Indiana University-Purdue University Indianapolis (IUPUI) / Currently, changes to endogenous opioid circuits in various pain modalities, including surgical and neuropathic pain, remain unclear. Dynorphin, which is released by prodynorphin-expressing neurons (Pdyn+ neurons), is the endogenous opioid ligand to kappa opioid receptors (KOR). Moreover, a recent study has shown an increase in prodynorphin (Pdyn) mRNA expression in the prelimbic cortex (PL) in a mouse model of chronic pain. However, alterations in the activity of PL Pdyn-expressing neurons (PLPdyn+ neurons) in postoperative and chronic pain have never been explored. Firstly, I found that the population of PLPdyn+ neurons consists of both pyramidal and inhibitory subtypes. Secondly, I found that one day after surgical incision of the mouse hind paw, the excitability of pyramidal PLPdyn+ neurons was increased in both male and female mice, while the excitability of inhibitory PLPdyn+ neurons was unchanged. However, when postoperative pain behavior subsided, inhibitory PLPdyn+ neurons were hyperexcitable in male mice, while pyramidal PLPdyn+ neurons were hypoexcitable in female mice. Lastly, I dissected electrophysiological changes to PLPdyn+ neurons in the spared nerve injury (SNI) model of chronic neuropathic pain. At both early and late stages of SNI pain development, increased excitability of pyramidal PLPdyn+ neurons was detected in both male and female mice. However, in both male and female mice, the excitability of inhibitory PLPdyn+ neurons decreased 3 days after SNI but was conversely increased when measured 14 days after SNI. My findings suggest that different subtypes of PLPdyn+ neurons manifest distinct alterations in the development of different pain modalities in a sex-specific manner.

The development of tissue culture electrophysiology has been hindered by these numerous technological problems. The function of this thesis was to identify and propose solutions to these technological problems and concurrently develop a system for prolonged intracellular GME monitoring (30 to 40 hours) of membrane parameters from cells grown in tissue culture. The technological problems identified in the present study and for which solutions have been proposed are: (1) Maintenance of a sterile environment for an open tissue culture vessel in which GME measurements can be made for periods exceeding 30 to 40 hours.; (2) Elimination of environmental electrical interference which normally interferes with GME recording and data displaying.; (3) Design of an optical instrumentation system that is compatible with microelectrode penetration needs and tissue culture cell observation.; (4) Elimination of building and peripheral instrument vibrational influences at the microelectrode recording site.; (5) A method of microelectrode preparation that would produce consistent glass microelectrodes meeting the peculiar needs of tissue culture electrophysiology.; (6) Design of a culture recording chamber which would permit: (a) culturing of cells using standard tissue culture techniques.; (b) easy access of cells for microelectrode penetration.; (c) continuous optical observation.; (d) continuous temperature regulation of cultured cells for periods exceeding 30 to 40 hours.; (e) shielding against electrical field disturbances during microelectrode recording.; (f) rapid perfusion of media to the chamber without causing harsh turbulence allowing continuous intracellular recording via glass microelectrode even during perfusion of substances.; (g) microelectrodes penetration of cells over a wide range of angles.; (7) Design of a complete intracellular tissue culture electrophysiology monitoring system.; and (8) Development of explant and culturing procedures to provide cell substrate to test the effectiveness of the tissue culture electrophysiology system design.

<p>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 </p> <p>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 </p> <p>was explained by Wistars utilizing the previously learned task-rule, whereas the P rats did not. </p> <p>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 </p> <p>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.</p>

Dopamine cells within the ventral tegmental area of the brain are involved in motivation and reward. Drugs of abuse target these dopamine cells altering their activity and plasticity resulting in addiction. While dopamine cell activity is primarily involved in addiction, the GABA neurons in the VTA have also been shown to have an indirect role. By decreasing the activity of the inhibitory GABA inputs onto dopamine neurons abusive drugs can indirectly increase dopamine cell activity resulting in addictive behaviors. However, although GABA neurons are important in the perception of reward, much less is known about how the excitatory inputs to these cells are regulated and possibly altered by drugs of abuse. Using transgenic mice expressing GFP attached to the GAD promoter, GABA cells were located and patched using whole cell voltage clamp and EPSCs were measured. High frequency stimulation induced LTD of the excitatory inputs to GABA neurons. The endocannabinoid analogue R- methanandamide also induced LTD at these excitatory synapses. These results suggest that endocannabinoids could potentially regulate the activity of GABA cells and as a result the activity of dopamine neurons. The endocannabinoid receptor involved is likely CB1, but not TRPV1 as only the CB1 antagonist AM-251 blocked this high frequency stimulus-induced LTD. Future research could then determine if the pathways involved in this LTD could potentially be altered by drugs of abuse contributing to addiction.

