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Item The Addicted Phenotype: Protein Phosphorylation Status and Dopamine Receptor Responsiveness(2007-05-22) Edwards, Scott; Self, DavidUp-regulation of cAMP/PKA signaling by drugs of abuse may contribute to escalation and relapse, possibly by differentially altering dopamine receptor-responsiveness in the mesolimbic dopamine system. To investigate this hypothesis, our initial studies measured alterations in cAMP-dependent and -independent protein phosphorylation in vivo produced by chronic cocaine and heroin self-administration, changes in mesolimbic protein phosphorylation compared to individual differences in the propensity for escalating cocaine self-adminstration, and, ultimately, dopamine receptor-mediated regulation of relapse to cocaine seeking in withdrawal. Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviors, but whether differential alterations develop in the biochemical response to cocaine is less clear. In Chapter 2, we studied cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify regulation related to the context of behavioral reinforcement, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 hrs of intravenous cocaine administration, and subjected to western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including phosphorylation of the AMPA GluR1 receptor subunit at serine 845 in striatal and amygdala subregions, and the NMDA NR1 receptor subunit at serine 897 in the CA1 subregion of hippocampus. Tolerance also developed to cAMPindependent GluR1 S831 phosphorylation in the prefrontal cortex. In contrast, sensitization to cocaine-induced phosphorylation of the pre-synaptic vesicle protein synapsin I at serine 9 developed in amygdala and hippocampal subregions, while cAMP-dependent phosphorylation of the dopamine-synthesizing enzyme tyrosine hydroxylase at serine 40 decreased in pre-synaptic striatal dopamine terminals in striatal subregions. Cocaineinduced phosphorylation of extracellular signal-regulated kinase (ERK) was dissociated from downstream phosphorylation of the transcription factor cAMP-response element binding protein (CREB) in many brain regions, and failed to develop either tolerance or sensitization with chronic administration, and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in selfadministering animals, while negative dose-related correlations were found primarily with passive yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in lieu of their potential impact on the development of cocaine addiction. In Chapter 3, we studied alterations in protein kinase A (PKA)-dependent and PKA-independent phosphorylation in multiple brain regions in rats undergoing either spontaneous or naltrexone-precipitated withdrawal (WD) from chronic intravenous heroin self-administration. Spontaneous WD from heroin self-administration produced region-specific increases in PKA-dependent GluR1S845 phosphorylation in the nucleus accumbens shell, basolateral amygdala, hippocampal CA1 and CA3 regions, and premotor cortex after 24 but not 12 hrs, and there were no changes in prefrontal cortex, nucleus accumbens core or caudate-putamen. Increased GluR1Item Cell-Free Formation of RNA Granules(2012-07-16) Han, Tina Wei; Mcknight, Steven L.Asymmetric RNA localization is a mechanism by which a cell can spatially and temporally regulate the translation of RNAs. This mechanism is essential for many developmental processes such as germ cell formation in Drosophila embryos, as well as establishment of cell polarity and synaptic plasticity in the brain. In many instances, asymmetric RNA localization is achieved through transport and sequestration by RNA granules. RNA granules are large, non-membrane bound ribonucleoparticles that have been observed in various biological contexts. Unfortunately, because RNA granules are highly heterogeneous and weakly associating aggregates, they can be difficult to study biochemically, which constitutes a major impediment for gaining a more detailed understanding of the mechanisms governing RNA granule assembly. Here we describe two in vitro models for studying RNA granule assembly. The first method is based on the precipitation activity of a 3,5-disubstituted isoxazole compound that can be used as a quick and efficient pharmacological tool to probe the function and regulation of RNA granules. The second method utilizes a three-dimensional protein-retaining hydrogel formed from a recombinant protein. Polypeptides of low amino acid complexity were found to be the sequence determinants of isoxazole precipitation and hydrogel retention. Next generation sequencing was used to identify RNAs that partitioned with granule components in both isoxazole