli in the PSNL chronic pain model. Further analyses of genes with altered expression after nerve injury, revealed candidates for future studies of chronic pain biomarkers. Interestingly, the Vglut2f/f;Ht-Pa-Cre mice developed an elevated itch behavior. To investigate more specific neuronal populations, we analyzed mice lacking Vglut2 in the Nav1.8 population, as inflammatory hyperalgesia, cold pain, and noxious mechanosensation have been shown to depend upon Nav1.8Cre positive sensory neurons. We showed that deleting Vglut2 in Nav1.8Cre positive neurons abolished thermal hyperalgesia in persistent inflammatory models and responses to noxious mechanical stimuli. We also demonstrated that substance P and VGLUT2-dependent glutamatergic transmission are co-required for the development of formalin-induced inflammatory pain and heat hyperalgesia in persistent inflammatory states. Deletion of Vglut2 in a subpopulation of neurons overlapping with the vanilloid receptor (TRPV1) primary afferents in the dorsal root ganglia resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. Substance P signaling and VGLUT2-mediated glutamatergic transmission in TRPV1 neurons was co-required for the development of inflammatory pain states. Analyses of an itch phenotype uncovered the pathway within TRPV1 neurons, with VGLUT2 playing a regulatory role and GRPR neurons, which are to plausible converge the itch signal in the spinal cord. These studies confirmed the essential role of VGLUT2-dependent glutamatergic transmission in acute and persistent pain states and identified the roles of specific subpopulations of primary afferent neurons. Additionally, a novel pain and itch transmission pathway in TRPV1/VGLUT2 positive neurons was identified, which could be part of the gate control of pain.

T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3. We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms. Perforated patch recordings demonstrated that activation of Galpha<q/11>-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. Further study of the inhibition of Cav3.3 channels by a specific Galpha<q/11>-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics. Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Galpha<q/11> signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Galpha<q/11>-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC). In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs. Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs. A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.

Epidemiological studies indicate that maternal vitamin D deficiency may be a candidate developmental risk factor for schizophrenia. For example, people born in winter/spring, urban environments and dark-skinned individuals whose parents migrated to cooler climates are all at increased risk of developing schizophrenia later in life. The biological plausibility that a low prenatal level of vitamin D has an adverse impact on the developing brain has been studied using a developmental vitamin D (DVD) deficient rat model. These animals display molecular and anatomical abnormalities in brain development and alterations in behaviour as adults. Compared with control rats, neonatal DVD-deficient rat brains are different in shape; displaying a thinner cortex and larger lateral ventricles. Moreover, the brains appear to be less differentiated. At adulthood, DVD-deficient rats show an enhanced sensitivity to novelty-, antipsychotic- and psychomimetic- induced locomotion. These observations have lead to the hypothesis that dopamine and/or glutamate neurotransmission may be altered in DVD-deficient rats. Thus, the main aim of this thesis was to further characterise the dopamine and glutamate neurotransmitter systems in DVD-deficient rats. DVD-deficiency resulted in sex and age specific changes in dopamine signalling. At birth, DVD-deficient rats showed altered dopamine metabolism in the forebrain providing the first report of altered dopamine function after DVD-deficiency. Female DVD-deficient rats displayed a post-adolescent (at 3 months of age) enhanced response to amphetamine-induced locomotion. Accompanying this behavioural sensitivity were decreased levels of dopamine 1 and 2 receptor density in the nucleus accumbens. The altered behaviour in female DVD-deficient rats was not associated with increased dopamine release in the prefrontal cortex, caudate putamen or nucleus accumbens. Although a similar increase in the behavioural sensitivity to amphetamine was not observed in male DVD-deficient adult rats, increases in the density of the dopamine transporter were observed in the caudate putamen and nucleus accumbens. However, when examined at a mature adult age (6 months) neither the enhanced response to amphetamine, receptor or transporter changes persisted. These results suggest that after puberty a transient change in dopamine receptor signalling manifests as an altered response to amphetamine under certain environmental and experimental conditions. Glutamate signalling was probed with the N-methyl-D-aspartate receptor antagonist MK-801. Adult male DVD-deficient rats showed an enhanced locomotor response to MK-801 and this persisted when examined at a mature adult age. Female DVD-deficient rats showed an enhanced response but this was only observed at the mature adult age examined. No behavioral differences were observed prior to adolescence. This behavioural sensitivity did not appear to be due to altered dopamine release after MK-801 in the prefrontal cortex and caudate putamen. Taken together, male DVD-deficient rats develop a locomotor sensitivity to MK-801 at an earlier age than DVD-deficient females. This behavioural alteration is not associated with altered dopamine function. The combined results from the studies in this thesis present a complex phenotype that suggests altered dopamine and glutamate interactions in DVD-deficient rats that are dynamic; demonstrating both age and sex specific traits. I speculate that the development of these behavioural and neurochemical alterations in DVD-deficient rats follows a similar temporal profile to the symptomology observed in schizophrenia patients. Both behavioural sensitivities to amphetamine and MK-801 are observed in schizophrenia in addition to a delayed onset of symptoms in females. This provides further support for a role of vitamin D in the developing brain and suggests that a transient deficiency can result in long-term behavioural and neurochemical alterations. Together this suggests that the DVD-deficient rat model may be an informative model for exploring the developmental vitamin D deficiency hypothesis of schizophrenia.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.