Blast-induced traumatic brain injury (bTBI) has been a health concern in both military and civilian populations due to recent military and geopolitical conflicts. Military service members are frequently exposed to single and repeated blasts throughout their training and deployment. As a result of blast exposures, military personnel report symptoms of various neurological and neurosensory deficits. Our group has previously reported decreased long term potentiation (LTP) following either single or repeated bTBI in a rat organotypic hippocampal slice culture (OHSC) model. LTP is a neuronal correlate for learning and memory and is a neurological metric that can be used to evaluate blast injury severity and the efficacy of therapeutic interventions. In the first aim of this thesis, we characterized LTP deficits following repeated bTBI to develop tolerance criteria for blast exposures. We did so by varying the blast injury severity, the inter-blast interval between blasts, and the recovery period following blast exposure. We determined that LTP deficits were compounded as a result of repeated mild bTBI. LTP deficits were attenuated with increasing inter-blast intervals and with increasing recovery periods after injury. Even after three repeated mild bTBIs, LTP spontaneously recovered after 6 days. In the second aim, we investigated the pathological changes in OHSCs following repeated blast exposures. Following injury, we observed robust microglial activation, evidenced by increased expression of the pro-inflammatory marker, CD-68, and decreased expression of the anti-inflammatory marker, CD-206. We also observed increased expression of MIP-1α, IL-1β, MCP-1, IP-10, and RANTES and decreased expression of IL-10 in the acute period after both single and repeated bTBI. Following partial depletion of microglia prior to injury, injury induced LTP deficits were significantly reduced. Lastly, treatment with a novel drug, MW-189, immediately after a repeated bTBI prevented LTP deficits. In the third aim, we investigated changes in inflammatory markers like cyclooxygenase (COX) and tested the efficacy of COX or prostaglandin receptor (EP3R) inhibitors in attenuating LTP deficits. We observed that expression of COX-2 increased 48 hours following repeated blast injury; however, COX-1 expression was unchanged. Following repeated bTBI, EP3R expression was upregulated and cyclic adenosine monophosphate (cAMP) concentration was decreased. Treatment of blast injured OHSCs with a COX-1 specific inhibitor, SC-560, a COX-2 specific inhibitor, rofecoxib, a pan-COX inhibitor, ibuprofen, or an EP3R inhibitor, L-798,106 improved LTP deficits. Delayed treatment with L-798,106 and ibuprofen also improved LTP deficits. Our data suggests that bTBI induced neuroinflammation may be partially responsible for the functional deficits that we have observed in blast-injured OHSCs. Additionally, we also conclude that COX and EP3R inhibition may be viable therapeutic strategies to reduce bTBI induced neurophysiological deficits. In the final aim, we investigated bTBI induced changes to the electrophysiological network of OHSCs. Following blast exposure, sham and injured OHSCs were administered increasing concentrations of bicuculline, a GABAA receptor antagonist. Doing so revealed an increase in connectivity and clustering coefficients in sham slices compared to injured slices. This suggested that the underlying neuronal network of injured slices was dysfunctional. Biologically, this dysfunction could be explained by the decreased expression of GABAA receptor α1 and α5 subunits. A loss of GABAA receptor expression or function may explain the electrophysiological network disruptions that we observed. More work will be required to determine how blast exposure decreases the expression of GABAA receptors and how these receptors may contribute to network deficits. This thesis has expanded upon the tolerance criteria for repeated blast exposures. These studies have also further characterized the pathological changes in microglial activation and explored promising therapeutic pathways that could be used to attenuate functional deficits. Lastly, this thesis has also provided novel ways to interrogate neuronal networks following blast injury, revealing subtle deficits that will need to be explored in more detail.

