Voltage-gated calcium channels are ubiquitously expressed in neurons and are of vital importance to proper cellular functioning. Calcium entry into cells via activation of voltage-gated calcium channels controls a wide variety of cellular functions including neurotransmitter release, muscle excitation-contraction coupling, gene regulation and activation of signaling cascades. Regulation of calcium channels via activation of G protein-coupled receptors is a prominent mechanism of calcium current inhibition and neurotransmitter release. An intriguing characteristic of this modulatory pathway is its voltage dependence whereby the degree of calcium current inhibition varies depending on the membrane voltage of the cell, and is susceptible to activity-dependent relief by trains of action potentials. Previously, it has been suggested that in chick ciliary ganglion neurons, somatostatin inhibits calcium current in a voltage dependent manner. Interestingly, the specific characteristics of the inhibition varied depending on the recording configuration used to collect data. Thus, I measured the voltage-dependence of somatostatin-mediated calcium current inhibition in individual ciliary ganglion neurons using the whole-cell and perforated patch configuration of voltage clamp recordings. The results indicate that the cytoplasmic dialysis that occurs during whole-cell recordings enhances the voltage dependence of calcium current inhibition and suggests that there is a greater concentration of activated G protein subunits in this configuration. While much is known about step depolarization-evoked calcium current and the kinetic changes that accompany G protein-mediated inhibition, relatively little is known about the effects of kinetic slowing on AP-evoked calcium current. Therefore, I used a modification of action potential waveforms was used to determine the effect of G protein activation on the kinetics of single action potential-evoked calcium current. The results demonstrate that kinetic slowing does not alter the time course of action potential-evoked calcium current and suggests that modulated channels may not contribute to AP-evoked calcium current.

Although considerable progress has been made we do not yet fully understand the behavioral and neurobiological bases of nicotine reinforcement, and without this knowledge treatment strategies aimed at reducing smoking remain deficient. This dissertation provides an original perspective on nicotine reinforcement, which arises from substantial evidence of complex interactions between nicotine and nonpharmacological stimuli. The present experiments tested the hypothesis that nicotine reinforcement derives from at least two sources: 1) the primary reinforcing properties of nicotine, an action that requires response-dependent drug administration, and 2) the more prominent ability of nicotine to enhance behavior maintained by salient non-nicotine stimuli, an action that does not require a contingent relationship between drug administration and reinforced operant responding. Although novel for nicotine, this hypothesis has origins in an extensive literature on the reinforcing properties of psychostimulant drugs. Empirical support for the application of this hypothesis to nicotine reinforcement will be presented. By investigating the interaction between nicotine and nonpharmacological stimuli within the context of drug self-administration in rats, the present research has generated new insights into the paradox of how nicotine, an apparently weak primary reinforcer, can sustain the robust behavior observed in self-administration and in smoking. Hypotheses generated by these data provide important direction for future investigations into the neurobiology of nicotine reinforcement.

ASSOCIATION OF THE 5-HTTLPR WITH PROLACTIN RESPONSE TO CITALOPRAM IN A COMMUNITY POPULATION Eloise Peet, BS University of Pittsburgh, 2004 The serotonin transporter (5-HTT) is a key mechanism regulating magnitude and duration of serotonergic transmission in the central nervous system, and is the site of action of selective serotonin reuptake inhibitors (SSRIs) used for treating psychiatric conditions. Variation in treatment response to SSRIs has been correlated with a common bi-allelic length polymorphism in the 5-HTT-promoter region (5-HTTLPR), known to modulate transcriptional efficiency of the 5-HTT gene in vitro. The alleles, designated long (l) or short (s), result in one of three possible genotypes: l/l, l/s, or s/s. The (s) allele has been hypothesized to have a dominant functional effect, and has been associated with decreased transporter transcription efficiency and poorer therapeutic response to antidepressants. Acute transporter blockade with SSRIs rapidly increases central nervous system serotonin levels, leading to hypothalamic receptor stimulation and the release of several hormones, including prolactin. The specific aim of this study is to characterize the prolactin response to acute 5-HTT-reuptake blockade according to 5-HTTLPR genotype, to further elucidate the effect of this polymorphism on serotonin transporter function in vivo. This study has been designed to test the hypothesis that, when compared to subjects with the l/l genotype, subjects with either the s/l or s/s genotype will experience a blunted prolactin response following acute administration of the highly selective reuptake inhibitor Citalopram. To accomplish this goal, a cohort of 206 community volunteers were intravenously administered a weight-adjusted dose of Citalopram. Each subject was genotyped for the 5-HTTLPR, and blood samples were obtained for prolactin and Citalopram levels immediately before and at regular intervals for 2.5 hours after the Citalopram injection. Results: Citalopram-induced prolactin response, reported as prolactin area under the curve (PRL AUC), was significantly associated with 5-HTTLPR (F = 3.08, p = 0.048). Among individuals with the s/s genotype, PRL AUC response (M±SD: 84.2 ± 51.8 ng/ml * 150min) was significantly lower (p = 0.014) than the l/l group (246.0 ± 40.2 ng/ml * 150 min). The difference in PRL AUC between subjects with the l/l genotype and the l/s group (172.5 ± 41.3 ng/ml * 150 min) was not significant (p = 0.21); the difference in PRL AUC between subjects with the l/s genotype and the s/s group also was not significant (p = 0.23). When results were analyzed as the maximum change in prolactin, the Citalopram-induced PRL MAX was similarly associated with 5-HTTLPR (p = 0.034). Conclusions: Subjects with the 5-HTTLPR s/s genotype exhibit significantly lower prolactin response in response to the SSRI Citalopram than subjects carrying two copies of the l allele. The s allele does not have a dominant effect on the prolactin response to Citalopram in a non-patient population.

