Olfactory perception affects the food choice of most living species. A strong set of data have been accumulated to demonstrate that the olfactory bulb, the first relay of olfactory information, as a metabolic sensor. Mitral cells of the olfactory bulb project to central processing areas such as the piriform cortex. Our laboratory has recently determined that a subpopulation of mitral cells can modulate their firing frequency in response to changes in extracellular glucose concentration, thus acting as glucose sensors. Glucose sensors are found throughout the brain in the hypothalamus, the brainstem, amygdala, septum and hippocampus. Glucose has been extensively studied in non-neuronal tissues given its essential role as the main cellular fuel. In glucose sensing neurons, however, where glucose acts as a signal, this process remains largely unknown. My dissertation project focused on providing further evidence of glucose-sensing in the olfactory system, specifically the olfactory bulb and the anterior piriform cortex. I hypothesized that glucose-sensing happened via the insulin-dependent glucose transporter type 4. I first mapped the presence of the glucose transporter type 4 in the different cellular layers of the olfactory bulb and the piriform cortex. mRNA of the glucose transporter type 4 and the voltage-gated potassium channel Kv1.3 were present in the mitral cell layer of the olfactory bulb. In the piriform cortex, the glucose transporter type 4, Kv1.3, and insulin receptors exhibited a broad diversity of distribution. Neurons in the different layers of the piriform cortex expressed one of the three proteins, two of them, or the three proteins co-expressed together. Both mitral cells in the olfactory bulb and pyramidal neurons in the piriform cortex had glucose-sensing properties whereby glucose modulated the electrical behavior of these cells. Mitral cells increased or decreased their firing frequency in response to low glucose (1 mM) while the electrical activity of pyramidal neurons was dependent on extracellular glucose concentration. Switching glucose concentration from high (10 mM) to low (0.5 mM) decreased the instantaneous frequency in pyramidal neurons. Switching glucose concentration from moderate (5 mM) to low (1 mM) revealed two subpopulations of pyramidal neurons that either decreased their instantaneous frequency or were unresponsive to the change in glucose concentration. Pyramidal neurons were responsive to insulin as well, and both mitral cells and pyramidal neurons required glucose metabolism to sense glucose as demonstrated in vitro by using the glucose analog 2-deoxyglucose, or alloxan, a glucokinase inhibitor. Furthermore, I bilaterally implanted cannulas into the anterior piriform cortex of rats, micro-injected insulin (172 nM), glucose (10 nM), or a small peptide Kv1.3 inhibitor margatoxin (0.1 nM). Animals were then subjected to an olfactory habituation/dishabituation paradigm. Results showed that insulin and glucose reduced olfactory discrimination but blocking Kv1.3 improved olfactory habituation and discrimination. My work is the first to demonstrate the presence of the glucose transporter type 4 in the anterior piriform cortex, it is also the first to provide insights into the glucose sensing transduction cascade in the olfactory system, a process that appears to be modulated by insulin and glucose metabolites. The results of this study help provide a better fundamental understanding of the physiological regulation of olfactory perception in relationship with the metabolic status. This study might also pave the way towards identifying a potential therapeutic target to control overeating; a main cause of obesity in western countries. / A Dissertation submitted to the Program in Neuroscience in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / July 18, 2018. / Food intake, Glucose, Metabolism, Olfactory, Sensing / Includes bibliographical references. / Debra Ann Fadool, Professor Directing Dissertation; Michael Blaber, University Representative; Michael Meredith, Committee Member; Thomas C. S. Keller, Committee Member; Michael Overton, Committee Member.