The single largest cause of compound attrition during drug development is due to inadequate tools capable of predicting and identifying protein interactions. Several tools have been developed to explore how a compound interferes with specific pathways. However, these tools lack the potential to chronically monitor the time dependent temporal changes in complex biochemical networks, thus limiting our ability to identify possible secondary signaling pathways that could lead to potential toxicity. To overcome this, we have developed an in silico neuronal-metabolic model by coupling the membrane electrical activity to intracellular biochemical pathways that would enable us to perform non-invasive temporal proteomics. This model is capable of predicting and correlating the changes in cellular signaling, metabolic networks and action potential responses to metabolic perturbation. The neuronal-metabolic model was experimentally validated by performing biochemical and electrophysiological measurements on NG108-15 cells followed by testing its prediction capabilities for pathway analysis. The model accurately predicted the changes in neuronal action potentials and the changes in intracellular biochemical pathways when exposed to metabolic perturbations. NG108-15 cells showed a large effect upon exposure to 2DG compared to cyanide and malonate as these cells have elevated glycolysis. A combinational treatment of 2DG, cyanide and malonate had a much higher and faster effect on the cells. A time-dependent change in neuronal action potentials occurred based on the inhibited pathway. We conclude that the experimentally validated in silico model accurately predicts the changes in neuronal action potential shapes and proteins activities to perturbations, and would be a powerful tool for performing proteomics facilitating drug discovery by using action potential peak shape analysis to determine pathway perturbation from an administered compound.

Spatial navigation in humans correlates with activity of cells in hippocampus that respond when we traverse specific locations in our environment. Hippocampal pyramidal cells in rodents called "place cells" may contribute to episodic memory by encoding location in physical space. Place cells display plasticity by "remapping" or altering their firing rates and patterns of activity in response to changes in spatial environment. Impaired remapping may underlie age-related deficits in spatial memory tasks. Using in vivo high-density electrophysiology to record place cell activity in awake, behaving rats, we tested the hypothesis that CA3 neuron hyperactivity in aged animals could be normalized by pharmacotherapy. Results show that acute, systemic administration of low dose levetiracetam and sodium valproate ameliorates deficits in the aged hippocampal network by reducing firing rates, decreasing place field area, and increasing the spatial selectivity of CA3 place cells. We then tested the hypothesis that place cell activity, field area, and spatial selectivity may be an indicator for therapeutic enhancement of spatial memory in young adult rats. The results demonstrate that α5IA enhances hippocampal-dependent spatial memory as measured by the location novelty recognition task in rats, consistent with the previously established action of α5IA as an enhancer of spatial memory in the water maze test. Electrophysiological recordings on the same animals carried out in parallel demonstrate that α5IA increases place cell firing rates, reduces field area, and increases spatial selectivity. Together, these results suggest that reducing place field area and enhancing spatial selectivity correlate with the age-independent therapeutic improvement of spatial memory. The increase in place cell firing rates by α5IA likely results from its known action as a negative allosteric modulator of α5-subunit-containing receptors (α), which are located extrasynaptically at the base of dendritic spines on CA1 and CA3 pyramidal cells. Thus, to potentially target extrasynaptic tonic inhibition in the hippocampus, we synthesized and validated two α specific miRNAs as a platform for future attempts to improve spatial memory in young adult and aging animals via molecular genetics.

