Given the dramatic rise in obesity and the diseases linked with it, it is becoming increasingly important to understand the mechanisms that underlie the body's ability to recognize fat. The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that relays the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to activate trigeminal ganglionic neurons has remained unexplored. In general, my research has provided the first evidence of fatty acids activating the somatosensory system by increasing the intracellular calcium concentration and generating receptor potentials. Other experiments were focused on identifying fatty acids-responsive pathways in acute isolated trigeminal neurons involved with fat somatosensory perception. My results revealed that fatty acids-activated pathways involved the release of intracellular calcium stores in subpopulations of trigeminal neurons. By using pseudorabies virus as a "live-cell" tracer, a subpopulation of lingual-innervated trigeminal neurons was labeled. Quantitative real-time polymerase chain reaction on individual neurons showed several transient receptor potential channel markers were expressed in these labeled neurons, which indicated the identification of lingual-innervated neurons was successful. This technique helped resolve the problem of trigeminal neurons being a mixed population of cells, and confirmed the role of the release of intracellular calcium stores in fatty acid-activated pathways. Using patch clamp recording, I discovered that the linoleic acid activated signaling pathway involved the activation of G protein and phospholipase C. I further began to characterize the downstream conductance that is activated by linoleic acid in rat trigeminal neurons. Using perforated patch clamp recording, I have recorded linoleic acid-induced currents that exhibited many of the properties of transient receptor potential-like channels, suggesting that this "cellular sensor" is probably playing an important role in the somatosensory perception of fat. Taken together, this dissertation research has revealed the ability of fatty acids to act as effective tactile stimuli and identified several elements of the fatty acids-activated signaling pathway involved in the somatosensory perception of fat.

Stroke is a leading cause of death and disability worldwide. Although rapid recognition and prompt treatment have dropped mortality rates, most stroke survivors are left with permanent disability. Approximately 87% of all strokes result from the thromboembolic occlusion of the cerebrovasculature (ischemic strokes). Potential stroke therapeutics have included anti-inflammatory drugs, as well as many other targets with the goal of mitigating the acute and chronic inflammatory responses typically seen in an ischemic stroke. While these approaches have had great success in preclinical studies, their clinical translation has been less successful. Master inflammatory cytokines, such as IL-1, are of particular interest. IL-1’s isoforms, IL-1α and IL-1β, were long thought to have similar function. While IL-1β has been extensively studied in stroke, the role of IL-1α during post stroke inflammation has been overlooked. Because IL-1 inhibitors have been unsuccessful in clinical application, we reasoned that IL-1α may provide previously unknown benefits to the brain after injury. We hypothesized that IL-1α could be protective or even accelerate reparative processes in the brain such as producing new blood vessels (angiogenesis) or neurons (neurogenesis). To test that IL-1α is protective after stroke, we tested IL-1α’s protective effects on primary cortical neurons in in vitro models of stroke. We showed that IL-1α was directly protective on primary cortical neurons in a dose-dependent fashion. We then performed mouse middle cerebral artery occlusion stroke studies to determine the safety of giving IL-1α in vivo. These studies showed that administering IL-1α acutely was neuroprotective. However, intravenous (IV) administration of IL-1α resulted in transient, hemodynamic changes following drug delivery. To minimize these systemic effects, we administered IL-1α intra-arterially (IA) directly into the stroke affected brain tissue, allowing us to significantly lower the concentration of administered IL-1α. In comparison to IV, IA IL-1α showed greater histological protection from ischemic injury as well as improved functional recovery following stroke, all without systemic side effects. To test that IL-1α could aid in neurorepair following stroke, we tested IL-1α’s ability to help damaged blood vessels repair in vitro. We found that IL-1α significantly increased brain endothelial cell activation, proliferation, migration, and capillary formation. We tested IL-1α’s proangiogenic properties in vivo by administering IL-1α three days following stroke. Delayed administration allowed us to separate IL-1α’s acute neuroprotective effects from potential subacute angiogenic effects. We found that mice receiving IL-1α performed significantly better on behavioral tests and also showed greater vascularization within the penumbra two weeks following stroke. We also found that IL-1α treated animals showed more endothelial activation than vehicle treated animals. Finally, our studies showed that IL-1α treated animals showed increased early-phase neurogenesis with evidence of increased proliferation at the subventricular zone suggesting that IL-1α’s beneficial effects are even more far-reaching than previously thought. In conclusion, our experiments suggest that the inflammatory cytokine IL-1α is neuroprotective and neuroreparative in experimental ischemic stroke and worthy of further study as a novel stroke therapy.

