The green algae Chlamydomonas reinhardtii senses light through two photosensory proteins, channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2). The initial discovery of these two photoreceptors introduced a new class of light-gated ion channels. ChR2 is an inwardly-rectified ion channel that is selective for cations of multiple valencies. Similar to microbial-rhodopsin ion pumps, ChR2 has a seven transmembrane domain motif that binds the chromophore all-trans retinal through a protonated Schiff base linkage. Physiologically, ChR2 functions to depolarize the membrane which initiates a signaling cascade triggering phototactic response. This fundamental property has been pivotal in pioneering the field of optogenetics, where excitable cells can be manipulated by light. ChR2 reliably causes neuronal spiking with high spatial and temporal control. Moreover, the recent discovery of new chloride-conducting channelrhodopsins (ChloCs) has further expanded the optogenetic toolbox. Although structurally similar to microbial-rhodopsin ion pumps, ChR2 undergoes more complex conformational rearrangements that lead to ion conductance. Currently, the molecular basis for ChR2 gating remains unresolved. Revealing the specific structural interactions that modulate ChR2 function have important implications in understanding the intricacies of ion transport and molecular differences between ion pumps, channels, and transporters. Here we describe a combined computational and experimental approach to elucidate the mechanism of ion conductance, channel gating, and structure-function relationship of ChR2. Our results have contributed to expanding our understanding of the fundamental properties of ion channels.

Amyotrophic Lateral Sclerosis (ALS) is a debilitating and fatal neurodegenerative disease affecting upper and lower motor neurons. Though studied for over two decades since the first ALS-associated genetic mutation was discovered, researchers have yet to uncover the pathological processes that lead to progressive degeneration of motor neurons in ALS, or to develop effective treatments. One prominent hypothesis proposes that excitotoxicity caused by increased motor neuron firing plays a role in ALS pathogenesis. While prior studies reported increased action potential firing in early postnatal ALS-model motor neurons in vivo, it remains unknown whether the increased activity stems from increased intrinsic excitability of ALS motor neurons or from increased excitatory drive, and whether these changes are transient or persist into adulthood, when ALS symptoms emerge. In this thesis, I circumvented the difficulties in standard measurement of electrophysiological properties of adult spinal motor neurons in vivo by relying on the visualization of the axon initial segment, a subcellular structure known to undergo compensatory structural changes in response to perturbations in excitatory input. I discovered that cultured motor neurons derived from stem cells of the SOD1G93A mouse model of ALS display shortened axon initial segments and hypoexcitable electrophysiological properties. The shortening of the axon initial segment is compensatory, as ALS motor neurons receive increased numbers of excitatory inputs and manifest increased spontaneous activity. Remarkably, similar shortening of the axon initial segment was detected in early presymptomatic spinal motor neurons in vivo. The shortened axon initial segment persists into the symptomatic stages and is particularly pronounced in motor neurons containing p62 immunoreactive aggregates and neurons exhibiting swollen mitochondria, two signs of stress and neurodegeneration in the disease. Based on these observations I propose that early in the presymptomatic stages of the disease, spinal motor neurons recruit excessive excitatory inputs, resulting in their increased activity that is in part compensated by shortening of the axon initial segment. This state persists and becomes even more pronounced in motor neurons exhibiting biochemical changes preceding neurodegeneration. While these observations support the potential role for excitotoxic stress in spinal ALS motor neurons, I paradoxically observed the opposite phenotype in ALS-vulnerable cranial motor neurons in the brainstem of the SOD1G93A animals, raising the possibility that the cellular stress that drives the neurodegeneration in ALS is motor neuron subtype specific.