Indiana University-Purdue University Indianapolis (IUPUI) / Several pathological disorders are characterized by maladaptive decision-making (Dalley & Robbins, 2017). Decision-making tasks, such as Delay Discounting (DD), are used to assess the behavioral manifestations of maladaptive decision-making in both clinical and preclinical settings (de Wit, Flory, Acheson, Mccloskey, & Manuck, 2007). DD measures cognitive impulsivity and broadly refers to the inability to delay gratification (Hamilton et al., 2015). How decisions are made in tasks that measure DD can be understood by assessing patterns of behavior that are observable in the sequences of choices or the statistics that accompany each choice (e.g. response latency). These measures have led to insights that suggest strategies that are used by the agent to facilitate the decision (Linsenbardt, Smoker, Janetsian-Fritz, & Lapish, 2016). The current set of analyses aims to use individual trial data to identify the neural underpinnings associated with strategy transition during DD. A greater understanding of how strategy change occurs at a neural level will be useful for developing cognitive and behavioral strategies aimed at reducing impulsive choice. The rat dorso-medial prefrontal cortex (dmPFC) has been implicated as an important brain region for recognizing the need to change strategy during DD (Powell & Redish, 2016). Using advanced statistical techniques, such as demixed principal component analysis (dPCA), we can then begin to understand how decision representations evolve over the decision- making process to impact behaviors such as strategy change. This study was the first known attempt at using dPCA applied to individual sessions to accurately model how decision representations evolve across individual trials. Evidence exists that representations follow a breakdown and remapping at the individual trial level (Karlsson, Tervo, & Karpova, 2012; Powell & Redish, 2016). Furthermore, these representational changes across individual trials have previously been proposed to act as a signal to change strategies (Powell & Redish, 2016). This study aimed to test the hypothesis that a ‘breakdown’ followed by a ‘remapping’ of the decision representation would act as a signal to change strategy that is observable in the behavior of the animal. To investigate the relationship between trials surrounding the breakdown and/or subsequent remapping of the decision representation and trials surrounding strategy changes, sequences of trials surrounding the breakdown and/or remapping were compared to sequences of 9 trials surrounding the strategy-change trial. Strategy types consisted of either exploiting the immediate lever (IM-Exploit), delay lever (DEL-Exploit), or exploring between the two lever options (Explore). Contrary to the hypothesis, an overall relationship between breakdown and remapping trial sequences were not associated with change-trial sequences. In partial support of the hypothesis however, at the 4-sec delay when the subjective value of the immediate reward was high, a relationship between breakdown sequence and strategy change sequence was detected for when the animal was exploiting the delay lever (e.g. DEL-Exploit strategy). This result suggests that a breakdown in decision representation may act as a signal to prompt strategy change under certain contexts. One notable finding of this study was that the decision representation was much more robust at the 4-sec delay compared to the 8-sec delay, suggesting that decisions at the 4-sec delay contain more context that differentiate the two choice options (immediate or delay). In other words, the encoding of the two choice options was more dissociable at the 4-sec delay compared to the 8-sec delay, which was quantified by measuring the average distance between the two representations (immediate and delay) on a given trial. Given that Wistar rats are equally likely to choose between the immediate and delay choice alternatives at the 8-sec delay (Linsenbardt et al., 2016), this finding provides further support for current prevalent theories of how animals use a cognitive search process to mentally imagine choice alternatives during deliberation. If context which differentiates choice options at the 8-sec delay is less dissociable, it is likely that the cognitive search process would be equally likely to find either choice option. If the choice options are equally likely to be found, it would be assumed that the choice alternatives would also be equally likely to be chosen, which is what has been observed in Wistar rats at the 8-sec delay.

Humans hold the remarkable capacity to attend to a single person’s voice when many people are talking. Nevertheless, individuals with hearing loss may struggle to tune into a single voice in these types of complex acoustic situations. Current hearing aids can remove background noise but are unable to selectively amplify a single person’s voice without first knowing to whom the listener aims to attend. Studies of multitalker speech perception have demonstrated an enhanced representation of attended speech in the neural responses of a listener, giving rise to the prospect of a brain-controlled hearing aid that uses Auditory Attention Decoding (AAD) algorithms to selectively amplify the target of the listener’s attention as decoded from their neural signals. In this dissertation, I describe experiments using non-invasive and invasive electrophysiology that investigate the encoding and decoding of speech representations that inform our understanding of the influence of attention on speech perception and advance our progress toward brain-controlled hearing devices. First, I explore the efficacy of AAD in improving speech intelligibility when switching attention between different talkers with data recorded non-invasively from listeners with hearing loss. I show that AAD can be effective at improving intelligibility for listeners with hearing loss, but current methods for AAD with non-invasive data are unable to detect changes in attention with sufficient accuracy or speed to improve intelligibility generally. Next, I analyze invasive neural recordings to more clearly establish the boundary between the neural encoding of target and non-target speech during multitalker speech perception. In particular, I investigate whether speech perception can be achieved through glimpses, i.e. spectrotemporal regions where a talker has more energy than the background, or if the recovery of masked regions is also necessary. I find that glimpsed speech is encoded for both target and non-target talkers, while masked speech is encoded for only the target talker, with a greater response latency and distinct anatomical organization compared to glimpsed speech. These findings suggest that glimpsed and masked speech utilize separate encoding mechanisms and that attention enables the recovery of masked speech to support higher-order speech perception. Last, I leverage my theory of the neural encoding of glimpsed and masked speech to design a novel framework for AAD. I show that differentially classifying event-related potentials to glimpsed and masked acoustic events is more effective than current models that ignore the dynamic overlap between a talker and the background. In particular, this framework enables more accurate and stable decoding that is quicker at identifying changes in attention and capable of detecting atypical uses of attention, such as divided attention or inattention. Together, this dissertation identifies key problems in the neural decoding of a listener’s attention, expands our understanding of the influence of attention on the neural encoding of speech, and leverages this understanding to design new methods for AAD that move us closer to the development of effective and intuitive brain-controlled hearing assistive devices.

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