The positive symptoms of schizophrenia primarily result from an excess of high affinity D2-like receptors (i.e. D2High receptors). First-generation antipsychotics, such as haloperidol, are D2-like antagonists that can cause severe extrapyramidal effects. Aripiprazole, a dopamine and serotonin partial agonist, has fewer side effects, making it tolerable for adults and children. Extrapyramidal effects (e.g. Parkinsonism, dystonia, and akathisia) are among the most problematic side effects produced by antipsychotic compounds, which likely result from an excess of D2-like receptors in the dorsal striatum. In order to examine the effects of repeated antipsychotic treatment on dopamine system functioning, this thesis compared the molecular effects of repeated haloperidol and aripiprazole administration on D2High receptors, as well as various indices of dopamine second messenger system functioning. Preadolescent and adult rats were pretreated with haloperidol or aripiprazole for 11 consecutive days. After either a 4- or 8-day drug abstinence period dorsal striatal tissue was extracted. [35S]GTPγS binding assays were conducted to assess the effects of repeated haloperidol and aripiprazole treatment on the efficacy and potency of D2-like receptors. PKA subunits and components of the Akt pathway were measured using Western Blots. Results showed that repeated treatment with haloperidol or aripiprazole did not significantly affect D2-like receptor efficacy or potency in young or adult rats. In both age groups, haloperidol significantly increased the expression of PKA-Cα, PKA-Cβ, and PKA-RII, but not p-PKA. Haloperidol also significantly increased PKA-Cβ and PKA-RII levels relative to aripiprazole. Repeated administration of haloperidol significantly increased p-GSK-3β levels in young and adult rats, but neither haloperidol nor aripiprazole significantly affected GSK-3β, Akt, or p-Akt levels. Overall, the results of this thesis indicate that repeated aripiprazole and haloperidol treatment differentially affects D2 signaling pathways in the dorsal striatum. Aripiprazole has less extreme or prolonged effects on D2 receptor signaling pathways than haloperidol, as evidenced by the lack of post-treatment upregulation in the cAMP and Akt pathways. Upregulation of D2-like receptors and, in turn, upregulation of proteins in the cAMP and Akt pathways may be partially responsible for the side effects induced by long-term antipsychotic treatment.

Personality neuroscience is a rapidly expanding field of study fueled by a growing interest in understanding the structural brain correlates of individual differences in personality. Data on the structural brain correlates of personality are especially lacking from large-scale studies, and are nearly nonexistent in the adolescent age group. Furthermore, the role of sex differences in structural brain changes associated with personality are rarely considered. To address this gap in knowledge, this thesis investigates the structural brain correlates of personality and sex differences in structure at age fourteen. A large sample of adolescents (N = 2000) were drawn from the IMAGEN project. Data on adolescents’ puberty status, IQ, and personality were collected through adolescent-reported questionnaires and interviews. The structural brain correlates of personality were examined utilizing personality variables from the NEO Five Factor Inventory (NEO-FFI) and Voxel-Based Morphometry (VBM). Our results showed few correlations between any dimension of the NEO-FFI and regional grey matter volume (GMV). In the total sample, a negative correlation was found for agreeableness and bilateral supplementary motor area (SMA), which was also present in the male subsample. The female sample showed a significant negative correlation between extraversion and the right SMA, and a positive correlation in the left cerebellum. A non-linear effect of extraversion positively correlated with the right precuneus in females. The present study suggests personality traits are not strongly reflected in GMV during adolescence. This thesis includes a discussion on future directions and suggestions for assessing the brain correlates of personality.