GRIN2A and GRIN2B encode the GluN2A and GluN2B subunits of the NMDA receptor, a subtype of ionotropic glutamate receptor that displays voltage-dependent block by Mg2+ and a high permeability to Ca2+. These receptors play important roles in synaptogenesis, synaptic transmission and synaptic plasticity, as well as contributing to neuronal loss and dysfunction in several neurological disorders. Recently, individuals with a range of childhood onset epilepsies, intellectual disability and other neurodevelopmental abnormalities have been found to carry heterozygous gene-disrupting or protein-altering point mutations in GRIN2A and GRIN2B. This thesis addresses the hypothesis that these point mutations cause key functional disturbances to NMDA receptor properties that contribute to neurodevelopmental disorders. To test this hypothesis, a group of related mutations were selected for functional assessment in heterologous systems: four missense mutations affecting residues in or near the subunit pore regions, all of which are associated with epilepsy and intellectual disability. To model the impact of gene disrupting mutations in GRIN2A, a preliminary analysis of the functional consequences of GluN2A haploinsufficiency in a genetically modified rat was also performed. Three of the four missense mutations were found to be associated with profound alterations in fundamental NMDA receptor properties: compared to wild type, GluN2AN615K was found to reduce Mg2+ block, GluN2BN615I and GluN2BV618G to cause potentiation by Mg2+, and GluN2AN615K and GluN2BN615I showed reduced conductance. GluN2AR586K was not found to influence the parameters assessed. When GluN2AN615K was expressed alongside wild type subunits in the same NMDA receptor, it was found to have a dominant negative effect. Finally, I established successful gene targeting in a new rat Grin2A knock-out model, and observed that heterozygous neurons had lower GluN2A protein expression and current density, making a good model to study human epilepsies associated with loss of a GRIN2A allele. This thesis provides evidence that three missense mutations in GRIN2A and GRIN2B affect physiologically important properties of the NMDA receptor, and that GluN2A haploinsufficiency influences important neural properties in vitro. This is consistent with these mutations causing disease and highlights these and related mutations as potential therapeutic targets in the future.

The nuclei of the midline thalamus are an important part of the brain’s limbic system. Previous work has described the presence of dopamine D2 receptors in the midline thalamus in humans, non-human primates, and rodents. A similar body of literature has also demonstrated dopaminergic innervation of the midline thalamus across these species. However, little is known regarding a) the source of dopaminergic innervation to the midline thalamus in rodents and b) the function of D2R in the midline thalamus in any species. I begin this thesis with a review of the literature examining the anatomy, electrophysiological properties, and role in behavior of the paraventricular nucleus of the thalamus (PVT), a region where D2R mRNA and protein is expressed. I next describe a series of three sets of experiments aimed toward examining the anatomical, electrophysiological, and behavioral role of D2R in the PVT in mice. In the first set of experiments, I used anatomical methods to show that D2R are particularly enriched in neurons of the PVT. I focused on D2R-expressing PVT neurons specifically and show their afferent and efferent projections throughout the brain. In addition, I describe a set of experiments aimed to establish a dopaminergic innervation to the PVT. In the second set of experiments, I used electrophysiological methods to study D2R-expressing PVT neurons. Here, I establish that tonic firing in D2R-expressing thalamic relay neurons in the PVT is inhibited by quinpirole, a D2R/D3R agonist, and increased by sulpiride, a D2R/D3R antagonist. In the third set of experiments, I assessed the behavioral function of D2R in PVT neurons since this has never been studied in any species. I directly manipulated PVT D2R in two directions: a) by overexpressing D2R, and b) by downregulating D2R. Here I show PVT D2R plays a role in both cocaine locomotor sensitization as well as contextual fear expression. Our findings demonstrate for the first time the role of D2R in the PVT and add to literature suggesting that the PVT is an important component of the neural circuitry underlying fear behavior and drug reward. I conclude this thesis with a discussion of the findings described in the three sets of experiments as well as a proposal for future experiments.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and the presence of stereotypic behaviors or restricted interests. To explore possible consequences of an excitatory/inhibitory (E/I) imbalance on the visual system in ASD, we investigated spatial suppression in 16 children with ASD and 16 neurotypical comparison children from 6 - 12 years of age using a visual motion processing task during high-density electroencephalography (EEG) recording in order to derive the N1 event related potential (ERP). Consistent with prior behavioral research, neurotypical participants displayed spatial suppression in conditions of large, high-contrast sinusoidal gratings as indexed by delayed N1 response latency. As predicted, children with ASD displayed weakened surround suppression, i.e. shorter N1 response latency to large, high-contrast sinusoidal gratings. However, this study also unexpectedly revealed that children with ASD showed longer N1 latencies in response to small, high-contrast sinusoidal gratings as compared to neurotypical control children. Although there were no statistically significant differences between children with ASD and NT children for N1 peak amplitude, there was a strong negative correlation between N1 amplitude represented in absolute values for large, high-contrast sinusoidal gratings and hyper-responsiveness item mean scores on the Sensory Experiences Questionnaire for children with ASD, but not for NT children. As predicted, no significant differences were found within or between groups in the low-contrast experiment. Our results are indicative of weakened spatial suppression and deficits in contrast gain in children with ASD, suggestive of an underlying E/I imbalance in ASD.

