Intellectual Disability (ID) is a condition in which day-to-day cognitive, intellectual and adaptive functioning is negatively affected including poor performance in memory tests in human subjects. Patients with comorbid anxiety and depression demonstrated adverse memory when subject to a verbal learning test. Abnormal mutation in the human deaf1 gene has been previously reported to be associated with ID. Previous behavioral studies in mice with a brain specific conditional neuronal knock outs (NKO) of deaf 1 gene exhibited memory deficit and anxiety-like behavior. These deaf 1 NKO mice represent a convenient model for the study of the effects of ID on both in vivo and in vitro memory tests. Earlier studies in these deaf 1 NKOs have shown increased levels of anxiety in the Elevated Plus Maze and Open Field - Test along with contextual-memory deficits in Fear-conditioning experiments. In the intact animal, behavioral phenotyping experiments in mice such as Fear conditioning including contextual and cued fear conditioning measures the ability of the animal to learn, remember and associate an aversive experience to environmental cues. Studies in rodent brain slices involving Long-term potentiation (LTP) and long-term depression (LTD) have long been associated to reflect substrates for memory formation and memory loss respectively. While early-LTP (ELTP) typically lasts between 30-60 minutes, late-LTP (LLTP) lasts for hours; though there is much disagreement about the time courses. In vitro LTD was first reported in 1978 and since then has been studied in details. NMDA receptor (NMDAR) and metabotropic glutamate receptor (mGLUR) activation has been implicated in induction of both LTP and LTD among others. The CA1 region of the rodent hippocampus is the most widely explored area for LTP studies especially stratum radiatum (SR). In addition to the commissural fibers, SR receives Schaffer-collaterals (SC) and is an integral part of memory formation. Previous studies have reported that the CA1 region of the hippocampus expresses both NMDAR-LTD and mGluR-LTD. In the present study, we aim to establish whether these mice might show altered hippocampal Long-term Potentiation (LTP) and/or Long-term Depression (LTD) when brain slices from deaf1 NKO mice were subject to electrophysiological studies and if so, whether pharmacological interventions had any effect on it. Using electrophysiological techniques, hippocampal slices from DEAF1 KO mice were tested for possible alterations in LTP when compared to age-matched controls. Both early and late forms of LTP were examined, since these two types of LTP are medicated through different biochemical mechanisms. ELTP was unaltered in the NKO animals compared to their WT littermates. This experiment was followed by investigating LLTP. The control animals, as expected, exhibited a large LTP. The DEAF1 animals, in contrast, showed a paradoxical response to LLTP stimulation. Instead of the increase in response as observed in the control animals, slices from DEAF1 mice decreased to about 80% of baseline at 30 mins post train. This depression (LTD) became greater throughout the 3 hours of post-train recording, at the end of which the responses were approximately 25% of baseline. The mechanisms of this LTD were then explored with focus on glutamate receptors. Based upon existing knowledge in the literature, the possible roles of both NMDA receptors and mGlu receptors (mGluR1 and mGluR5) were explored. Treatment with D-AP5 - a selective NMDAR antagonist on slices from control animals showed no effect on the baseline evoked responses, but LLTP was blocked following D - AP5 treatment. In slices from DEAF1 NKO animals, AP5 did not affect the baseline evoked responses, but it reversed the expected LTD to a robust LLTP. Next, the involvement of mGlu receptors, known to play a role in LTD, was tested. In controls, there was once again no effect on baseline activity but LLTP at both 30 mins and 180 mins P.T was significantly enhanced as compared to aCSF-only treated slices. In slices from DEAF1 mice, similar to the AP5 study, LY367385 did not affect the baseline response, but reversed LTD to LLTP. Following this, the effect of 40 µM MPEP (an mGluR5 antagonist) was tested, and produced similar results. Thus, three receptor antagonists known to impair the expression of LTD in wild-type animals not only prevented its appearance, but lead to a robust enhancement of the response in DEAF1 hippocampus. Further exploration of the mechanics of altered LTP was undertaken by using Synaptic Tagging and Capture (STC) in combination with pharmacology. Results of the STC experiments suggest that there could be a differential effect of plasticity-inducing stimulation on downstream protein targets. Finally, whole-cell patch recordings were performed to examine the biophysical characteristics of individual CA1 pyramidal neurons. Taken together, the results suggest that multiple mechanisms may be involved in the generation and expression of LTD in the DEAF1 mice.

