Neurovascular coupling is the relationship between neural activity and blood flow that allows the brain to exhibit increases in blood flow to areas of elevated neural activity during sensory stimulation. It is these localized changes in blood flow, collectively known as the hemodynamic response, that are detected by modern neuroimaging techniques such as functional magnetic resonance imaging (fMRI). Intact neurovascular coupling is imperative to neural health as de-coupling of neural activity from blood flow modulations has been implicated in many neurodegenerative diseases such as Alzheimer's disease, dementia, traumatic brain injury, and ischemic stroke. Despite the importance of neurovascular coupling for both fMRI interpretation and neurological disease, the mechanisms underlying the control of blood flow in the brain remain poorly understood. While previous studies have proposed a range of different cellular mechanisms capable of mediating vascular changes in the brain, it remains difficult to reconcile these mechanisms with a unified theory that is also consistent with the complex spatiotemporal features of the hemodynamic response. The goal of this dissertation is to study the vascular components of the hemodynamic response and the cellular mechanisms that orchestrate them. Using novel high-speed multi-spectral optical imaging of the exposed rodent somatosensory cortex, a detailed characterization of the cortical hemodynamic response is conducted. These observations guide cellular level two-photon microscopy of neural and glial cell activity. The precise spatiotemporal characteristics of the neurovascular response elucidated in these in vivo studies are then used to construct and constrain a conceptual framework for the signaling and actuation pathways that orchestrate the hemodynamic response. To test this framework, targeted light-dye treatment and optogenetic stimulation are used to selectively activate or deactivate targeted signaling pathways. The findings of this research strongly suggest that at least two different mechanisms control the sensory-evoked blood flow response, the first of which critically depends on the vascular endothelium.

Many synapses within the central nervous system are highly sensitive and responsive to ethanol. Although the regulation of postsynaptic receptors by alcohol is well studied, the mechanisms underlying the presynaptic effects of alcohol to alter neurotransmitter release remain relatively unexplored. This dissertation addresses whether alcohol-induced changes in transcriptional activity can promote synaptic vesicle fusion and therefore, neurotransmitter release. To identify a transcriptional pathway by which ethanol can regulate neurotransmitter release, we first investigated the effects of acute alcohol on the expression of genes encoding for synaptic vesicle fusion machinery proteins that form the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex. The proteins in this complex reside on the vesicle membrane (synaptotagmin 1 and synaptobrevin/vesicle-associated membrane protein, which is also known as VAMP) and the plasma membrane (syntaxin 1 and synaptosomal associated protein of 25 kDa, which is also known as SNAP-25), and their interactions within the SNARE complex trigger vesicle fusion and neurotransmitter release. We found that ethanol treatment of mouse cortical neurons increased the mRNA and protein expression levels of a subset of SNARE complex proteins, including synaptotagmin 1 (Syt1) and one of the isoforms of synaptobrevin, VAMP2, but not the other isoform, VAMP1. The gene induction of Syt1 and Vamp2 by alcohol occurs via activation of the transcription factor heat shock factor 1 (HSF1), while HSF1 transcriptional activity had no effect on Vamp1 mRNA levels. We then investigated whether ethanol altered neurotransmitter release in cortical neurons, using whole-cell voltage clamp electrophysiology. We found that alcohol increased gamma-aminobutyric acid (GABA) release via HSF1, but had no effect on glutamatergic synaptic vesicle fusion. Collectively, these data indicate that alcohol induction of HSF1 transcriptional activity triggers a specific coordinated adaptation in GABAergic presynaptic terminals that ultimately results in increased GABA release. This molecular mechanism could explain some of the transient changes in synaptic function that occur after alcohol exposure, and may underlie some of the enduring effects of chronic alcohol drinking on local circuitry.