The resting length of respiratory muscles must be altered during changes in posture in order to maintain stable ventilation. Prior studies showed that although the vestibular system contributes to these adjustments in respiratory muscle activity, the medullary respiratory groups receive little vestibular input. Additionally, previous transneuronal tracing studies and physiological experiments demonstrated that propriospinal interneurons in the C1-C2 spinal cord send projections to the diaphragm motor pool. The present study tested the hypothesis that C1-C2 interneurons mediate vestibular influences on diaphragm activity. Recordings were made from 145 C1-C2 neurons that could be antidromically activated from the C5-C6 ventral horn, 60 of which had spontaneous activity, during stimulation of vestibular receptors using electric current pulses or whole-body rotations in vertical planes. The firing of 19 of 31 spontaneously active neurons was modulated by vertical vestibular stimulation; the response vector orientations of most of these cells were closer to the pitch plane than the roll plane, and their response gains remained relatively constant across stimulus frequencies. Virtually all spontaneously active neurons responded robustly to electrical vestibular stimulation, and their response latencies were typically shorter than those for diaphragm motoneurons. Nonetheless, respiratory muscle responses to vestibular stimulation were still present after inactivation of the C1-C2 cord using large injections of either muscimol or ibotenic acid. These data suggest that C1-C2 propriospinal interneurons contribute to producing posturally-related responses of respiratory muscles, although additional pathways are also involved in generating these responses.

The supplementary eye field (SEF) is a region of cortex located on the dorsomedial shoulder of the frontal lobe, considered to be involved in the control of eye movements. SEF neurons show spatially selective activity during visually- and memory-guided saccades. The selectivity exhibited by SEF neurons has been described as being related to an eye- or head-centered reference frame. We have previously shown that SEF neurons exhibit selectivity in an object-centered reference frame: neurons will fire selectively when saccades are directed to one end of a bar or another, irrespective of the absolute location of the bar in space. It is not well known how SEF neurons display selectivity for object-centered locations. In order to better understand the mechanism of this phenomenon, we performed three studies. In the first study, we asked how SEF neurons encode locations in both egocentric and object-centered reference frames. We recorded from single SEF neurons while monkeys performed tasks requiring spatial representation in either eye-centered or object-centered reference frames. Different SEF neurons encoded locations in eye-centered coordinates only, object-centered coordinates only, or in complex combinations of the two. In the second study, we tested whether object-centered selectivity is an innate property of SEF neurons or whether it is acquired through learning. We recorded the activity of SEF neurons before and after training monkeys to perform an object-centered task. Some SEF neurons exhibited object-centered selectivity before training. Following training, this number was increased, as was the intensity of object-centered spatial selectivity. In the third study, we investigated whether the object-centered selectivity seen in SEF neurons during performance of an object-centered task is reduced during performance of a non-object-centered task. We recorded from SEF neurons while monkeys performed either an object-centered task or a color matching task with an object as a target. An equivalent number of neurons showed object-centered selectivity in both tasks, but the strength of selectivity was slightly higher during performance of the object-centered task. We conclude from the results of these studies that neurons in the SEF are critically involved in the dynamic representation of locations using multiple spatial reference frames.