abstract: Music training is associated with measurable physiologic changes in the auditory pathway. Benefits of music training have also been demonstrated in the areas of working memory, auditory attention, and speech perception in noise. The purpose of this study was to determine whether long-term auditory experience secondary to music training enhances the ability to detect, learn, and recall new words. Participants consisted of 20 young adult musicians and 20 age-matched non-musicians. In addition to completing word recognition and non-word detection tasks, each participant learned 10 nonsense words in a rapid word-learning task. All tasks were completed in quiet and in multi-talker babble. Next-day retention of the learned words was examined in isolation and in context. Cortical auditory evoked responses to vowel stimuli were recorded to obtain latencies and amplitudes for the N1, P2, and P3a components. Performance was compared across groups and listening conditions. Correlations between the behavioral tasks and the cortical auditory evoked responses were also examined. No differences were found between groups (musicians vs. non-musicians) on any of the behavioral tasks. Nor did the groups differ in cortical auditory evoked response latencies or amplitudes, with the exception of P2 latencies, which were significantly longer in musicians than in non-musicians. Performance was significantly poorer in babble than in quiet on word recognition and non-word detection, but not on word learning, learned-word retention, or learned-word detection. CAEP latencies collapsed across group were significantly longer and amplitudes were significantly smaller in babble than in quiet. P2 latencies in quiet were positively correlated with word recognition in quiet, while P3a latencies in babble were positively correlated with word recognition and learned-word detection in babble. No other significant correlations were observed between CAEPs and performance on behavioral tasks. These results indicated that, for young normal-hearing adults, auditory experience resulting from long-term music training did not provide an advantage for learning new information in either favorable (quiet) or unfavorable (babble) listening conditions. Results of the present study suggest that the relationship between music training and the strength of cortical auditory evoked responses may be more complex or too weak to be observed in this population. / Dissertation/Thesis / Doctoral Dissertation Speech and Hearing Science 2017

No description available.

This study investigated the cellular basis for the enhanced ganglionic responsiveness to NK1 agonists in the spontaneously hypertensive rat (SHR) in comparison to their normotensive counterpart, the Wistar-Kyoto (WKY) rat. Rats for in vivo studies were anesthetized with pentobarbital and treated with chlorisondamine (10.5 μmol/kg). Extracellular recordings from the external carotid nerve showed a greater responsiveness of decentralized SHR superior cervical ganglia (SCG) to intravenous injection of SP (32 nmol/kg). Blood pressure and heart rate were increased in SHRs, whereas WKY rats responded with a decrease in blood pressure and only slight tachycardia. Membrane properties of SCG neurons, as shown by intracellular microelectrode recordings, were similar between strains. Picospritzer application of the NK1 agonist GR-73632 (100 μM, 1 s) evoked slow depolarization and increased neuron excitability. Spontaneous firing was evoked only in some neurons. Depolarization amplitudes were similar between strains; however, the NK1 agonist depolarized a greater number of neurons in hypertensive rats. In conclusion, SHRs are more responsive to ganglion stimulation by NK1 agonists due to a greater number of responsive cells within the SCG rather than an enhanced responsiveness of individual neurons.

Indiana University-Purdue University Indianapolis (IUPUI) / The medial prefrontal cortex (mPFC) is a cortical brain region involved in the evaluation and selection of motivationally relevant outcomes. mPFC-mediated cognitive functions are impaired following acute alcohol exposure. In rodent models, ethanol (EtOH) doses as low as 0.75 g/kg yield deficits in cognitive functions. These deficits following acute EtOH are thought to be mediated, at least in part, by decreases in mPFC firing rates. However, these data have been generated exclusively in anesthetized rodents. To eliminate the potentially confounding role of anesthesia on EtOH modulated mPFC activity, the present study investigated the effects of acute EtOH injections on mPFC neural activity in awake-behaving rodents. We utilized three groups: the first group received 2 saline injections during the recording. The second group received a saline injection followed 30 minutes later by a 1.0 g/kg EtOH injection. The last group received a saline injection followed 30 minutes later by a 2.0 g/kg EtOH injection. One week following the awake-behaving recording, an anesthetized recording was performed using one dose of saline followed 30 minutes later by one dose of 1.0 g/kg EtOH in order to replicate previous studies. Firing rates were normalized to a baseline period that occurred 5 minutes prior to each injection. A 5-minute time period 30 minutes following the injection was used to compare across groups. There were no significant differences across the awake-behaving saline-saline group, indicating no major effect on mPFC neural activity as a result of repeated injections. There was a significant main effect across treatment & behavioral groups in the saline-EtOH 1.0 g/kg group with reductions in the EtOH & Sleep condition. In the saline-EtOH 2.0 g/kg, mPFC neural activity was only reduced in lowered states of vigilance. This suggests that EtOH only causes gross changes on neural activity when the animal is not active and behaving. Ultimately this means that EtOH’s impact on decision making is not due to gross changes in mPFC neural activity and future work should investigate its mechanism.