Proper functioning of the voltage gated sodium channel, NaV1.5, is essential for maintenance of normal cardiac electrophysiological properties. Changes to the biophysical properties of sodium channels can take many forms and can affect the peak component of current carried during phase zero of the action potential; the “persistent” or “late” current component conducted during the repolarizing phases of the action potential; the availability of the channel as seen by changes in window current; and the kinetics of channel transitions between closed, opened and inactivated states. Mutations in NaV1.5 that alter these parameters of channel function are linked to a number of cardiac diseases including arrhythmias such as atrial fibrillation. In addition, mutations in many of the auxiliary proteins that form part of the sodium channel macromolecular complex have likewise been associated with diseases of the heart. Mutations in regions of the sodium channel responsible for interactions with these auxiliary proteins have also been linked to various dysfunctional cardiac states. Indeed, a large number of disease causing mutations are localized to the C-terminal domain of NaV1.5, a hotspot for interacting proteins. Using a transgenic mouse model, we show that expression of a mutant sodium channel with gain-of-function properties conferring increased persistent current, is sufficient to cause both structural and electrophysiological abnormalities in the heart driving the development of spontaneous and prolonged episodes of atrial fibrillation. The sustained and spontaneous atrial arrhythmias, an unusual if not unique phenotype in mice, enabled explorations of mechanisms of atrial fibrillation using in vivo (telemetry), ex vivo (optical voltage mapping), and in vitro (cellular electrophysiology) techniques. Since persistent sodium current was the driver of the structural and electrophysiological abnormalities leading to atrial fibrillation, we subsequently pursued studies exploring the mechanisms of persistent sodium current. Prior work of heterologously expressed sodium channels identified calmodulin as a regulator of persistent current. Mutation of the calmodulin binding site in the C-terminus of the cardiac sodium channel caused increased persistent current when the channel was expressed heterologously. The role of calmodulin in the regulation of the sodium channel in cardiomyocytes has not been definitively determined. We created transgenic mice expressing human sodium channels harboring a mutation of the calmodulin binding site. Using whole cell patch clamping, we found, in contrast to previously reported findings, that ablation of the calmodulin binding site did not induce increased persistent sodium current. Instead, loss of calmodulin binding stabilized the inactivated state by shifting the V50 for steady-state inactivation in the hyperpolarizing direction. Furthermore, loss of calmodulin binding sped up the transition to the inactivated state demonstrated by a significantly shortened tau of inactivation. In contrast to studies performed in heterologous expression systems, our findings thus suggest that in heart cells, calmodulin binding increases availability, similar to its role in regulating NaV1.4 channels. The studies were then expanded to explore the role of other interacting proteins, fibroblast growth factor (FGF) homologous factors (FHF), in the presence and absence of calmodulin binding. Using whole cell patch clamping, we found that a mutation (H1849R) of the sodium channel causing decreased FHF binding affinity leads to a rightward shift in steady-state inactivation and a slowed rate of inactivation of INa. A third mutant channel, with concurrent decreased FHF and calmodulin binding affinity similarly results in a rightward shift in steady-state inactivation suggesting a dominant effect of the H1849R mutation. Persistent current was not elevated in either of these mutant channels. Importantly, the methodology that we report enables us and other groups to carry out studies of human sodium channels in the native environment of NaV1.5. Our investigation into calmodulin’s role, which yielded conclusions distinct from prior findings in heterologous expression systems, demonstrates the value of this approach.

In 2008, The American College of Cardiology/American Heart Association/Heart Rhythm Society issued revised guidelines for automatic implantable cardioverter-defibrillators (AICD). We hypothesized that these guidelines have influenced AICD implantation rates.

The nature and assessment of executive function are areas of active research. Many current assessments of executive function are complex, have limited reliability and validity, and suffer from task impurity, meaning other cognitive processes may indirectly influence task performance. Additionally, measures may be culture, language, or education bound limiting their use in certain populations (Miyake, Emerson, & Friedman, 2000; Miyake, Friedman, et al., 2000; Strauss, Sherman, & Spreen, 2006; Stuss, 2007). The purpose of this project was to develop a novel set of executive function measures to address issues with current clinical measures. The new measures 1) can be used in an ERP environment, 2) use the same stimulus set to address task impurity and 3) use simpler cognitive operations of inhibition, set-shifting, and updating, identified in previous research by Miyake et al., (2000). Twenty-nine undergraduate participants at the University of South Florida were administered currently used clinical measures of executive function theorized to engage in inhibition, set-shifting, and updating and the set of the novel tasks. ERP data was collected during the administration of the novel tasks. Behaviorally, conditions theorized to engage executive function resulted in slower response reaction time than control conditions. Additionally, behavioral results indicated that performance on novel tasks were differentially related to different clinical EF tasks. ERP differences were observed between both Go/No-Go conditions (inhibition) and among N-back conditions (updating). Results suggest the novel executive function tasks are tapping into different cognitive processes and may be a viable tool for studying executive function in the future.

