Dopamine mechanisms in the central nervous system are important in the control of both normal and abnormal motor function. The recent observations in both animal and human studies, that melatonin, the principal hormone of the pineal gland, may have a role in the control of movement and the pathophysiology of movement disorders, have given rise to the concept that melatonin may have a modulatory influence on central dopaminergic neurotransmission. This study makes use of three animal behavioural models as well as a biochemical model of central dopaminergic function to further investigate the concept. Results from studies using the biochemical model, which investigated the effect of melatonin on dopamine and apomorphine stimulation of dopamine-sensitive adenylate cylase, suggest that melatonin is neither a competitive antagonist nor agonist at the D₁ receptor level, although the possibility of physiological stimulation or antagonism is not excluded. In behavioural studies, prior melatonin mg/kg administration (1 and 10 (8M) ip) inhibited apomorphine induced stereotypy and locomotor activity in normal rats, and apomorphine-induced rotational behaviour in 6-hydroxydopamine and quinolinic acid lesioned rats. The possibility that these results may have physiological significance is borne out by the observation that, under enviromental lighting conditions that are associated with raised endogeous melatonin levels, apomorphine- induced stereotypy and locomotor activity is attenuated. The general conclusion is that melatonin has an inhibitory influence on central nervous system dopaminergic function, suggesting therefore, that the pineal gland and melatonin may have a role in the pathophysiology and treatment of movement and behavioural disorders associated with dopaminergic dysfunction

The effects of a range of neuronal excitants were examined on the firing of central neurones of the cerebral cortex, ventrobasal thalamus, dentate gyrus and dorsal and ventral horns of the spinal cords of urethane anaesthetized rats. These responses were pharmacologically characterized on the basis of their susceptibilities to a number of antagonists and from these results, inferences were made concerning probable receptor mechanisms employed by the agonists. Throughout these experiments the technique of iontophoresis was found to be an ideal one for evaluating the effects of agonists and antagonists on single neurones. Neurones in the cortex, thalamus and Renshaw cells of the spinal cord were readily excited by acetylcholine. These responses were elicited also by both nicotinic and muscarinic cholinomimetics. Excitations produced by acetylcholine and acetyl-β-methylcholine were antagonized by atropine and those of acetylcholine and nicotinic agonists were blocked by nicotinic antagonists. The results may be interpreted as revealing a difference between excitatory cholinergic receptors in the rat and in the cat; the nature of these receptors is discussed. to The excitatory responses of ventrobasal thalamic neurones iontophoretically applied amino acids related to glutamate and aspartate could be blocked both by glutamate diethylester and α-aminoadipate. These two antagonists were found to possess different mechanisms of action however, as the ranking orders of susceptibility of the agonists differed for each antagonist. An analysis of these orders led to the proposal that more than one and possibly as many as three different receptors for the excitatory amino acids exist on central neurones. A number of additional compounds were tested for an evaluation of their antagonistic properties against the amino acid induced responses, and these results are discussed in light of possible steric requirements of the receptors. Granule cells of the dentate gyrus were excited by the amino acids and by their synaptic responses to stimulation of perforant path and commissural inputs. A differential effectiveness of glutamate diethylester and α-aminoadipate was suggestive that two distinct excitatory amino acid receptors, both of which appear to be of synaptic significance, coexist on the same neurones. The effects of octopamine were compared with those of catecholamines on neurones of the cortex and dorsal horn of the spinal cord. Both excitation and depression of neuronal firing was observed with octopamine and these responses appeared not to be correlated with those effected by the catecholamines. A further separation of the actions of octopamine and the catecholamines was evident when the amine induced responses were compared in the presence of the antagonists, propranolol and α-flupenthixol. These blocking compounds were effective in attenuating the effects of the catecholamines, but had no effect upon the octopamine induced changes in firing rate. The results suggest that receptors sensitive to octopamine and which appear to be pharmacologically distinct from those previously categorized as catecholamine receptors, may exist on central neurones of the rat. On the basis of the present findings, it was evident that when the technique of iontophoresis is combined with standard neurophysiological methods of identifying central neurones by their responses to synaptic stimulation, valuable information can be obtained concerning the nature of the synaptic transmitters employed by these cells. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Calcium (Ca2+) is a universal second messenger controlling a wide variety of cellular reactions and adaptive responses. All the versatility of a Ca2+ signaling requires that the concentration of Ca2+ ions in the cytoplasm be highly regulated. Generation of Ca2+ mobilizing signals in cells involves regulation by multiple components controlling Ca2+ release from the internal stores, Ca2+ influx across the plasma membrane, elicitation of Ca2+ sensitive processes and finally the removal of Ca2+ from the cells. Inositol-1, 4, 5-trisphosphate receptors (IP3Rs) and ryandine receptors (RyRs) are the most studied Ca2+ release channels located on the internal stores. Previous studies have shown ryanodine receptors (RyRs) play a key role in the process of Ca2+ signaling participating in the oscillatory patterns of controlling the release of Ca2+ from ER and regulating the influx of Ca2+ by coupling with plasma Ca2+ channels. Although recent progress deciphered the behavior and function of RyRs in regulation of Ca2+ signal, it still remains mysterious in understanding the molecular mechanism of its regulation and its connection with plasma membrane Ca2+ channels in neuronal cells. Here this study aimed to utilized the most cutting-edge RNA interference techniques, along with well-characterized pharmacological regulators of RyRs, to better characterized the role of RyRs is our neuronal cell line model NG115-401L. Our first main goal of this project was to develop an effective protocol that could selectively knockout or knockdown expression levels of the RyR1 gene in NG115-401L cells. After testing different siRNA primers including their combination with different transfection reagent, the result shows a significant silencing effect to the RyR1 mRNA expression levels. In the second part of this study, we used a group of pharmacological agents with well-known regulatory actions on RyRs to characterize the functional roles of the RyRs expressed in NG115-401L cells. All four agonists which are ryanodine, caffeine, CMC and PCB 95 show their abilities to activate the RyRs, increase [Ca2+]iand induce the influx of Ca2+ via SOC. After transfected NG115-401L cells by siRNA, the Ca2+ release and influx signals were highly diminished suggesting RyR1 gene was successfully knocked down and the successfully knocked down and the Ca2+ mobilization mediated by RyR1 was decreased greatly. Finally in order to study the effects of the regulation of Ca2+ by RyR modulators and RyR gene knockdown on cell growth patterns and cell viability, the NG115-401L cells were exposed to various concentrations of RyR regulators and siRyR1 primer for different time periods. The siRNA transfection showed the least effect on cell growth, as compared with pharmacological agents that modulate RyR function. Considering we achieved high levels of gene knockdown and its low cytotoxity, our result suggests that siRNA silencing for RyRs may become a promising gene therapeutic target in the future.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-‐gated sodium channels (VGSCs) are critical for the initiation and propagation of electrical signals in neurons; consequently they are significant regulators of neuronal excitability. They are exquisitely tuned and aberrations in their activity can lead to pathophysiological conditions. This dissertation highlights the roles of two prominent brain isoforms of VGSCs, Nav1.1 and Nav1.6. These isoforms have distinct localization in the brain. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment (AIS) of GABAergic neurons, while Nav1.6 is found at the distal AIS and nodes of Ranvier of both GABAergic and excitatory neurons. Several mutations have been identified in Nav1.1 and recently mutations in Nav1.6 have been discovered in patients with distinct epileptic phenotypes that respond poorly to current anti-epileptics. There is a need to better understand mechanistically how mutations in these channel isoforms lead to epilepsy in order to identify more efficacious treatment strategies. Therefore, the aims of this dissertation were to 1) examine the differential biophysical properties of Nav1.1 and Nav1.6, 2) determine the biophysical consequences of epilepsy-­associated mutations in Nav1.1 and Nav1.6 and examine the effects of cannabinoids on wildtype and mutant channel activity and 3) test the effects of selective inhibition of Nav1.1 versus Nav1.6 on epileptiform activity. To address these aims, whole­‐cell electrophysiology and mutlielectrode array recordings were used. The results demonstrate that 1) Nav1.1 and Nav1.6 have important differences in their biophysical properties that may be important in the fine­‐tuning of neuronal excitability, 2) epilepsy-­‐associated mutations in Nav1.1 and Nav1.6 alter several biophysical properties of the channels but have differential effects on resurgent current generation suggesting a divergence in the mechanism by which they induce epileptogenesis and cannabidiol can inhibit aberrant channel activity and reduce neuronal excitability and 3) pharmacological inhibition of Nav1.6, but not Nav1.1, abolishes epileptiform activity. Overall, this dissertation provides insight into the distinct contributions of Nav1.1 and Nav1.6 to physiological and pathophysiological firing activity and their ability to be targeted for therapeutic purposes. This knowledge is critical for understanding the potential role of VGSCs in epilepsy syndromes and identifying possible drug targets for more efficacious treatment strategies.

Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our results reveal a novel trypsin-initiated escape response in fish. These studies should enhance our understanding of fish communication in general and alarm behavior in particular. Furthermore, since pain receptors in other animals are also PAR2, our finding may be useful in exploring pathways of pain reception.

A "complex" system typically has a relatively large number of dynamically interacting components and tends to exhibit emergent behavior that cannot be explained by analyzing each component separately. A biological neural network is one example of such a system. A multi-agent model of such a network is developed to study the relationships between a network's structure and its spike train output. Using this model, inferences are made about the synaptic structure of networks through cluster analysis of spike train summary statistics A complexity measure for the network structure is also presented which has a one-to-one correspondence with the standard time series complexity measure sample entropy.

Background: Light-adaptation is a multifaceted process in the retina that helps adjust the visual system to changing illumination levels. Many studies are focused on the photochemical mechanism of light-adaptation. Neural network adaptation mechanisms at the photoreceptor synapse are largely unknown. We find that large, spontaneous Excitatory Amino Acid Transporter (EAATs) activity in cone terminals may contribute to cone synaptic adaptation, specifically with respect to how these signals change in differing conditions of light. EAATs in neurons quickly transport glutamate from the synaptic cleft, and also elicit large thermodynamically uncoupled Cl- currents when activated. We recorded synaptic EAAT currents from cones to study glutamate-uptake events elicited by glutamate release from the local cone, and from adjacent photoreceptors. We find that cones are synaptically connected via EAATs in dark ; this synaptic connection is diminished in light-adapted cones. Methods: Whole-cell patch-clamp was performed on dark- and transiently light-adapted tiger salamander cones. Endogenous EAAT currents were recorded in cones with a short depolarization to -10mV/2ms, while spontaneous transporter currents from network cones were observed while a local cone holding at -70mV constantly. DHKA, a specific transporter inhibitor, was used to identify EAAT2 currents in the cone terminals, while TBOA identified other EAAT subtypes. GABAergic and glycinergic network inputs were always blocked with picrotoxin and strychnine. Results: Spontaneous EAAT currents were observed in cones held constantly at -70mV in dark, indicating that the cones received glutamate inputs from adjacent photoreceptors. These spontaneous EAAT currents disappeared in presence of a strong light, possibly because the light suppressed glutamate releases from the adjacent photoreceptors. The spontaneous EAAT currents were blocked with TBOA, but not DHKA, an inhibitor for EAAT2 subtype, suggesting that a / non-EAAT 2 subtype may reside in a basal or perisynaptic area of cones, with a specialized ability to bind exocytosed glutamate from adjacent cones in dark. Furthermore, these results could be artificially replicated by dual-electrode recordings from two adjacent cones. When glutamate release was elicited from one cone, the TBOA-sensitive EAAT currents were observed from the other cone. Conclusions: Cones appear to act like a meshwork, synaptically connected via glutamate transporters. Light attenuates glutamate release and diminishes the cone-cone synaptic connections. This process may act as an important network mechanism for cone light adaptation. / by Matthew JM Rowan. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Rhythmic movementssuch as swimming, flying, and walkingare ubiquitous in nature. Intrinsically active neural networks called central pattern generators (CPGs) provide the feedforward signals to actuate these movements, but the preferred movement frequency is often equivalent to the resonant frequency of the musculoskeletal system. Sensory feedback is essential to synchronize the neural and musculoskeletal systems to the mechanical resonant frequency, a phenomenon called resonance tuning. In this dissertation, we use a simple computational model of rhythmic movement to understand how the configuration of sensory feedback affects both the sensitivity of resonance tuning to parameter variation and the resiliency of resonance tuning to perturbation. Although previous studies have shown that resonance tuning is limited to frequencies that are above the intrinsic CPG frequency, we demonstrate that this limitation is only valid with negative feedback and with endogenously bursting CPG neurons. Specifically, we show that with positive feedback, resonance tuning occurs at frequencies that are below the intrinsic CPG frequency. Moreover, when the synaptic connections within the CPG are required for bursting activity, resonance tuning occurs both above and below the intrinsic CPG frequency with negative feedback and does not occur with positive feedback. Using Floquet analysis, we then demonstrate that perturbations decay more quickly when resonance tuning is realized with positive than with negative proportional feedback. Finally, we evaluate how the intrinsic CPG frequency, feedback gain, and mechanical damping affect the stability and range of resonance tuning with negative and positive feedback. Overall, these results indicate that the configuration of sensory feedback dramatically affects both the parameter space in which resonance tuning occurs and the stability of the resultant periodic motion.