Stroke has been a major source of morbidity and mortality for centuries. Eight-five percent of all strokes are ischemic in nature, meaning they are caused by the occlusion of a major cerebral artery. Despite extensive research to develop effective treatments for ischemic stroke, therapeutic options remain limited. Apoptosis (also termed "programmed cell death") is a process by which a stressed or damaged cell commits "suicide". In stroke, runaway apoptosis contributes to stroke neurodegeneration and neurological decline for days to weeks after disease onset. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis that are activated in distinct molecular pathways, but their impact in stroke is poorly defined. Direct evidence for caspase activation in stroke and the functional relevance of this activity has not been previously characterized. For this dissertation, we developed an unbiased technique for in vivo trapping of active caspases in rodent models of ischemic stroke. We isolated active caspase-9 as a principal contributor to ischemic neurodegeneration in rodents (Rattus norvegicus and Mus musculus). Caspase-9 is the initiator caspase for the intrinsic cell death pathway. Intranasal delivery of a novel, cell membrane-penetrating inhibitor for caspase-9 confirmed the pathogenic relevance of caspase-9 activity in stroke. Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. Caspase-6 is an effector caspase and a member of the intrinsic death pathway that has never been implicated in stroke until now. Coincidentally, we discovered that caspase-6 is specifically activated within the axonal compartment. The temporal and spatial pattern of activation demonstrates that neuronal caspase-9 activity induces caspase-6 activation, which mediates axonal loss in the early stages of stroke (+/- 24 hours). We developed a novel inhibitor for caspase-6, based on a catalytically inactive clone, which demonstrated neuroprotective and axoprotective efficacy against ischemia. Collectively, these results assert that selective inhibition of caspase-9 and caspase-6 is an effective translational strategy for stroke. The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function. Thus, caspases are logical therapeutic targets for stroke. However, they have never been clinically evaluated due to a paucity of ideal drug candidates. This dissertation outlines fresh insights into the mechanisms of stroke neurodegeneration and offers novel caspase-based therapeutic strategies for clinical evaluation.

Stroke has been a major source of morbidity and mortality for centuries. Eight-five percent of all strokes are ischemic in nature, meaning they are caused by the occlusion of a major cerebral artery. Despite extensive research to develop effective treatments for ischemic stroke, therapeutic options remain limited. Apoptosis (also termed "programmed cell death") is a process by which a stressed or damaged cell commits "suicide". In stroke, runaway apoptosis contributes to stroke neurodegeneration and neurological decline for days to weeks after disease onset. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis that are activated in distinct molecular pathways, but their impact in stroke is poorly defined. Direct evidence for caspase activation in stroke and the functional relevance of this activity has not been previously characterized. For this dissertation, we developed an unbiased technique for in vivo trapping of active caspases in rodent models of ischemic stroke. We isolated active caspase-9 as a principal contributor to ischemic neurodegeneration in rodents (Rattus norvegicus & Mus musculus). Caspase-9 is the initiator caspase for the intrinsic cell death pathway. Intranasal delivery of a novel, cell membrane-penetrating inhibitor for caspase-9 confirmed the pathogenic relevance of caspase-9 activity in stroke. Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. Caspase-6 is an effector caspase and a member of the intrinsic death pathway that has never been implicated in stroke until now. Coincidentally, we discovered that caspase-6 is specifically activated within the axonal compartment. The temporal and spatial pattern of activation demonstrates that neuronal caspase-9 activity induces caspase-6 activation, which mediates axonal loss in the early stages of stroke (<24 hours). We developed a novel inhibitor for caspase-6, based on a catalytically inactive clone, which demonstrated neuroprotective and axoprotective efficacy against ischemia. Collectively, these results assert that selective inhibition of caspase-9 and caspase-6 is an effective translational strategy for stroke. The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function. Thus, caspases are logical therapeutic targets for stroke. However, they have never been clinically evaluated due to a paucity of ideal drug candidates. This dissertation outlines fresh insights into the mechanisms of stroke neurodegeneration and offers novel caspase-based therapeutic strategies for clinical evaluation.