The molecular mechanisms of alternative forms of cell death are becoming increasingly common, particularly in the case of neurodegenerative diseases. Proteases pathways, such as calpains and caspases, and their interactions with the mitogen-activated protein kinase (MAPK) signaling pathways have not been well-explored, particularly in the context of a pro-toxic role for the extracellular-related kinase (ERK). HT22 cells lack ionotropic glutamate receptors, but are still sensitive to high concentration of extracellular glutamate, which depletes glutathione and causes oxidative toxicity in an ERK-dependent manner. Using a purified clone of HT22 cells which were consistently responsive to glutamate-induced toxicity in an ERK-dependent manner, we have found that caspase-1- and -2-like inhibitors were effective at blocking cell death. Delayed addition of these inhibitors remained protective when added up to 3 hours after glutamate exposure. Both inhibitors decreased ERK phosphorylation at 9 hours following glutamate treatment, a timepoint where the second rise in ERK activation has been found previously. Contrary to previous reports using pharmacological calpain inhibitors, we have found that molecular inhibition of calpains by overexpression of calpastatin, the endogenous calpain inhbitor, was effective at delaying cell toxicity in glutamate treated HT22 cells. Evidence for calpain activation was demonstrated using two separate assay systems. In an in vitro enzymatic assay, calpains were found to be activated in the glutamate-induced toxicity of HT22 cells, and that this activation appears to be biphasic in nature, with the early activation MEK-independent and the late activation MEK-dependent. Furthermore, the endogenous calpain target PARP was cleaved to a fragment consistent with calpain-mediated cleavage at late timepoints following glutamate exposure. The appearance of this fragment, consistent with the late MEK-dependent activation, was also dependent on MEK activity. These results indicate for the first time that calpains are indeed activated and involved in cell death execution, that they can be at least in part regulated by MEK activity. Taken together, we have found that glutamate-induced oxidative stress in HT22 cells represents a form of cell death where caspase-1- or -2-like proteases may function upstream of ERK activation, but that ERK activation is required at least in part for a further, downstream calpain activation.

In the brain, serotonin (5-hydroxtryptamine, 5-HT) is synthesized in the raphe nucleus. Raphe serotonergic projections modulate neurotransmissions throughout the brain influencing mood and behavior. The serotonin transporter (SERT; SLC6A4) clears 5-HT from the synapse for degradation or reuse, thus regulating levels of 5-HT and limiting its actions on 5-HT receptors. Dysfunction in 5-HT modulation of neurotransmission is associated with mood and developmental disorders including anxiety, depression, and autism and there is genetic evidence for increased risk for depression in individuals possessing polymorphisms in SLC6A4 as well as genes which interact with SLC6A4. ITGB3 encodes integrin β3, a cell adhesion molecule which has been implicated as a modulator of peripheral serotonergic systems via genetic and functional interactions with SLC6A4, as well as in regulation of synaptic plasticity and maturation. In the brain, integrin β3 couples to integrin αv to form a functional receptor, making integrin αvβ3 an interesting target for regulation of neural 5-HT systems. Immunohistochemical experiments revealed integrin β3 localization in serotonergic neurons, colocalized with SERT. Examination of genetic interactions utilizing an Itgb3-/+ x Slc6a4-/+ mouse model revealed reduced SERT expression, and an anxiety- and depression-like phenotype compared to wildtype littermates. Further experimentation of the functional interaction between integrin αvβ3 and SERT via pharmacological targeting of integrin αvβ3 revealed integrin αvβ3 regulation of SERT uptake activity. These studies highlight integrin β3 as a potential modulator of brain 5-HT systems and subsequently 5-HT mediated behavioral phenotypes.

Insights into the organization of vertebrate nervous systems have often arisen through systematic examinations of specific behaviors and their possible neural substrates. This approach is even more effective when diverse groups can be assessed in order to identify commonalities in innervation, anatomy, or nervous system representation. Throughout this collection of investigations, we have examined a wide-ranging group of semi-aquatic vertebrates possessing peripheral nervous system specializations related to mechanotransduction. These included a range of insectivores such the American water shrew (Sorex palustris), the smallest homeothermic diver with an elaborate array of whiskers. We also analyzed the behavior and central nervous system representations of the tentacled snake (Erpeton tentaculatus) in the process of identifying the sensory function of the unique paired facial appendage. More extensive observations were collected from two members of the order Crocodylia the Nile crocodile (Crocodilus niloticus) and the American alligator (Alligator mississippiensis) with particular attention devoted to their integumentary sensory organs (ISOs). These ubiquitous dome-shaped protuberances speckle the jaws of all crocodilians and are also found on the bodies scalation of members of the families Crocodilidae and Gavialidae, yet their precise function and the behaviors they mediate have remained ambiguous. We suggest that the ISOs impart an exquisite level of tactile sensitivity, even exceeding that of the primate fingertip, thereby providing a sophisticated sense of touch to an otherwise armored body surface and draw comparisons between the sensory system organization of mammalian and reptilian taxa.