and hydrogel models and were found to be enriched in mRNAs known to be constituents of neuronal transport granules for dendritic localization. The overrepresented gene ontology categories for these RNAs included cell adhesion, extracellular matrix, and synaptic proteins. The average length of the 3’UTR of these RNAs was found to be longer than the 3’UTRs of RNAs excluded from the cell-free RNA granule preparations. These two in vitro models for studying RNA granule assembly offer a novel approach to identify candidate targets recruited to RNA granules by specific RNA-binding proteins. [Keywords: RNA granules, low complexity sequence, hydrogel, isoxazole]Item EEG signal analysis for seizure detection(Texas Tech University, 1996-05) Qin, DongyingBiological tissue obeys Ohm's law for small current densities [6]. Bioimpedance can be measured with four electrodes: two for current injection and two for voltage measurement. The magnitude of the impedance is given by the ratio of the magnitude of the measured voltage and the magnitude of the injected current. There seems to be a slow increase in measured impedance several minutes prior to the onset of seizure activity. This result may be caused by ionic (Ca^^) shifts prior to seizure formulation [13]. However, whether the result is common for most epilepsy patients is still unknown. Besides, there are also some difficulties in determining a suitable warning threshold, and the procedures for bioimpedance measurement are more complicated than that for an EEG [13]. An EEG is the recording of electrical cerebral potentials [6]. AU chemical and physical processes that take place in living cells produce electrical energy. The energy can be recognized by changes in potential of the cell membrane [11]. The potential of one cell is very small, but when a larger group of cells acts simultaneously, the potential is higher and can be recorded with suitable amplification. Many techniques have been developed for EEG signal analysis. Some techniques have been used for seizure detection or prediction, but there have been no conclusive findings. In this thesis, EEG analysis is used as a tool for seizure detection or prediction. Several different signal analysis techniques are examined. The objective is to determine which technique is the best for seizure detection or prediction.Item Effects of Regional Deletion of Rab3A-interacting Molecule and PTEN on Brain Function(2013-04-16) Haws, Michael; Eisch, Amelia J; Powell, Craig M; Huber, Kimberly; Goldberg, MatthewThis dissertation describes experiments designed to delete/knockdown molecules from targeted neuronal populations to study brain region-specific behavioral functions. To this end, I utilized two different conditional knockdown techniques to study the role of the presynaptic active zone molecule Rab3A-interacting Molecule (RIM1) and the phosphatase and tensin homologue on chromosome 10 (PTEN). The cre-lox system was used to eliminate RIM1 from the hippocampal dentate gyrus and area CA3, while adeno-associated virus expressing PTEN-directed interference RNA was injected into the basolateral amygdala to knockdown PTEN in local pyramidal neurons. In the case of RIM1, I hypothesized that deletion of RIM1 from the dentate gyrus or from area CA3 would replicate a subset of the learning and memory deficits found in RIM1à-/- mice. Though the conditional RIM1 knockout mice were not completely selective for the dentate gyrus or for area CA3, both conditional knockouts induced a different behavioral abnormality present in RIM1à-/- mice. My results help narrow the potential brain regions involved in key RIM1à-/- mice behavioral aberrations. In the case of PTEN, I hypothesized that deletion of PTEN specifically in the basolateral amygdala (BLA) would cause increased anxiety and neuronal hypertrophy. Knockdown of PTEN in the BLA did not induce anxiogenesis though it did increase soma volume, dendritic caliber, spine size, mushroom:thin spine ratio, and the frequency of spontaneous miniature excitatory post-synaptic currents. These findings are in contrast to previous findings of increased spine density with PTEN knockdown. This difference likely represents the more sensitive techniques employed in the present studies to ascertain dendritic spine type and density. Though PTEN knockdown had synaptic effects I did not observe any behavioral effects. However, limitations in viral knockdown of PTEN transcripts or viral infection rate may be responsible for the lack of effect. Indeed, limitations exist for both the transgenic and viral approaches used which proved to be challenging obstacles to designing experiments, interpreting data and coming to more extensive concrete