No description available.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and is a core component of the neuromuscular junction, facilitating fast synaptic transmission leading to muscle contraction. Mutations to the human adult muscle nAChR lead to various forms of congenital myasthenic syndrome (CMS), a disease characterized by progressive fatigable muscle weakness. A central channel pore constriction formed by a ring of five leucine residues (L9’) forms part of the nAChR channel gate. CMS-causing mutations in the L9’ residues lead to a form of CMS that results in longer channel opening times and a delayed signal decay. To understand better how L9’ mutations in the human adult muscle nAChR influence channel function, I used two-electrode voltage clamp electrophysiology to perform a comprehensive mutant screen of all L9’ residues in each subunit of the human adult muscle nAChR. This resulted in a total of 76 unique mutations: 19 L9’ mutations consisting of every possible natural amino acid substitution in each subunit (α, β, ε, δ). The results of this screen show that while the polarity and size of a substituted residue contribute to its effect on channel function, increasing the polarity of the side chain typically has a more potentiating effect on channel function than does a change in size. The subunit in which the mutation is expressed also tailors the effect of a given mutation on channel function, with several δL9’ mutations producing qualitatively different effects than equivalent mutations in other subunits. Because the majority of L9’ mutations resulted in a gain-of-function, I originally postulated that interactions between L9’ and surrounding residues stabilize the resting state with the elimination of such interaction through mutations destabilizing the resting state to promote channel gating. Using a double mutant cycle, I explored interactions between the L9’ and adjacent non-L9’ residues but found that there are only weak or no interactions that contribute to channel function. Instead, my data support the hypothesis that the nAChR operates via a hydrophobic gating mechanism, and that adjacent L9’ residues are driven together by the hydrophobic effect to form a closed pore. L9’ mutations that either increase the polarity or decrease residue size likely reduce the hydrophobic driving forces that stabilizes the resting state, thus leading to an enhancement in channel function.

Reproducibility of results in biomedical research is an area of concern that should be paramount for all researchers. Importantly, this issue has been examined for experiments concerning cardiac electrophysiology. Specifically, multiple labs have found differences in results when comparing cardiac conduction velocity (CV) between healthy mice and mice that were heterozygous null for the gap junction (GJ) forming protein, Connexin 43. While the results of the comparison study showed differing extracellular ionic concentrations of the perfusates, specifically sodium, potassium, and calcium ([Na+]o, [K+]o, and [Ca2+]o), there was a lack of understanding why certain combinations of the aforementioned ions led to specific CV changes. However, more research from our lab indicates that these changes can predict modifications to a secondary form of cardiac coupling known as ephaptic coupling (EpC). Therefore the work in this dissertation was twofold, 1) to examine the effects of modulating EpC through perfusate ionic concentrations while also modulating GJC and 2) to investigate the effects of modulating all three of the main ions contributed with cardiac conduction (Na+, K+, Ca2+) and the interplay between them. Firstly I designed and tested changes from the use of 3D printed bath for optical mapping procedures. After verification that the bath did not modify electrophysiological or contrile parameters, I studied the effects of physiologic changes to EpC determinants ([Na+]o and [K+]o) on CV during various states of GJ inhibition using the non-specific GJ uncoupler carbenoxolone (CBX). Multiple pacing rates were used to further modify EpC, as an increased pacing rate leads to a decrease in sodium channel availability through modification of the resting membrane potential. with no to low (0 and 15 µM CBX) GJ inhibition, physiologic changes in [Na+]o and [K+]o did not affect CV, however increasing pacing rate decreased CV as expected. When CBX was increased to 30 µM, a combination of decreasing [Na+]o and increasing [K+]o significantly decreased cardiac CV, specifically when pacing rate was increased. Next, the combinatory effects of cations associated with EpC (Na+, K+, and Ca2+) were tested in to examine how cardiac CV reacts to changes in perfusate solution and how this may explain differences in experimental outcomes between laboratories. Briefly, experiments were run where [K+]o was varied throughout an experiment and the values for [Na+]o and [Ca2+]o were at one of two specific values during an experiment. 30 µM CBX was added to half of the experiments to see the changes in the CV-[K+]o relationship with GJ inhibition. With unaltered GJ coupling, elevated [Na+]o maintains CV during hyperkalemia. Interestingly, both [Na+]o and [Ca2+]o must be increased to maintain normal CV during hyperkalemia with reduced GJ coupling. These data suggest that optimized fluids can sustain normal conduction under pathophysiologic conditions like hyperkalemia and GJ uncoupling. Taken as a whole, this dissertation attempts to shed light on the importance of ionic concentration balance in perfusate solutions on cardiac conduction. / Ph. D. / The use of fluid replacement therapy was first used during the outbreak of Blue Cholera in the 1830s. However, after the development of basic fluids for intravenous fluid therapy, there have been very few changes in the fluid recipes. This same principle can be applied to cardiac research, where blood substitute perfusates are used during experimentation. However, there have been disagreements in experimental outcomes between various labs running matching studies which only varied in choice of perfusate solution. Therefore, one of the goals of this dissertation was to explore how changing ionic concentrations in cardiac perfusate solutions affected cardiac electrophysiological parameters. To fully appreciate changes in cardiac conduction, we also had to investigate changes to gap junctional coupling (GJC), which is the canonical determinant of cardiac conduction. Gap junctions are low resistance pathways which allow direct cell-to-cell coupling, which leads to synchronized cardiac conduction and contraction. However, there have been recent studies that have found a secondary form of cardiac coupling, known as ephaptic coupling (EpC), which is controlled through extracellular ionic concentrations, especially sodium, potassium, and calcium ([Na⁺]₀, [K⁺]₀, and [Ca²⁺]₀ respectively) and extracellular nano-domains known as the perinexus. We first investigate making small physiologic changes to [Na⁺]₀ and [K⁺]₀, while also inhibiting GJs to find the relationship between EpC and GJC. The results indicated that these EpC modulators could indeed modulate conduction, but only after GJs were sufficiently inhibited. However, results from this study disagreed with historical work indicating that [K⁺]₀ had a biphasic relationship with CV. Therefore, we then examined the effects of [Na⁺]₀ and [Ca²⁺]₀ on the CV-[K⁺]₀ relationship. Interestingly, it was found that inclusion of [Na⁺]₀ and [Ca²⁺]₀ had varying effects, depending on the level of GJ in the hearts. Specifically, hyperkalemia (high levels of potassium) v is associated with decreases cardiac CV. With a full complement of GJs it was found that increased [Na⁺]₀ was able to maintain cardiac CV at control levels. However, with inhibited GJ coupling, both increased [Na⁺]₀ and [Ca²⁺]₀ were needed to maintain conduction. This indicated that increasing EpC during GJ inhibition could be a possible safety mechanism for cardiac CV. The data in this dissertation aim to provide information to the importance of perfusate composition when regarding scientific data.