The abuse of and addiction to drugs of abuse, such as tobacco, alcohol, opioids, and illicit drugs, are growing global problems that affect the welfare of individuals and societies worldwide. The National Institute of Drug Abuse estimates the annual cost of substance abuse to be over $740 billion in costs related to drug intoxication, withdrawal and relapse. A primary challenge in the treatment of substance abuse is the tendency of users to relapse following acute or extended periods of abstinence; on average over 60% of substance abusers will return to drug use within a year of receiving treatment, many relapsing following stressful life events. Central to the successful treatment of drug addiction is understanding the cellular mechanisms by which relapse episodes occur. Current data suggest that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) is an important event underlying stress-induced reinstatement to drug-seeking in a rodent model of stress-induced relapse. In conjunction with immunohistochemistry and pharmacological treatments, we used this behavioral model of stress-induced relapse to evaluate PACAP and PACAP type-1 receptor (PAC1-R) signaling in stress-induced reinstatement to cocaine seeking. Activation of the PAC1 receptor appears to be critical to stress-induced reinstatement, as the selective PAC1-R agonist, maxadilan, produced reinstatement behaviors in the absence of stress. Moreover, BNST pretreatment with either mitogen activated protein kinase-ERK (MEK) or endocytosis inhibitors to block extracellular signal-related kinase (ERK) signaling attenuated stress-induced reinstatement. Furthermore, BNST phosphorylated ERK (pERK) expression, mediated by PAC1-R activation, is substantially potentiated in cocaine-experienced animals after stressor exposure, in a manner that is dependent on endosomal signaling and MEK activity. These data suggest that the activation of a PAC1 signaling cascade is a key event underlying stress-induced reinstatement. Furthermore, this data may suggest a permanent change in the BNST PACAP system (sensitization) following cocaine exposure.

Fear and anxiety disorders are potentially crippling conditions that often stem from past experience of trauma and chronic stress. One clear feature of these disorders is the failure to use proximate spatial and contextual information presented in the environment to regulate reflexive physiological threat responses. The central nervous system networks that govern spatial navigation and contextual learning and memory are a series of complex circuitries in which the hippocampus is integrally involved. Deficits in hippocampal function have been linked to severe anterograde and mild retrograde amnesia of semantic and episodic memory, and specific deficits in contextual processing. These deficits manifest as failure to distinguish between the details of contexts that help predict for danger or safety and can thus lead to the overexpression of threat responses that compose the behavioral symptoms of fear disorders. The dentate gyrus (DG) is a subdivision of the hippocampus that serves as the first filter of excitatory flow through the hippocampus. The DG is hypothesized to function in “pattern separation” or the dissociation of similar inputs into dissimilar outputs. Failure in this domain leads to generalization between contexts, a common feature of pathology. Pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor are associated with multiple behavioral disorders such as post-traumatic stress disorder, schizophrenia, and bipolar disorder. Mutations in the PAC1 receptor gene are associated with hypervigilance, and modified amygdalar and hippocampal activity. These results are mirrored by rodent studies where central PACAP infusion causes anxiety-like behavior, pain hypersensitivity, anorexia, and reinstatement of drug-seeking. PAC1 receptor transcript is found in high abundance in granule cells of the dentate gyrus and potentiation of DG synapses is impaired in PAC1 knockout mice. PACAP is known to have effects of long-duration, such as those in injury repair, growth, and development, but it also can affect ion channel physiology to control neuronal excitability through several parallel intracellular signaling cascades including those dependent on adenylyl cyclase, phospholipase C, and extracellular signal regulated kinase. Accumulated evidence suggests that recruitment of extracellular signal regulated kinase can be through either adenylyl cyclase-, phospholipase C-, or a receptor endocytosis-dependent mechanism. The experiments described in this dissertation address the role of PACAP in the DG in regulating expression of fear behavior, the effects of PACAP on the excitability of DG granule cells, and the signaling pathways and ion channels responsible for these effects. We found that PACAP infused into the DG amplifies expression of fear to a context but does not affect fear acquisition. Electrophysiology studies demonstrate that treating DG neurons with PACAP increases their excitability, and that parallel signaling mechanisms recruit extracellular signal regulated kinase to drive this excitability. Furthermore, these effects on excitability are attenuated by blocking a persistent inward sodium current. This work represents novel regulation of the DG and its impacts on behavior and identifies a current that likely participates in modulating granule cell excitability in multiple domains. In aggregate, this research traces the path from ligand, to receptor and intracellular signaling, to neurophysiology in order to propose a comprehensive description of behavioral regulation by these processes.

