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My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

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.

The two neuron types that initially degenerate with Alzheimer's Disease are the cholinergic projections from the septum to the hippocampus and from the substantia innominata to the cortex, and the somatostatinergic neurons in the hippocampus and cortex. The functional relationship between these two types of neurons was investigated using folic acid, a neuro-excitant, and cysteamine, a somatostatin depleter. Folic acid causes a neuron to fire at a much higher rate than normal (Spector, 1971). Folic acid was injected into either the septum or the substantia innominata, and the long-term effect of the resulting acute hyperactivity of the cholinergic neurons on somatostatin neurons was measured as somatostatin-like immunoreactivity in the hippocampus and cortex. Glutamic acid decarboxylase activity, a marker for gamma-amino butyric acid (GABA) neurons, was also measured because it has been shown to decrease in the cortex after injection of folic acid into the substantia innominata. The administration of folic acid to the cholinergic neurons did not have a significant long-term effect on somatostatin-like immunoreactivity nor glutamic acid decarboxylase activity; therefore, a hyperactivity of the cholinergic neurons did not result in degeneration of GABAergic nor somatostatinergic neurons. Cysteamine causes a short-term depletion of somatostatin. Cysteamine was injected subcutaneously and the effect of an acute decrease of brain somatostatin on the cholinergic neurons was studied by measuring high affinity choline uptake, an indicator of cholinergic activity. Administration of cysteamine had no measured effect on high affinity choline uptake in the hippocampus or frontal cortex; therefore, a depletion of somatostatin did not effect cholinergic activity. The assay for high affinity choline uptake was tested by injection of pentobarbital, a drug known to decrease high affinity choline uptake. We detected a decrease in high affinity choline uptake after pentobarbital administration, indicating that if cysteamine were decreasing high affinity choline uptake, the assay would have detected it. / Master of Science

The central nervous system is largely responsible for receiving sensory information from the environment and determining motor output. Yet, centrally-derived behavior and sensation depends on the optimal maintenance of the cells, tissues, and organs that feed and support these functions. Most of visceral regulation occurs without conscious oversight, making the spinal cord a key site for integration and control. How the spinal cord modulates output to our organs, or sensory information from them, is poorly understood. The overall aim of this dissertation was to better understand spinal processing of both visceral sensory information to and sympathetic output from the spinal cord. I first established and validated a HB9-GFP transgenic mouse model that unambiguously identified sympathetic preganglionic neurons (SPNs), the spinal output neurons for the sympathetic nervous system. Using this model, I investigated the electrophysiological similarities and diversity of SPNs, and compared their active and passive membrane properties to those in other animal models. My results indicate that while many of the same characteristics are shared, SPNs are a heterogeneous group that can be differentiated based on their electrophysiological properties. Since descending monoaminergic pathways have particularly dense projections to sympathetic regions of the spinal cord, I next examined the modulatory role that the monoamines have on spinal sympathetic output. While each neuromodulator tested had a unique signature of action, serotonin and norepinephrine appeared to increase the excitability of individual SPNs, while dopamine had more mixed actions. Since many autonomic reflexes are integrated by the spinal cord, I also questioned whether these reflexes would be similarly modulated. I therefore developed a novel in vitro spinal cord and sympathetic chain preparation, which allowed for the investigation of visceral afferent-mediated reflexes and their neuromodulation by monoamines. This preparation exposed a dichotomy of action, where sympathetic and somatic motor output is generally enhanced by the monoamines, but reflexes mediated by visceral input are depressed. Utilizing the spinal cord and sympathetic chain preparation, I also investigated how the spinal cord modulates visceral sensory information. One of the most powerful means of selectively inhibiting afferent information from reaching the spinal cord is presynaptic inhibition. I hypothesized that both spinal visceral afferents and descending monoaminergic systems would depress transmission of visceral afferents to the spinal cord. My results demonstrated that activity in spinal visceral afferents can lead to spinally generated presynaptic inhibition, and that in addition to depressing synaptic transmission to the spinal cord, the monoamines also depress the intrinsic circuitry that generates this activity-dependent presynaptic inhibition. Taken together, my results indicate that descending monoaminergic pathways act to limit the amount of visceral sensory information reaching the central nervous system and increase sympathetic output, resulting in an uncoupling of output from visceral sensory input and transitioning to a feed-forward, sympathetically dominant control strategy. This combination offers complex modulatory strategies for descending systems.

The versatility of Ca2+ as a messenger regulating a myriad of signalling events requires that the concentration of Ca2+ ions in the cytoplasm be highly regulated. Capacitative Ca2+ entry (CCE) or store-operated Ca2+ (SOC) entry, whereby the depletion of intracellular Ca2+ stores induces the influx of Ca2+ across the plasma membrane, plays a crucial role in Ca2+ signalling. Despite the recent advances in elucidating the entry pathway, its molecular identity, biophysical properties and store-depletion signal remains undefined. Thapsigargin (TG), a sarcoplasmic/endoplasmic reticulum Ca2+ A TPase pump (SERCA), inhibitor induces passive depletion of the internal Ca2+ stores and triggers CCE. The universality of this signal has been widely accepted and TG has proven to be a valuable tool in studying CCE. The neuronal cell line NG 115 -401 L lacks the TG activated Ca2+ influx pathway. Agonists of the ryanodine receptor (RyR); chlorom- cresol (CMC), polychlorinated biphenyl 95 (PCB), ryanodine, caffeine, and that of the inositol-1 ,4 ,5-trisphosphate receptor (IP3R), bradykinin, effectively couple to the activation of Ca2+ influx in these cells. The Ca2+ influx signal due to these agonists can be inhibited by SOC blockers such as La3+, Zn2+, Ni2+ and SF&F 96365. Thapsigargin, CMC and PCB95 share the same Ca2+ releasable pools in the 401 L cells. Our data thus suggests that the channels present in the 401 L cells are likely to be receptor-activated channels rather than the store-depletion activated channels. Cell viability studies show that thapsigargin (25 nM) can decrease viability by 75% within 24 hrs and the RyR agonist caffeine decreased viability to <60% within 24hrs. CMC, PCB95 and ryanodine also were cytotoxic at higher doses. Nuclear fragmentation patterns and activation of caspase-3 in thapsigargin and caffeine-treated cells suggest the induction of apoptosis within 12 hrs of treatment. The treated cells were shown to generate nitric oxide, a potential apoptosis inducing agent.

Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE) following traumatic brain injury, accounts for 20% of symptomatic epilepsy. Reorganization of mossy fibers within the hippocampus is a common pathological finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus where they form synapses with pyramidal cells. During TLE, mossy fibers are observed to innervate the inner molecular layer where they synapse onto the dendrites of other dentate granule cells, leading to the formation of recurrent excitatory circuits. To date, the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown. Recent focus has centered on the involvement of tropomycin-related kinase receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β) inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2 (CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation of GSK3β should therefore increase CRMP2 activity. To determine the involvement of CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it was demonstrated that increased neurite outgrowth following GSK3β inactivation is CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the transience of GSK3β-inactivation. Based on previous work, it was hypothesized that activity-dependent mechanisms may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI. While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it did not differ significantly from vehicle-treated animals. Therefore, the necessity of CRMP2 in mossy fiber sprouting following TBI remains unknown.