conclusions. Transgene expression is often not as selective as desired. Virus injections may not localize to target region or may not infect enough neurons. Understanding and characterizing these and other limitations is vital.Item Exploring a Functional Disconnect Between Nestin-Expressing Type-1 Cells and Adult Hippocampal Neurogenesis(2010-05-14) DeCarolis, Nathan Anthony; Eisch, Amelia J.The subgranular zone of the hippocampal dentate gyrus generates new neurons throughout adulthood. The process of adult neurogenesis is well-described, but the source of proliferating progenitors is unknown. One potential source is the Type-1 cell, which is morphologically reminiscent of embryonic radial stem cells and expresses protein markers like nestin. However, there is no direct evidence that Type-1 cells are the source of neurogenesis. For this doctoral research, I asked two questions to gain more insight into the role of Type-1 cells in adult The subgranular zone of the hippocampal dentate gyrus generates new neurons throughout adulthood. The process of adult neurogenesis is well-described, but the source of proliferating progenitors is unknown. One potential source is the Type-1 cell, which is morphologically reminiscent of embryonic radial stem cells and expresses protein markers like nestin. However, there is no direct evidence that Type-1 cells are the source of neurogenesis. For this doctoral research, I asked two questions to gain more insight into the role of Type-1 cells in adult GL-YFP but not Nes-YFP mice showed recovery of YFP+ progenitors, suggesting that GL-YFP cells are stem-like while Nes-YFP cells are progenitor-like. This correlative and causative evidence that nestin-expressing Type-1 cells are not the source of neurogenesis significantly advances our understanding of the neurogenic process.Item Investigating the female mate preference brain : identifying molecular mechanisms underlying variation in mate preference in specific regions of a swordtail (Xiphophorus nigrensis) brain(2011-05) Wong, Ryan Ying; Hofmann, Hans A.; Cummings, Molly E.; Ryan, Michael J.; Crews, David; Zakon, Harold H.Choosing with whom to mate is one of the most important decisions a female makes in her lifetime and inter-individual variation of these preferences can have important evolutionary consequences. In order to get a complete understanding of why and how females choose a mate, we must identify factors that can contribute to variation of female mate choice. Many decades of research sought to understand ultimate mechanisms of female mate choice with proximate mechanisms receiving a lot more attention in recent years. For my thesis, I identify intrinsic and extrinsic factors that correlate with individual variation of female Xiphophorus nigrensis mate preference. I provide evidence that a female’s size (e.g. age and sexual experience) as well as male behavioral displays can predict female mate preference. Using genes associated with female mate preference (neuroserpin, neurologin-3), I identify four brain regions (Dl, Dm, HV, POA) that show significant differences in gene expression between females exhibiting high preference for males relative to females displaying little mate preference. Neuroserpin and neuroligin-3 gene expression within these brain regions are also positively correlated with female mate preference behavior. Two of these brain regions (Dm and Dl) integrate multisensory information and are found in the putative teleost mesolimbic reward circuitry; the other two regions (HV and POA) are involved in sexual behaviors. With the implication of the reward circuitry, I assess whether there are changes in dopamine synthesis (via tyrosine hydroxylase, TH) in dopaminergic brain regions associated with the degree of mate preference. I do not find evidence of rapid changes (within 30 minutes) of TH expression (i.e. dopamine synthesis) in dopaminergic brain regions related to variation in female mate preference. Collectively my results suggest that mate preference behavior in the brain may be coordinated not just through regions associated with sexual response but also through forebrain areas that may integrate primary sensory information, with no associated changes of a proxy for dopamine synthesis in dopaminergic brain regions.Item Living in a plant : brain and behavioral traits of acacia ants(2014-12) Amador Vargas, Sabrina; Mueller, Ulrich G.Acacia ants evolved obligate protective mutualisms with acacia trees, which they defend against herbivores, food parasites and encroaching vegetation. In this mutualism, the fitness of one partner entirely depends on the other. Other ant species are parasitic on acacia trees; they nest on the tree, harvest food