Sensory perception adapts to diverse environment. Although studies in the last few decades have started to address the question of how sensory systems transduce signals, how these systems cross-modulated is largely unknown. In this thesis, I study mechanosensation in the C. elegans touch receptor neurons (TRNs) to understand how sensory systems are modulated and adapt to the environment. I find that the touch sensitivity in the TRNs is modulated by both mechanical and non-mechanical factors. The mechanical factors are transduced directly by a secondary mechanosensory system in the TRNs, and the non-mechanical factors are detected by other neurons and relayed to the TRNs by neuropeptides. Both pathways converge through a common mechanism to regulate the surface expression of the MEC-4 mechanotransduction channels, which are needed for sensing touch. I then explore the consequences of modulation, and show that modulation by mechanical and non-mechanical factors adjusts the balance between the sensitivity to strong mechanical stimuli that predict dangers and sensitivity to weak stimuli that are usually not associated with danger. Such a balance maintains sensitivity to biologically-relevant mechanical stimuli while reducing unnecessary responses to weak stimuli, thus increasing the ability to survive under different conditions. I used neuronal-enhanced RNAi and mosaic analysis to discover two convergent signaling pathways, the integrin/focal adhesion signaling and insulin signaling, that modulate anterior touch sensitivity. Additional genes and pathways are also needed for optimal touch sensitivity in the TRNs, including the RAS/MAPK pathway, Rho-GTPases, cytoskeleton genes, and 43 other genes that cause lethality when mutated. The integrins/focal adhesion proteins act cell-autonomously in the TRNs to detect the mechanical environment. The focal adhesion proteins modulate force sensitivity and subsequent calcium signaling, and they are needed for long-term sensitization of touch sensitivity in response to sustained background vibration. Such sensitization maintains normal touch sensitivity under background vibration by partially counteracting the effect of habituation. This sensitization does not require the MEC-4/MEC-10 transduction channel, suggesting that the integrins may act as secondary force sensors. Insulin signaling, however, responds to non-mechanical signals that reduce touch sensitivity by decreasing the expression of insulin-like neuromodulators, including INS-10 and INS-22. The reduced touch sensitivity facilitates the completion of other tasks such as chemotaxis under background mechanical stimuli, thus increasing the chance of survival by escaping stressful conditions. Both insulin signaling and integrin signaling converge on AKT-1 and DAF-16, which modulate touch sensitivity by regulating the transcription of mfb-1, an E3 ubiquitin ligase expressed in the TRNs. MFB-1 regulates the amount of MEC-4 channel on the plasma membrane, thus modulating touch sensitivity. Together, these results describe an integrated pathway that transduces both mechanical and non-mechanical signals to modulate touch sensitivity through a common mechanism. These modulation mechanisms maintain optimal sensitivity to mechanical stimuli while avoiding unnecessary responses.

The obesity epidemic is imposing enormous costs on individuals and on developed and developing societies. Ultimately, obesity arises from a sustained imbalance in the energy balance equation from either excessive energy consumption or significantly reduced activity. Here we report on findings from two fRMI studies, each of which examines one side of the energy balance equation. In our first study, the Passive Viewing of Foods, we examined the effects of acute exercise on self-report measures of appetite suppression and on neural activity resulting when normal BMI subjects viewed blocks of high calorie or low calorie food cues. We found that acute exercise suppressed self-reported appetite and reduced the activation of two key brain areas relative to appetite regulation: the dorsal anterior cingulate (dorsal ACC), a frontal attention processing area, and the nucleus accumbens, a central reward processing area. Moreover, we conducted functional connectivity analysis to examine other areas of the brain that were positively or negatively correlated with these two areas when viewing high calorie food cues following exercise. The functional network identified was broadly distributed and included increased coupling with the putamen, insula, operculae, inferior frontal gyrus, and superior parietal lobule and decreased coupling with the amygdala and orbital frontal cortex, among other areas. We believe this is the first study of exercise induced appetite suppression that used whole brain analysis and functional connectivity to show both absolute reductions of activity in the dorsal ACC and nucleus accumbens as well as a distributed functional network with differential coupling and de-coupling. These findings help identify a functional network that mediates appetite suppression as a result of acute exercise. In our second study, the Categorical Food Stroop, we deploy a novel Stroop-like paradigm that used the same high and low calorie food cue exemplars to examine the effects of the food cues on cognitive interference and the cognitive control effect of conflict adaptation. In the study, subjects categorized the high and low calorie food cue word targets, which were overlain on veridical images of the same food cue exemplars. Relative to interference, we observed that normal BMI subjects took 18 ms longer to categorize high calorie words overlain on low calorie images (high calorie incongruent trial) than they did to categorize low calorie words on high calorie distractors (low calorie incongruent trial). Relative to conflict adaptation, a measure of cognitive control over response inhibition when there are conflicting response options, we observed a significant overall effect of conflict adaptation but then showed that only one calorie characteristic (high calorie incongruent trials following high calorie incongruent trials, HH trials) was significantly contributing to the overall conflict adaptation. To our knowledge, this was the first categorical food Stroop and the first study to identify the role of caloric characteristic in modulating cognitive control relative to response selection in food-related decisions. Our neural observations showed that the increased interference in incongruent trials is associated with activation in the supramarginal gyrus, superior parietal lobule and the superior lateral occipital cortex. The high cognitive control HH trials compared to the low cognitive control trials activated the parahippocampal gyrus, the right amygdala, the orbital frontal cortex, the superior parietal lobule, the angular gyrus, and temporal-occipital gyrus. The parahippocampal gyrus and superior parietal lobule were used as seeds in functional connectivity analysis and revealed a high degree of overlap in their distributed functional networks mediating high cognitive control trials. The findings shed new light on both the high calorie stimulus specificity of cognitive control in normal subjects and the distributed functional network that mediates the effects of the cognitive control in food related decisions.