Aberrations in conduction or repolarization are established prerequisites for arrhythmogenesis. The following dissertation investigates how reducing either ephaptic (EpC) or gap junctional (GJ) coupling between cardiomyocytes can modulate cardiac conduction, repolarization, or the relationship between these two phenomena. Our lab has previously demonstrated that EpC can be modified in the ventricular epicardium using ionic and osmotic challenges to the Langendorff-perfused heart. In the first series of experiments, we show that reducing EpC via treatment with mannitol or hyponatremia can unmask conduction deficits that are otherwise below the resolution of detection in Scn5a+/- mice. Interestingly, we also observe that combination of the two treatments resolves severe conduction delay due to hyponatremia in the heterozygous animal. These data suggest it may be valuable to pursue the use of mannitol or hyponatremia as novel diagnostics for sodium channel loss of function diseases. The importance of extracellular perfusate is also highlighted by the second investigation, which evaluates how sodium and calcium concentration modulate repolarization in the context of hyperkalemia, a common comorbidity of hospitalized patients that increases the risk of arrhythmia. Calcium may potentially play a role in modulating APD adaptation to pacing rate in the context of this disease state, though more research is needed to clarify the exact mechanism of this effect. Finally, we investigate the relationship between conduction and repolarization in the epicardium, and conclude that this relationship does not appear to be dictated by the degree of cell-cell coupling in the myocardium, but instead is driven by endogenous gradients of action potential duration within the tissue. Taken together, these data demonstrate ways in which both conduction and repolarization are sensitive to modulations of EpC, though we also find that the relationship between these two phenomena is not influenced by such changes in electrical coupling. / Doctor of Philosophy / The ability of the heart to function as a pump is dependent on the successful coordination of electrical activity throughout the heart. Disruptions to this intricate electrical system result in cardiac arrhythmias, which in turn prevent the heart from effectively perfusing the body with oxygenated blood. The present dissertation investigates ways in which we can modulate cell-cell communication within the heart, and how this may in turn influence disease states with a high propensity for arrhythmia. We show that reducing electrical coupling between cells using simple interventions like reducing serum sodium or increasing osmolarity may be a viable technique for diagnosing "concealed" disease states (i.e. disease states that are asymptomatic for much of a patient's life). We then explore ways in which elevated serum potassium, known as hyperkalemia, may alter the heart's ability to recover from electrical activation (repolarization). Finally, we show that the relationship between cardiac activation and repolarization is not as dependent on cell-cell communication as was once thought. Taken together, this dissertation provides evidence that transiently disrupting cell-cell communication may hold promise for development of diagnostics for some congenital cardiac diseases, and yet does not appear to disrupt the relationship between electrical conduction and repolarization across the heart.