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.

Indiana University-Purdue University Indianapolis (IUPUI) / Nearly 18% of children are diagnosed with developmental disabilities. Autism spectrum disorders (ASDs) and attention deficit hyperactivity disorder (ADHD) are increasingly common developmental disabilities, but neither is well understood. ADHD and ASD are both prevalent in the genetic disorder Neurofibromatosis type 1 (NF1) which impairs the Ras-MAPK/ERK pathway through mutation of the neurofibromin gene (NF1+/−). More broadly, syndromic forms of developmental disorders are often caused by mutations of proteins in pathways interconnected with Ras including TSC1/2, FMR1, and SynGAP. Of NF1 patients, around 30-50% are diagnosed with ASDs and more than 60% with ADHD. These studies are the first to show that male mice haploinsufficient for the Nf1 gene (Nf1+/−) exhibit deficits in behavioral inhibition in multiple contexts, a key feature of ADHD. They exhibit hyperactivity and impulsivity in an open field, delay discounting task, and cliff avoidance reaction test, rescuable through treatment with the clinically effective ADHD drug, guanfacine (α2A adrenergic receptor agonist). Previous experiments in our lab identified social deficits including deficits in consolidation of social memory. Using optogenetics and awake behaving electrode recordings, we explored the role of the cortico-striatal-limbic circuitry in impulsivity and in social deficits in male Nf1+/− mice. Manipulation of the prefrontal cortex, nucleus accumbens, or basolateral amygdala through optogenetics rescued social deficits. These studies are the first to record brain activity in a preclinical model of NF1 during impulsive behavior, finding broad spectrum changes across slow, delta, theta, and gamma oscillatory frequencies and decreased synchrony of the prefrontal cortex and nucleus accumbens during a delay discounting task. Overall, Nf1+/− male mice with deletion of a single NF1 gene recapitulate cognitive phenotypes of NF1 patients and are a useful model system to identify alterations in neural circuitry associated with ASD and ADHD.

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At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.

Large conductance Ca2+-activated K+ channels (BK channels) are expressed ubiquitously in both excitable and non-excitable cells and are important for a range of physiological functions. BK channels gate K+ efflux in response to concurrent depolarized membrane voltage and increased intracellular Ca2+ to modulate action potential shape and duration in neurons, regulate contractility in smooth muscle, and control fluid secretion by epithelial cells in the airway and gut. In addition, mutations in the human BK channel gene (KCNMA1) are linked to neurological disease, such as epilepsy and paroxysmal dyskinesia. Thus, BK channel modulators may provide treatment avenues for BK channelopathies. It will be important to expand our arsenal of BK channel-selective activators and inhibitors and to grow our understanding of their molecular mechanisms of action. Discovery of new channel modulators will further lead to a greater understanding of BK channel structure and function. To better understand the basic structure-function relationship of BK channel gating in response to increased intracellular Ca2+ concentration, in this work I initially investigate structural determinants of BK channel activation in response to conformational changes following Ca2+ binding. I analyze crystal structures of the BK channel cytosolic Ca2+-sensing domain (CSD), also known as the “gating ring”, formed by the C-terminal domains of each of the four identical pore-forming subunits. In the Ca2+-bound state, N449 from the adjacent subunit contacts the bound Ca2+ ion, forming a “Ca2+ bridge.” Mutating N449 to alanine eliminates this coordinate interaction, and using electrophysiology, I found that BK channels with the N449A mutation exhibit a shift in the voltage required for half maximal activation (V1/2) towards more positive voltages. Using size-exclusion chromatography, I observed that the purified BK channel CSD with the N449A mutation shows reduced gating ring oligomerization in response to Ca2+ compared to the wild-type CSD. To further probe molecular determinants of BK channel gating and increase our arsenal of BK channel gating modulators, I optimized a fluorescence-based high throughput screening approach to discover compounds with BK channel inhibitor activity with 99.73% confidence. Through this approach I discovered that the -opioid receptor agonist, loperamide, is a potent BK channel inhibitor. Loperamide (LOP) reduced the open probability of channels at depolarized voltages, but not at very negative voltages when the voltage-sensor is at rest. I observed a weak voltage dependence of loperamide inhibition, consistent with loperamide binding shallow within the inner cavity to block the channel pore. I quantified the inhibitory effect of LOP using an allosteric model in which LOP blocks conduction through open channels and binds with 45-fold higher affinity to the open state over the closed state. These data suggest that loperamide may represent a new class of “use-dependent,” open channel blockers. Together this work describes an approach to understanding BK channel structure and function with the goal of identifying and developing novel therapeutics for the treatment of BK-related diseases. / Biochemistry

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Fluoroscopy is considered the gold standard for locating catheters during cardiac electrophysiology (EP) procedures. However, fluoroscopy emits ionizing radiation which can lead to adverse health effects when exposed to in high doses (World Health Organization, 2016). Electroanatomic mapping (EAM) systems display the three-dimensional location of EP catheters and measure the local electrical activity of the heart. They can minimize a physician’s reliance on fluoroscopy and can help reduce radiation exposure during a case (Casella, 2011). EAM systems are diagnostic medical devices that inform the placement of ablation therapy and must accurately locate catheters to be deemed safe. Test methods to determine EAM system accuracy should be compared back to a gold standard, such as fluoroscopy. Fluoroscopy only provides a two-dimensional image of the catheter location, which is not a suitable ground truth for measuring the three-dimensional accuracy of EAM systems. X-Ray Reconstruction of Electrode Locations (XRROEL) calculates the true three-dimensional catheter location by performing a coordinate transform on two-dimensional fluoroscopy images. This thesis outlines the development and validation of the XRROEL method in a porcine animal model, and describes how XRROEL can be applied to optimize the location accuracy of electroanatomic mapping system algorithms.