Operant (instrumental) conditioning is a laboratory method for investigating voluntary behavior and involves training a particular response, such as pressing a lever, to earn a reinforcer. Operant behavior is generally divided into two categories: actions and habits. Actions are goal-directed and controlled by response-outcome (R-O) associations. Habits are stimulus-driven and controlled by stimulus-response associations (S-R). Behavior is determined to be goal-directed or habitual by whether or not it is sensitive (action) or insensitive (habit) to reinforcer/outcome devaluation. Many brain regions have been linked to the learning and/or expression of actions and/or habits. This dissertation investigates a few different brain regions in goal-directed and habitual behavior, and determines more specific roles for the prelimbic cortex, infralimbic cortex, prelimbic cortex to dorsomedial striatum pathway, and Crus I/II of the cerebellum. Chapter two investigates the prelimbic and infralimbic cortices in goal-directed behavior. We trained rats on a two-response paradigm, where one response was extensively-trained, and a second response was minimally-trained in a separate context. This maintained both responses as goal-directed. In experiment 1, inactivation of the prelimbic cortex at time of test resulted in an attenuation of responding, but only for the minimally-trained response. This implicates the prelimbic cortex in the expression of goal-directed behavior, but only when that goal-directed behavior is minimally-trained. In experiment 2, we repeated the procedure with infralimbic cortex inactivation and found an attenuation of the extensively-trained response. This implicates the infralimbic cortex in the expression of extensively-trained behavior that is goal-directed. The third chapter examines the role of the prelimbic cortex-to-dorsomedial striatal pathway in minimally-trained operant behavior. Both regions have been implicated in operant behaviors and have strong anatomical connections, but few studies have directly linked them together in the mediation of operant behaviors. After minimal instrumental conditioning, we silenced projections from the prelimbic cortex to the dorsomedial striatum and found that instrumental behavior was reduced, implicating this PL-DMS pathway in the expression of minimally-trained operant responding. The final chapter examines the role of Crus I/II of the cerebellar cortex in the expression of goal-directed and habitual behavior. The cerebellum is well-characterized as a mediator of motor coordination via its connections with the motor cortex. There is also evidence of connections between Crus I/II and non-motor regions of the prefrontal cortex. Additionally, recent studies have pointed towards a role for Crus I/II in non-motor function. In experiment 1, rats learned one minimally-trained and one extensively-trained response, and both responses were goal-directed. Inactivation of Crus I/II attenuated responding only in rats that had undergone reinforcer devaluation. Residual responding in rats that have undergone reinforcer devaluation is attributed to habit, suggesting that Crus I/II may be involved in habit expression. In a follow-up experiment, we extensively-trained a single response and verified that it was expressed as a habit. This time, Crus I/II inactivation at time of test had no effect. Overall, this complex pattern of results suggests the possibility that Crus I/II of the cerebellar cortex is only engaged in habit expression when two responses are trained, but further experiments will be necessary to verify this.

Objective: Internalizing problems are commonly diagnosed during adolescence, and are associated with distress, impairment, and negative mental health outcomes in adulthood. Thus, there is a critical need to characterize adolescents who are at the highest risk for escalating to clinical levels of internalizing problems while extending current literature and incorporating both biological and environmental predictors. This study aimed to characterized risk profiles for fourteen-year-old adolescents who developed clinical levels of internalizing (High Internalizing [HI]) problems by age nineteen, using brain, genetic, personality, cognitive, life history, psychopathology, and demographic measures. The study also examined whether there were functional and structural brain differences in three groups of adolescents on select regions of interest (ROIs) on the Faces Task, Stop Signal Task, and Modified Incentive Delay Task. Method: Participants were 91 adolescents who met clinical criteria for at least one Anxiety and/or Depressive Disorder by age 19 and 1,244 controls who varied in symptom level but did not reach clinically-diagnostic criteria. Ten-fold cross-validated logistic regression using elastic net regularization was used to identify risk profiles associated with high levels of internalizing symptomatology. To examine group differences in regions of interest on three fMRI tasks and in gray matter volume, ANCOVAs were conducted. The three groups were: 1) adolescents who never met HI criteria (Controls), 2) those who met HI criteria in middle adolescence (Middle Onset), and 3) those who met HI criteria in late adolescence (Late Onset). Results: Logistic regression identified 13 variables from personality, psychopathology, life events, and functional brain variables to predict High Internalizing symptoms (mean AUC 0.78, p<.0001). ANCOVAs showed there were several ROIs that demonstrated main effects of Time, and one main effect of Group during response inhibition in the left inferior frontal gyrus, triangular part (pars triangularis), with participants in the Middle Onset group showing increased activation levels compared with the Control group. There were no other significant main effects of Group or Time x Group interactions. Conclusions: These findings give insight into personality, psychopathological, and brain-related factors that are associated with high levels of internalizing symptoms, highlighting the importance of including biological variables in conjunction with psychosocial variables when examining risk factors for internalizing problems. Results also suggest an association between activation in frontal cortex and parietal lobe regions during response inhibition and higher internalizing symptoms in late adolescence. Between-group activation and volumetric ROI comparisons generally yielded main effects of time, confirming prior evidence that activation levels and GMV continue to change over the course of adolescence.