No description available.

The difficulty with movement initiation, or akinesia, is a cardinal symptom of Parkinson�s disease (PD) and the loss of dopaminergic cells, affecting the function of the basal ganglia, the thalamus and the motor cortex, has long been documented. From a broader perspective, it has been proposed that akinesia is caused by impaired function in different brain areas, inside and outside the basal ganglia, operating as a �behavioural arrest control system� (Klemm, 2001). Several neurotransmitters seem to modulate the activity of this system and, contrasting the well-known effects of dopamine, the involvement of adenosine has only recently emerged, particularly via A2A receptors. Adenosine plays an opposite role to dopamine in the brain: adenosine stimulation at A2A receptors inhibits movement (Ferre et al., 1991a; Hauber and Munkle, 1995; Rimondini et al., 1997), whereas A2A antagonists seem to promote movement (Kanda et al., 2000; Bara-Jimenez et al., 2003; Pinna et al., 2005). Although specific adenosine A2A and dopamine D2 receptors are known to antagonistically interact (Ferre et al., 1997; Fuxe et al., 1998; Ferre et al., 2001), little is known of the involvement of A2A receptors in regulating neural activity in the basal ganglia, a crucial point for the future use of A2A antagonists as adjuvant therapy in Parkinson�s disease. In fact, although it is generally accepted that akinesia results from altered function in the cortico-basal ganglia-cortical loop, as confirmed in several studies reporting changes in basal ganglia activity following dopamine depletion (Blandini et al., 2000; Bevan et al., 2002; Boraud et al., 2002), no study to date has systematically investigated electrophysiological changes in the basal ganglia during akinesia induced by adenosine receptor stimulation. Starting from a common behavioural effect, this study tries to bridge this gap by investigating and comparing, in two basal ganglia structures, the neural substrate of akinesia after acute dopamine D2 receptor blockade and adenosine A2A receptor stimulation. The external segment of the globus pallidus (GP, or simply globus pallidus in the rat) and the substantia nigra pars reticulata (SNr) were chosen as the recording sites because both nuclei are included into the �behavioural arrest control system� and seem to express somewhat complementary functions, as a respective key integrative station and main output of the basal ganglia. Dopamine function was manipulated by acute decrease in availability of dopamine binding sites in the brain, through specific dopamine D2 receptor blockade with systemic injections (1.0 and 1.5 mg/kg) of raclopride(3,5-dichloro-N-[(1-ethylpyrrolidin-2-y)methyl]-2-hydroxy-6-methoxy-benzamide), resulting in akinesia. Conversely, movement was inhibited by specific adenosine A2A receptor stimulation with systemic injections (2.5 and 5.0 mg/kg) of the drug CGS21680 (sodium-2-p-carboxyethylphenylamino-5-N-carboxamidoadenosine). In both situations, behaviour was assessed through specific akinesia tests. Single neuron activity before injection and changes in the firing frequency and firing pattern occurring after injection have been analysed and compared for each cell recorded from GP and SNr, during periods of behavioural rest. Synchronised firing between cell pairs has also been assessed. However, the small number of cell pairs showing correlated firing in each structure after systemic injection of drugs was not statistically relevant for further analysis and interpretation of synchronised firing during drug induced akinesia. In our experiments, both drugs inhibited movement, albeit somewhat differently, with lack of rigidity and �flat� body position after adenosine stimulation. Dopamine blockade decreased mean firing rate and dramatically altered the firing pattern in both investigated structures, generally increasing burst activity (increased percentage of spikes in bursts, mean number of bursts, mean number of spikes per burst, mean intra-burst firing frequency) and decreasing regularity of firing (increased coefficient of variation of the inter-spike intervals). Increased burst activity in the rat basal ganglia in an acute model of parkinsonian akinesia, following systemic raclopride injections, confirmed the importance of changes in the firing pattern in PD. The only electrophysiological effect of systemic A2A stimulation was decreased mean firing rate in the GP, a weak effect that could not propagate towards output stations of the basal ganglia. The lack of changes in the firing pattern, at both input and output levels of the basal ganglia, suggests a correlation with the lack of rigidity in adenosine-stimulation-induced akinesia.