Neurovascular coupling is the relationship between neural activity and blood flow that allows the brain to exhibit increases in blood flow to areas of elevated neural activity during sensory stimulation. It is these localized changes in blood flow, collectively known as the hemodynamic response, that are detected by modern neuroimaging techniques such as functional magnetic resonance imaging (fMRI). Intact neurovascular coupling is imperative to neural health as de-coupling of neural activity from blood flow modulations has been implicated in many neurodegenerative diseases such as Alzheimer's disease, dementia, traumatic brain injury, and ischemic stroke. Despite the importance of neurovascular coupling for both fMRI interpretation and neurological disease, the mechanisms underlying the control of blood flow in the brain remain poorly understood. While previous studies have proposed a range of different cellular mechanisms capable of mediating vascular changes in the brain, it remains difficult to reconcile these mechanisms with a unified theory that is also consistent with the complex spatiotemporal features of the hemodynamic response. The goal of this dissertation is to study the vascular components of the hemodynamic response and the cellular mechanisms that orchestrate them. Using novel high-speed multi-spectral optical imaging of the exposed rodent somatosensory cortex, a detailed characterization of the cortical hemodynamic response is conducted. These observations guide cellular level two-photon microscopy of neural and glial cell activity. The precise spatiotemporal characteristics of the neurovascular response elucidated in these in vivo studies are then used to construct and constrain a conceptual framework for the signaling and actuation pathways that orchestrate the hemodynamic response. To test this framework, targeted light-dye treatment and optogenetic stimulation are used to selectively activate or deactivate targeted signaling pathways. The findings of this research strongly suggest that at least two different mechanisms control the sensory-evoked blood flow response, the first of which critically depends on the vascular endothelium.

How animal behavior is controlled at the molecular and cellular levels is still largely mysterious. Here, I document my studies on the mechanisms controlling a simple behavior--chemotaxis--in the nematode worm Caenorhabditis elegans. My work focuses on a pair of amphid sensory neurons at the head end of the worm called ASEs. The neurons are exposed and respond to environmental chemical signals, and instruct downstream locomotory responses that cause the worm to move up or down chemical gradients. The ASE neurons are morphology similar and arranged symmetrically across the head. Yet, it has been known for some time that they show differences in which ionic signals they are primarily responsive to (Na+/, Cl/, K+) in regards to chemotaxis behavior. Furthermore, it had been observed that the ASEs also express distinct sets of genes, in particular, receptor-type guanylyl cyclases (rGCs). This thesis begins with my contribution to a study of the function of ASE asymmetry in chemotaxis. I, along with another graduate student, found that an additional four salt ions (Br-, Li+/, I /, Mg2+) are sensed by either ASER (right) or ASEL (left) neurons. Evidence is presented that this laterality in ion receptivity allows the nematode to discriminate right-sensed salt cues in the background of left-sensed cues and vice versa. We further investigated what role asymmetrically expressed rGCs might play in the regulation of chemotaxis. Using mutants for some of these genes, we found that, depending on the rGC, they confer chemotactic responsiveness to one, two, or several salts. Hence, asymmetry in ASE ion sensitivity is conferred, at least in part, by asymmetry in rGC expression. Next, I attempted to test whether rGCs act as direct salt receptors, or function further downstream to modulate signal transduction. To address this question, I used chimeras made with three different ASER-expressed rGCs, all of which have the same basic domain architecture. I performed domain-swap experiments where the extracellular domain of one rGC was exchanged with the intracellular domain of another in all possible combinations. I was able to show that the extracellular domain is the region that confers specificity to which ions these rGCs respond. Furthermore I carried out experiments to test the idea that rGCs act as heterodimers, by heterologously expressing two rGCs together in all amphids other than ASEs. By doing this, I was able to confer a new ion-sensitivity function to the cells; like ASE neurons, they could sense ions and elicit a chemotactic response. Together these independent lines of evidence suggest that rGCs permit amphids to sense certain salts, and may therefore be acting as salt receptors. Finally, in an investigation of some chemotaxis mutants which employed whole genome sequencing, a particularly interesting mutant was uncovered that encodes a previously undescribed cyclic nucleotide-gated channel (CNG), che-6. I characterize the potential role of che-6, and propose that it encodes a novel CNG that functions in salt chemotaxis behavior and most likely acts downstream of rGCs. Taken together, these data shed light on the mechanism of salt chemotaxis in nematodes, and provide an example of how genes govern basic behaviors in this relatively simplified animal. I discuss what remains to be understood in this system, and how it compares to chemosensory systems in other animal species. The results are also interpreted in the light of maximizing the sophistication of a nervous system that is cell number- and size-limited.