Studies on repeated exposure to opioids have been carried out for decades yet the mechanisms for certain phenomena such as tolerance are still not fully understood. Furthermore, different medications, such as frequently prescribed benzodiazepines, or different disease states, such as HIV, have their own effects and interactions with chronic opioid exposure that are not fully understood. The overall objective of this dissertation was to investigate the complexities of chronic opioid exposure and how different disease states and medications may modulate the effects of chronic opioids. Our findings demonstrate that the administration of diazepam, at doses that are not antinociceptive or have any motor effects, reverse both antinociceptive and locomotor tolerance to orally active opioids. These doses of diazepam did not potentiate the acute effects of these prescription opioids. We also found that HIV-1 Tat expression significantly attenuated the antinociceptive potency of acute morphine in non-tolerant mice while not significantly altering the antinociceptive tolerance to morphine. Consistent with this, Tat attenuated withdrawal symptoms among morphine-tolerant mice. Pretreatment with maraviroc, a CCR5 antagonist blocked the effects of Tat, reinstating morphine potency in non-tolerant mice and restoring withdrawal symptomology in morphine-tolerant mice. Protein array analyses revealed only minor changes to cytokine profiles whether morphine was administered acutely or repeatedly; however, 24 h post repeated morphine administration, the expression of several cytokines was greatly increased. Tat further elevated levels of several cytokines and maraviroc pretreatment attenuated these effects. With the understanding that gap junctions may be involved in both HIV-Tat effects on opioid antinociception as well as tolerance, we investigated the role of gap junctions in opioid antinociceptive tolerance. We observed that carbenoxolone, a gap junction inhibitor, administered systemically attenuated the development of opioid antinociceptive tolerance. Furthermore, we observed a small percentage of carbenoxolone in brain tissue compared to the amount found in blood, suggesting a peripheral site of action. Finally, we show preliminary evidence that in vivo administration of carbenoxolone is able to attenuate tolerance to morphine in DRG neurons.

The development of traumatic brain injury (TBI) therapeutics and effective translation to clinic remains stubbornly elusive despite the high prevalence of TBI within the United States and across the globe. Interventions must be devised around testable targets, appropriately timed to intercede on secondary results. Here, we have utilized temporal neuroproteomics as an ideal approach to inform on the complex biochemical processing in order to address the well-recognized temporal evolution of TBI pathobiology and interrogate a novel therapeutic target in a mild-moderate rat Controlled Cortical Impact (CCI) within perilesioned somatosensory cortex. First, our findings revealed 2047 proteins significantly impacted within the first two weeks following TBI. Subsequent artificial neural network analysis revealed a delayed-onset cluster of proteins highly enriched in GABAergic neurotransmission and ion transport to reveal the prototypical target potassium/chloride transport 2 (KCC2 or SLC12A5) for further investigation with the KCC2-specific pharmacologic CLP290. Our tested therapeutic window guided by post-translational processing preceding one-day prior to protein loss revealed effective CLP290 restoration of KCC2 localization. We further demonstrated recovery in functional and behavioral assessments with one-day administration paradigm supporting the effectiveness of CLP290 treatment after brain injury. To better understand the underlying mechanism of CLP290, we utilized proteomic and bioinformatic approaches to tease out the biological response to treatment. Results demonstrate recovery of PKCδ-mediated phosphorylation of KCC2 and recovery of transporter activity. Additionally, findings reveal preservation of tyrosine kinase by reversing ubiquitin-mediated proteasomal degradation. Our functional assessment of secondary injury insults two-weeks following TBI revealed recovery in seizure threshold, reduction in lesion expansion and a decrease in cell loss suggesting maintained recovery of KCC2 and restored E/I balance. In conclusion, the presented studies in these two chapters propose a novel approach for development of therapeutics for TBI and test the selective manipulation via pharmacological intervention. These findings are promising for the development and treatment of other neurological disorders.