rewards, do not defend their own tree, and occasionally try to steal food from other trees, usually inhabited by mutualistic ants. To understand the behavioral and anatomical effects of the interaction between ants and host trees, I integrated brain anatomy, morphology and field experiments to study parasitic and mutualistic species of Pseudomyrmex ants associated with acacia trees. In Chapter 1, I describe a previously unknown behavior of stealing food from other ant-defended acacia trees in the parasitic acacia ant P. nigropilosus, and I evaluate four strategies that may allow parasitic ants to overcome the usually effective defenses of the robbed mutualistic ants protecting a host tree. In Chapter 2, I study how colony size correlates with the degree of division of labor and brain anatomy of workers, focusing on a species of acacia ant lacking morphological castes among workers, P. spinicola. In Chapter 3, I study acacia-ant behavior of killing vegetation encroaching on a host tree. I document the interspecific differences among acacia ants in the size of the area around the host tree that workers clear from encroaching vegetation. I further test for interspecific variation in pruning behavior, and whether mandibular force correlate with worker pruning decisions. In Chapter 4, I test whether ant species that routinely leave the host tree to forage or to prune encroaching vegetation are better at orienting themselves when returning to their host tree, compared to ant species that rarely leave their host tree. This dissertation documents how the obligate protective mutualism of an ant with a tree has consequences for division of labor, navigational skills, behavioral specializations, head shape and brain anatomy of ant workers.Item Membrane Lipids and Synaptic Vesicle Trafficking in the CNS(2009-01-14) Wasser, Catherine Rebecca; Kavalali, Ege TMost vesicles within a synapse are dormant. The rest participate in synaptic neurotransmission, with a portion of these preferentially fusing first. Moreover, all synapses experience spontaneous neurotransmitter release which may originate from the random exocytosis of vesicles prepared to fuse immediately upon calcium influx; however, spontaneously fusing vesicles may be independent because they prefer spontaneous fusion. The functional separation argues that the compositions the synaptic vesicle membranes are somehow unique between pools. The first three chapters explore the role of cholesterol in synaptic transmission. We treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed augmented spontaneous neurotransmission. In contrast, the same treatments severely impaired responses evoked by action potentials and hypertonicity. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. Chapter five examines the role of sphingosine on neurotransmitter release. By adding sphingosine to hippocampal cultures, we found that sphingosine enhances neurotransmission in a synaptobrevin-2-dependent manner. Chapter six investigates the stability of actively recycling synaptic vesicles. We employed several approaches (fluorescent and ultrastructural imaging) to monitor not only the fate recycling vesicles, but also the origin and reuse of spontaneously fusing vesicles. We conclude that at rest, the total recycling pool remains active and resists spontaneous fusion up to at least six hours; while spontaneous fusion of spontaneously fusing vesicles is much faster. This argues that vesicles fusing spontaneously do not originate from the recycling pool. In chapter seven, we observe how modifying synaptic vesicle membranes might affect neurotransmitter release. By the uptake of horseradish peroxidase into vesicles followed by hydrogen peroxide perfusion, we induced free radical modification of vesicle membranes and found that modifying recycling pool vesicles increased spontaneous fusion and attenuated evoked release. Taken together, the results of each chapter appear to suggest that the fusion of action potential-dependent and-independent vesicles are regulated by different mechanisms, supporting the theory that some vesicles may be unique within a synapse.Item Neuroinflammation, TNF, and Ceramide Signaling: Putative Pathways for Neurotoxicity in Parkinson's Disease(2010-05-14) Martinez, Terina Nichole; Tansey, Malu G.Parkinson’s disease is a progressive neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra that innervate the striatum, and it is the loss of these neurons that causes the motor dysfunction that is associated with the disease. However, the mechanisms that contribute to the induction and perpetuation of dopaminergic neuronal cell death in Parkinson’s disease are multifaceted and poorly understood. Inflammation