While much research has suggested that emotional experiences change dramatically over the lifespan, less is known about what underlies these changes at a mechanistic level. Specifically, it is unclear whether age predicts differences in bottom-up reactivity to emotional events, or in the ability to exert top-down control over emotional responses. The present studies sought to address these gaps in the literature. Studies 1 and 2 compared the behavioral and neural correlates, respectively, of emotional reactivity to and regulation of emotional responses to social and non-social aversive stimuli in individuals aged 10-22. Study 1 additionally examined the interaction between individual differences in sensitivity to social rejection and age and how this impacts regulation of emotional responses to social stimuli. Across these studies, age predicted differences in neural and behavioral correlates of regulation but not reactivity. Study 3 broadened the sample age range to include children as young as 6 years and obtained results that were generally consistent with those of Studies 1-2. Study 4 examined the generalizability of the findings from Studies 1-3 by examining reactivity and regulation of appetitive, rather than aversive, responses in participants ranging from 6-22 years. Behavioral indices of reactivity and regulation correlated with age in Study 4, but neural effects of age were only found for regulation. Data from Study 4 additionally suggested links between the neural correlates of regulation of craving and body mass index.

Growing evidence indicates that intracellular signaling lipids control the trafficking and processing of amyloid precursor protein (APP) with major implications for pathological and behavioral manifestations associated with Alzheimer's disease (AD). One such lipid is phosphatidic acid (PA), which mediates membrane trafficking and is produced by a variety of enzymes, including phospholipase D (PLD). We previously demonstrated that a PLD isoform, PLD2, is required for the synaptotoxic and memory-impairing actions of amyloid beta in an AD mouse model. The role that the other PLD isoform, PLD1, plays in AD is unclear, although cell culture studies from other groups have suggested it modulates the trafficking of APP and presenilin 1, the catalytic subunit of gamma secretase. Here, we investigate the role of PLD1 in the biology of APP as well as in an AD mouse model. We report that removing PLD1, unlike PLD2, causes a dramatic decrease in brain levels of PA, indicating that PLD1 is a major source of PA in the brain. Additionally, removing PLD1 from primary neurons causes a redistribution of APP from endosomes, a primary station for amyloidogenesis, to the Golgi complex, while PLD1 overexpression produces the converse phenotype. Pld1 null mice harboring familial AD-linked (FAD) APP mutations exhibited decreased brain amyloid and an accumulation of APP COOH-terminal fragments. This finding was particularly evident in Pld1 null membrane rafts, whose lipidome was profoundly altered. Acute inhibition of PLD1 reduced APP processing by gamma secretase, consistent with a regulatory role of the lipid raft environment on gamma secretase, a complex highly active in membrane rafts. Finally, Pld1 nullizygosity rescued cognitive deficits in the transgenic model. Thus, PLD1 and its product PA control the metabolism of APP and emerge as potential drug targets for Alzheimer's disease therapy.