Gastrulation is a fundamental process during embryogenesis when the germ layers and shape of the animal are generated. Although the major cell movements during gastrulation have been characterized, the underlying cellular and molecular mechanisms are only beginning to be understood. Identifying new molecules regulating gastrulation and studying their interactions with known regulators of gastrulation will bring us closer to a full mechanistic understanding of this key developmental process. In this study, I investigated the potential roles of the adhesion G protein-coupled receptor (GPCR) family during gastrulation. Among 30 annotated or partially annotated adhesion GPCRs in the sequenced zebrafish genome, the four members comprising the Group IV adhesion GPCR subfamily exhibit distinct and dynamic expression patterns during embryogenesis. Therefore, I performed functional analyses of members from this subfamily during embryogenesis. Interfering with Gpr124 function caused defects in multiple tissues in the caudal region of the embryo, including the notochord and vasculature, which warrant future studies on the mechanism of Gpr124 function. Central to this thesis, I uncovered a role for Gpr125 during convergence and extension (C&E) gastrulation movements and facial branchiomotor neuron (FBMN) migration. Consistent with a potential role during gastrulation, I showed gpr125 is expressed maternally and at blastula and gastrula stages. Excess Gpr125 in wild-type embryos impaired C&E movements and the underlying cellular and molecular polarities. Whereas interfering with Gpr125 function alone did not affect development, it exacerbated the C&E and FBMN migration defects of embryos with reduced Wnt/planar cell polarity (PCP) signaling. At the cellular level, Gpr125 depletion enhanced the disruption of polarized cell behaviors in embryos heterozygous and homozygous for trilobite/vang-like 2 (tri/vangl2) Wnt/PCP gene. At the molecular level, Gpr125 recruited Dishevelled (Dvl), the signaling hub of Wnt/PCP pathway, to the cell membrane, which fulfils the prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered with one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. This study reveals a role for Gpr125 in Wnt/PCP mediated processes and provides mechanistic insight into Gpr125 function and Wnt/PCP signaling.

GABAA receptors are pentameric, ligand-gated chloride channels that mediate the majority of fast inhibition in the brain. Because they are assembled from an array of 19 subunit subtypes, GABAA receptors are remarkable among neurotransmitter receptors for their potential diversity. Subunit composition determines the physiological and pharmacological properties of each isoform, so this diversity is both valuable for modulation of neuronal excitability and worthy of extensive study. It is clear that subunits do not assemble at random; rather, clear rules limit isoform production. The heterogeneity of receptor isoforms has been studied for decades, but there have been few comprehensive attempts to define the requirements for assembly and trafficking of functional pentamers. Here, we addressed this problem by expressing various combinations of GABAA receptor subunits in fibroblasts and using high-throughput screening techniques to determine what combinations successfully produce functional surface receptors. Furthermore, we assessed the stoichiometry and subunit adjacency of successfully assembled receptors. We confirmed previously-suspected rules and discovered previously-unknown determinants for assembly of common receptor isoforms, and we identified the efficient production of uncommon isoforms that should be sought in vivo. Given that GABAA receptors are essential for neuronal inhibition, it is perhaps unsurprising that numerous epilepsy-associated mutations have been identified in GABAA receptor subunit genes. However, the vast majority of idiopathic generalized epilepsies (IGEs) are polygenic, and for that reason there have been several recent efforts to detect variants in epileptic cohorts. The ultimate goal of these studies is to improve future diagnosis and treatment of IGEs, but a chasm remains between that goal and current knowledge. It is impossible to comprehensively characterize all variants; however, it may be possible to construct a framework that allows us to predict which variants in certain channels are likely to be deleterious. As such, the second part of this dissertation presents thorough characterizations of some reported monogenic mutations as well as our first efforts to use high-throughput screening to classify previously unstudied variants identified in epileptic cohorts.