has been shown to contribute to cytotoxicity in animal models of Parkinson’s disease, and increased levels of inflammatory cytokines have been observed in the cerebral spinal fluid and striatum of Parkinson’s disease patients. We have previously demonstrated that blocking soluble tumor necrosis factor (TNF) signaling with dominant-negative TNF inhibitors attenuates the loss of dopaminergic neurons in models of Parkinson’s disease, but which signaling pathways downstream of TNF mediate this effect remain undetermined. Here, I show that TNF-dependent ceramide signaling contributes to dopamine neuron cytotoxicity by compromising mitochondrial membrane potential, inducing endoplasmic reticulum stress and activating caspase signaling in vitro. My data demonstrate that TNF-induced cytotoxicity is partially ceramide-dependent, as TNF-induced cytotoxic effects are attenuated with two different pharmacological inhibitors of sphingomyelinase, an enzyme that hydrolyzes active ceramide from inactive sphingomyelin pools. Collectively, my data support a model whereby low-dose TNF and concomitant low TNF receptor1 occupancy activates downstream ceramide signaling and metabolism, culminating in caspasedependent cytotoxic cell death of dopaminergic neurons. My data and the data associating ceramide biology and metabolism with Parkinson’s disease warrants future studies examining the potential neuroprotective effects of inhibition of sphingomyelinase in animal models of Parkinson’s disease, and may eventually lead to improved therapy for patients who suffer from Parkinson’s disease.Item Regulation of Excitatory Neurotransmission, Synaptic Plasticity, and learning by Cyclin-Dependent Kinase 5(2009-06-17) Hawasli, Ammar Hamami; Bibb, James A.Cyclin-dependent kinase 5 has been implicated in many physiological and pathological processes in the central nervous system. To better understand Cyclin-dependent kinase 5's roles in the adult brain, we developed and studied several conditional Cyclin-dependent kinase 5 knockout model systems. Soon after conditional loss of Cyclin-dependent kinase 5, mice displayed improved hippocampal learning and enhanced synaptic plasticity in the hippocampal Schaffer collateral pathway. The genetically enhanced mice displayed increased N-methyl-D-aspartate receptor-mediated currents and elevated levels of the NR2B N-methyl-D-aspartate receptor subunit. The enhancement in synaptic plasticity was directly attributed to the increased current through NR2B-containing receptors. NR2B levels were elevated in Cyclin-dependent kinase 5 knockout mice due to an impairment in the calpain-mediated degradation of NR2B. Consistently, Cyclin-dependent kinase 5 directly facilitated the degradation of NR2B cytoplasmic-tail in vitro. Cyclin-dependent kinase 5, NR2B, and calpain coimmunoprecipitated in vivo and directly bound one another in vitro. NR2B inhibited Cyclin-dependent kinase 5 activity in vitro, indicating a potential feedback mechanism. These findings suggested that Cyclin-dependent kinase 5 interacts directly with NR2B and calpain to facilitate the degradation of NR2B, thereby attenuating synaptic plasticity. In addition to regulating functional plasticity, Cyclin-dependent kinase 5 also plays roles in structural plasticity and presynaptic function. Cyclin-dependent kinase 5 facilitated the calpain-mediated degradation of spectrin in vitro. Spectrin degradation and depolymerized actin levels were decreased in conditional Cyclin-dependent kinase 5 knockout hippocampus. These results implicate Cyclin-dependent kinase 5 dendritic in spine dynamics which is critical for synaptic plasticity. Loss of Cyclin-dependent kinase 5 also led to a presynaptic enhancement in post-tetanic potentiation and a deficit in paired-pulse facilitation, which are consistent with an increase in probability of synaptic vesicle release, due to increased numbers of vesicles in the readily releasable pool or altered sensitivity to presynaptic calcium. Finally, chronic Cyclin-dependent kinase 5 loss produced increases in behavioral and neuronal excitability followed by electrographic abnormalities in vivo and reduced brain weight. These findings suggest that the enhancement in excitatory neurotransmission which initially led to improvements in learning and plasticity preceded excessive excitability and subsequent neuropathology. Consequently, Cyclin-dependent kinase 5 regulates excitatory neurotransmission, synaptic plasticity.Item Role of Cholesterol 24-Hydroxylase in Hippocampal Long-Term Potentiation(2009-06-18) Ramirez, Denise Marie O'Donnell; Russell, David W.The mammalian brain contains a disproportionately large percentage of the body's cholesterol, steady-state levels