The work we present in this thesis is a series of studies of how heterogeneities in coupling affect the synchronization of coupled neural oscillators. We begin by examining how heterogeneity in coupling strength affects the equilibrium phase difference of a pair of coupled, spiking neurons when compared to the case of identical coupling. This study is performed using pairs of Hodgkin-Huxley and Wang-Buzsaki neurons. We find that heterogeneity in coupling strength breaks the symmetry of the bifurcation diagrams of equilibrium phase difference versus the synaptic rate constant for weakly coupled pairs of neurons. We observe important qualitative changes such as the loss of the ubiquitous in-phase and anti-phase solutions found when the coupling is identical and regions of parameter space where no phase locked solution exists. Another type of heterogeneity can be found by having different types of coupling between oscillators. Synaptic coupling between neurons can either be exciting or inhibiting. We examine the synchronization dynamics when a pair of neurons is coupled with one excitatory and one inhibitory synapse. We also use coupled pairs of Hodgkin-Huxley neurons and Wang-Buzsaki neurons for this work. We then explore the existance of 1:n coupled states for a coupled pair of theta neurons. We do this in order to reproduce an observed effect called quantal slowing. Quantal slowing is the phenomena where jumping between different $1:n$ coupled states is observed instead of gradual changes in period as a parameter in the system is varied. All of these topics fall under the general heading of coupled, non-linear oscillators and specifically weakly coupled, neural oscillators. The audience for this thesis is most likely going to be a mixed crowd as the research reported herein is interdisciplinary. Choosing the content for the introduction proved far more challenging than expected. It might be impossible to write a maximally useful introductory portion of a thesis when it could be read by a physicist, mathematician, engineer or biologist. Undoubtedly readers will find some portion of this introduction elementary. At the risk of boring some or all of my readers we decided it was best to proceed so that enough of the mathematical (biological) background is explained in the introduction so that a biologist (mathematician) is able to appreciate the motivations for the research and the results presented. We begin with a introduction in nonlinear dynamics explaining the mathematical tools we use to characterize the excitability of individual neurons, as well as oscillations and synchrony in neural networks. The next part of the introductory material is an overview of the biology of neurons. We then describe the neuron models used in this work and finally describe the techniques we employ to study coupled neurons.

Spinal cord injuries (SCI) sever communication between supraspinal centers and the central pattern generator (CPG) responsible for locomotion. Because the CPG is intact and retains the ability to initiate locomotor activity, it can be accessed electrically and pharmacologically. The goal of this thesis was to identify and characterize a novel spinal cord surface site along the sacral dorsal column (sDC) for electrically evoking locomotor-like activity in the neonatal rat spinal cord. Stimulation of the sDC robustly activated rhythmic left-right alternation in flexor-related ventral roots that was dependent on the activation of high-threshold C fiber afferents. The C fibers synapsed onto spinal neurons, which project to the lumbar segments as part of a pathway dependent on purinergic, adrenergic, and cholinergic receptor activation. In ventral roots containing only somatic efferents, rhythmic activity was rarely recruited. However, in ventral roots containing both autonomic and somatic efferents, sacral dorsal column stimulation recruited autonomic efferent rhythms, which subsequently recruited somatic efferent motor rhythms. The efferent rhythms revealed a half-center organization with very low stimulation frequencies, and the evoked alternating bursts entrained to the stimuli. Similar entrainment was seen when sDC stimuli were applied during ongoing neurochemically-induced locomotor rhythms. The rhythmic patterns evoked by sDC stimulation operated over a limited frequency range, with a discrete burst structure of fast-onset, frequency-independent peaks. In comparison, neurochemically-induced locomotor bursts operated over a wide frequency range and had slower time to peaks that varied with burst frequency. The overall findings support the discovery of an autonomic efferent pattern generator that is recruited by sacral visceral C fiber afferents. It is hoped that this research will advance the understanding of afferent activation of the lumbar central pattern generator and potentially provide insight useful for future development and design of neuroprosthetic devices.