Social interactions between individuals, particularly within groups, constitute a vital aspect of animal survival across nearly all species. In particular, higher level, evolutionarily preserved processes and behaviors such as pro-sociality (e.g., recognition of another’s emotional state) and group interactions (e.g., competitive foraging) have been widely studied in fields of ethology, psychology, and economics. While recent neuroethological studies have examined interactive behaviors at extremes of dyads in artificial environments or herds in nature, gaining a full understanding of the neuronal mechanisms underlying these behaviors requires new approaches that examine freely behaving individuals within groups. Additionally, while there is mounting evidence pointing to the role that the dorsomedial prefrontal cortex (dmPFC) and the dACC (dorsal anterior cingulate cortex) play as a hub of the ‘social brain,’ the single-neuronal processes driving pro-social behaviors and naturalistic group interactions remain unknown. In this dissertation, I begin by reviewing previous work that has guided the study of social interactive behaviors and their neuronal mechanisms in animal models. Next, I present our novel findings and advances that combine a host of cutting-edge techniques to explore fundamental gaps in our understanding of pro-sociality and group interactions in male mice. Specifically, we find that 1) rodent dmPFC neurons differentially represent other- and self-experiences and drive prosocial ‘helping’ behaviors, and 2) the dmPFC uniquely drives competitive effort based on information about others during group foraging. I also discuss the impact of these results that may be translated to the clinic, where deficits in these behaviors are a hallmark of psychosocial illnesses such as autism spectrum disorders (ASD). Using a mouse Shank3 haploinsufficient model of ASD, I show that 3) proper Shank3 expression is necessary for prosocial decision making and that adult restoration of Shank3 expression reverses neuronal social-encoding imbalances in the dmPFC. Last, I forecast the future of social neuroethology by discussing recent technological advancements that will allow us to reveal the neural and molecular mechanisms of complex social behaviors emerging from groups of animals. Together, findings from this dissertation add to our fundamental understanding of the complex role that the dmPFC plays in social cognition and interactive behaviors. Data from these studies also reveal a remarkably rich neuronal process in the mouse prefrontal cortex that drives prosocial and competitive decision making in groups. The novel assays that I developed in these experiments also provide a unique framework to develop new treatments for psychosocial disorders in future studies. / 2024-02-03T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Problematic alcohol consumption develops concurrently with deficits in decision-making. These deficits may be due to alterations in dorsal medial prefrontal cortex (dmPFC) neural activity, as it is essential for the evaluation and implementation of behavioral strategies. In this study, we hypothesized that differences in cognitive control would be evident between Wistars and alcohol-preferring P rats. Cognitive control can be split into proactive and reactive components. Proactive control maintains goal-directed behavior independent of a stimulus whereas reactive control elicits goal-directed behavior at the time of a stimulus. Specifically, it was hypothesized that Wistars would show proactive control over alcohol-seeking whereas P rats would show reactive control over alcohol-seeking. Proactive control in our rodent model is defined as responding to distal task cues whereas reactive control is responding to proximal cues. This was tested in rodents performing a 2-way Cued Access Protocol (2CAP) that facilitates measurements of alcohol seeking and drinking. Congruent sessions were the typical, default 2CAP sessions that consisted of the CS+ being on the same side as alcohol access. These were compared with incongruent sessions where alcohol access was opposite of the CS+. Wistars exhibited an increase in incorrect approaches during the incongruent sessions, which was not detectable in P rats. A trial-by-trial analysis indicated that the increases in incorrect responses was explained by Wistars utilizing the previously learned task-rule, whereas the P rats did not. This motivated the subsequent hypothesis that neural activity patterns corresponding to proactive control would be observable in Wistars but not P rats. Principal Component Analysis indicated that neural ensembles in the dmPFC of Wistars exhibited decreased activity to the cue light in incongruent sessions whereas P rat ensembles displayed increased activity at timepoints associated with the onset and end of alcohol access. Overall, it was observed that P rats showed the most differences in neural activity at times relevant for alcohol delivery; specifically, when the sipper came into the apparatus and left. Conversely, Wistars showed differences prior to approach as evidenced by both differences in cue-related activity as well as differences in spatial-strategies. Together, these results support our hypothesis that Wistars are more likely to engage proactive cognitive control strategies whereas P rats are more likely to engage reactive cognitive control strategies. Although P rats were bred to prefer alcohol, differences in cognitive control phenotypes may have concomitantly occurred that are of clinical relevance.