of which are maintained within a narrow range to preserve membrane function. The brain is denied access to circulating lipoproteins by the blood-brain barrier and therefore relies on de novo cholesterol synthesis through the mevalonate pathway to meet the tissue's requirement for this essential lipid. A small amount of brain cholesterol is turned over daily in select neurons by cholesterol 24-hydroxylase, which catalyzes the production of the membrane-permeable oxysterol 24(S)-hydroxycholesterol and represents the major pathway of cholesterol catabolism in this organ. Mice lacking 24-hydroxylase have a decreased rate of brain cholesterol synthesis and exhibit deficiencies in spatial, associative, and motor learning. Hippocampal slices prepared from these mice are unable to support the induction of long-term potentiation, a type of synaptic strengthening thought to underlie learning and memory. The ability of 24-hydroxylase knockout slices to exhibit long-term potentiation can be restored by treatment with geranylgeraniol, an isoprenoid end-product of the mevalonate pathway. Mechanistic insight into the role of geranylgeraniol in long-term potentiation has been revealed by calcium imaging studies in neurons cultured from wild-type and 24-hydroxylase knockout embryos. Neurons from mice lacking 24-hydroxylase have specific defects in N-methyl-D-aspartate (NMDA) receptor function, a subtype of ionotropic glutamate receptor essential for long-term potentiation. The subunit composition of NMDA receptors located in various functional pools is normal in 24-hydroxylase knockout hippocampus, suggesting that geranylgeraniol does not affect expression of NMDA receptors. Localization studies of 24-hydroxylase show the enzyme is predominantly expressed in the endoplasmic reticulum throughout the soma and dendrites of selected hippocampal, cerebellar, and cortical neurons, consistent with a postsynaptic need for cholesterol turnover in neurons of brain regions important for learning and memory. These findings reveal that cholesterol turnover is important to produce a constant supply of geranylgeraniol, which in turn is necessary for the induction of long-term potentiation and presumably learning in mice.Item The Role of Notch1 in adult Hippocampal Neurogenesis and function(2009-09-04) Ables, Jessica Lynn; Eisch, AmeliaNeurogenesis occurs throughout life in the hippocampal subgranular zone (SGZ) and is potently stimulated by exercise, but the underlying mechanisms are still poorly defined. Notch1 is a master regulator of developmental neurogenesis, yet its role in adult hippocampal neurogenesis is unclear. To test the hypothesis that cell-intrinsic Notch1 is critical to both basal and exercise-induced SGZ neurogenesis, we generated Nestin-creERT2/R26R-YFP/Notch1loxP/loxP (Notch1 iKO) mice to inducibly ablate Notch1 in Nestin-expressing stem and progenitor SGZ cells. The total number of YFP+ SGZ cells increased over time in wild type littermates, but not in Notch1 iKO mice. Morphological and phenotypic analyses revealed that fewer YFP+ DG neurons were generated over time in Notch1 iKO mice due to smaller pools of YFP+ stem-like and progenitor cells. Likewise, neural progenitors isolated from Notch1 iKO mice were incapable of forming new neurospheres with extended passaging. While non-running Notch1 iKO mice had fewer YFP+ SGZ cells relative to wild type littermates, Notch1 iKO mice given 30 days access to a running wheel had equal number of YFP+ SGZ cells relative to controls, suggesting that running rescued total YFP+ SGZ cell number independent of Notch1. However, running did not rescue YFP+ stem-like cell number in Notch1 iKO mice, suggesting that the putative stem-like SGZ cells make little contribution to adult hippocampal neurogenesis in these conditions. From these data, we conclude that Notch1 in Nestin+ stem and progenitor cells is critical to maintain basal adult hippocampal neurogenesis, but is not critical for exercise-induced neurogenesis. Neurogenesis has also been implicating in depression and behavioral response to antidepressants. To determine if reduced neurogenesis contributed to depression- or anxiety-related behavior, we assessed several measures of depression and anxiety in Notch1 iKO mice. We found that Notch1 iKO mice did not differ from WT mice in their behavior, suggesting that reduced neurogenesis is not associated with mood disturbances.Item Role of the Transient Receptor Potential Channels in Modulating Prefrontal Cortical excitability and the behavioral responses to cocaine(2009-09-04) Fowler, Melissa Ann; Cooper, Donald C.Role of the transient receptor potential channels in modulating prefrontal cortical excitability and behavioral responses to cocaine. Drug addiction is a disease that is influenced by both genetic and environmental factors that result in altered excitability in the key brain regions associated with reward and decision-making. The prefrontal cortex (PFC) processes reward-related information; and pathologies in PFC excitability resulting from prolonged drug use may lead to the loss of control over drug intake associated with drug addiction. We show that layer 5 pyramidal neurons in the PFC exhibit a prolonged depolarizing response to Gq-coupled receptor activation, which produces a period of heightened excitability of the cell following brief bursts of action potential activity. This burst triggered delayed depolarization enables the cell to convert subthreshold inputs into persistent firing output and may be a way for the cell to hold information in a short term memory buffer. The delayed after-depolarization (dADP) is reduced by dopamine and chronic cocaine, which may serve to bias the cell towards very strong inputs, such as those associated with drug cues, while preventing the cell from responding to smaller, subthreshold inputs. The dADP is induced by activation of Gq-coupled receptors, such as metabotropic glutamate receptors or muscarinic acetylcholine receptors and is mediated by subsequent activation of a non-selective cation channel, which pharmacological data suggested to be a canonical transient receptor potential (TRPC) channel. We used in situ hybridization, immunoblots, and real-time PCR to examine the expression of the TRPC channels and found dense expression of TRPC5 in the pyramidal cell layers of the PFC. Using adeno-associated viral mediated knock-down of TRPC5 in the prefrontal cortex of TRPC5flx mice, we show that TRPC5 channels are necessary for induction of the dADP in the PFC. We show that loss of TRPC5 in the PFC increases the locomotor activating and rewarding effects of cocaine. Knock-out of TRPC1 channels, on the other hand, has no effect on the dADP and does not alter behavioral responses to cocaine, suggesting that TRPC5 homomultimeric complexes rather than TRPC1/5 heteromultimeric complexes underlie the dADP in the PFC. These studies identify the TRPC5 channels as important for modulating neuronal excitability in the PFC and the behavioral responses to cocaine.Item The development of a scale for the minimal brain dysfunction syndrome in adolescents(Texas Tech University, 1973-05) Chaney, James A.The study of psychological and social disturbances in children with brain dysfunction has grown rapidly in the last 25 years. Barsch (1962) cited a summary of research on brain injury by Klebanoff (1945) and pointed out that during the period from 1870-1945, there were only two studies of psychological disturbances in children with brain dysfunction. Less than a decade later Klebanoff, Singer, and Milensky (1954) made another summary of the literature and included a separate section on the studies of psychological disturbances in braininjured children.Item The effect of hippocampal lesions upon activity and learning(Texas Tech University, 1966-08) Jackson, William JamesNot availableItem The pharmacology of beta-endorphin binding sites in the caudal dorsomedial medulla(Texas Tech University, 1997-08) D'Souza, MabelThe purpose of this study is to characterize the binding properties of an endogenous opioid, p-endorphin, in the caudal dorsomedial medulla (CDMM). The CDMM contains an important cardioregulatory region, called the nucleus of solitary tract (nTS). The nTS is richh innervated w ith P-endorphin producing neurons (Joseph et al., 1983) and possesses p-opioid receptors (Xia and Haddad, 1991). There is conflicting evidence indicating there is also a population of 5-opioid receptors in this area (Dashwood et al., 1988; Xia and Haddad. 1991). Further evidence suggests that pendorphin also binds to a nonopioid receptor in the CNS (Houghten et al.. 1984). Therefore, the cardioregulatory actions of p-endorphin may be mediated by a number of receptor subtypes in the nTS. To date, there has been no pharmacological binding profile for p-endorphin binding sites in the nTS. The purpose of this study is to identify the receptor(s) to which P-endorphin binds to in the nTS. This P-endorphin binding profile may then provide an important link between P-endorphin's observed cardiovascular effects and a specific receptor subtype mediating these actions. The long term implications of this pharmacological profile may help provide important therapeutic applications toward the treatment of analgesia without the deleterious cardiovascular side effects commonly associated with opioids and may provide an alternative treatment to clinical hypertension.