<p> Astrogliosis is a hallmark of central nervous system (CNS) neuroinflammatory disorders such as multiple sclerosis (MS). Astrocytes can play both beneficial and detrimental roles in response to neuroinflammation, thus a detailed understanding of the underlying molecular mechanisms governing astrogliosis might facilitate the development of therapeutic targets. While astrocytes do not express voltage-gated sodium channel (VGSC) Nav1.5 in nonpathological human brain, they exhibit robust upregulation of Nav1.5 within acute and chronic MS lesions. We investigated the contribution of voltage-gated sodium channels to astrogliosis in an <i> in vitro</i> model of mechanical injury to astrocytes. Previous studies have shown that a scratch injury to astrocytes invokes dual mechanisms of migration and proliferation in these cells. Our results demonstrate that wound closure after mechanical injury, involving both migration and proliferation, is attenuated by pharmacological treatment with tetrodotoxin (TTX) and KB-R7943, at a dose that blocks reverse mode of the Na⁺/Ca²⁺ exchanger (NCX), and by knockdown of Nav1.5 mRNA. We also show that astrocytes display a robust [Ca²⁺] transient after mechanical injury and demonstrate that this [Ca²⁺] response is also attenuated by TTX, KB-R7943, and Nav1.5 mRNA knockdown. This study provides support for a contribution of VGSCs in the pathway leading to astrogliosis.</p><p> We present here evidence supporting a contribution of sodium channel Nav1.5 to astrogliosis in an <i>in vitro</i> model of glial mechanical injury. We further implicate fluctuations in [Ca²⁺] due to reverse operation of NCX, triggered by VGSC activity, as a mechanism by which Nav1.5 contributes to the response of astrocytes to mechanical injury. Our results establish a link between the activity of VGSCs and astrogliosis by way of alterations in [Ca²⁺] . Here we show, in an <i> in vitro</i> model of mechanical injury to astrocytes, that voltage-gated sodium channel (VGSC) Nav1.5, traditionally viewed as a cardiac sodium channel, contributes to the astrocytic response to the insult via triggering reverse mode of the Na⁺/Ca²⁺ exchanger (NCX).</p><p> We then investigated the temporal dynamics of astrocytic Nav1.5 channel expression in response to neuroinflammatory pathologies. We examined astrocytes from mice with monophasic and chronic-relapsing experimental autoimmune encephalomyelitis (EAE) by immunohistochemistry to determine whether Nav1.5 is expressed in these cells, and whether the expression correlates with severity of disease and/or phases of relapse and remission. Our results demonstrate that Nav1.5 is upregulated in astrocytes <i>in situ</i> in a temporal manner that correlates with disease severity in both monophasic and chronic-relapsing EAE. Furthermore, in chronic-relapsing EAE, Nav1.5 expression is upregulated during relapses and subsequently attenuated during periods of remission. These observations are consistent with the suggestion that Nav1.5 can play a role in the response of astrocytes to inflammatory pathologies in the CNS and suggest Nav1.5 may be a potential therapeutic target to modulate reactive astrogliosis <i> in vivo</i>. </p><p> Finally, we investigated whether Nav1.5 expression in astrocytes plays a role in the pathogenesis of EAE. We created a conditional knockout of Nav1.5 in astrocytes and determined whether this affects the clinical course of EAE, focal macrophage and T cell infiltration, and diffuse activation of astrocytes. We show that deletion of Nav1.5 from astrocytes leads to significantly worsened clinical outcomes in EAE, with increased inflammatory infiltrate in both early and late stages of disease, unexpectedly, in a sex-specific manner. Removal of Nav1.5 in astrocytes leads to increased inflammation in female mice with EAE, including increased astroglial response and infiltration of T cells and phagocytic monocytes. These cellular changes are consistent with more severe EAE clinical scores. Additionally, we found evidence suggesting possible dysregulation of the immune response &ndash; particularly regarding infiltrating macrophages and activated microglia &ndash; in female Nav1.5 KO mice compared to WT littermate controls. Together, our results show that deletion of Nav1.5 from astrocytes leads to significantly worsened clinical outcomes in EAE, with increased inflammatory infiltrate in both early and late stages of disease, in a sex-specific manner. </p><p>

Reelin, a large extracellular matrix protein responsible for migration and laminar positioning of neurons during brain development, has been implicated in the pathogenesis of schizophrenia and autism. There are extensive populations that have been identified in the adult mouse brain which contain cells secreting Reelin; previously these neurons were believed to be almost exclusively GABAergic. We used immunohistochemistry to reveal multiple groups of Reelin positive neurons that are not GABAergic. Specifically, we used Reelin and GABA antibodies or Vgat cre::Ai9 tdTomato to analyze whether Reelin positive cells are indeed GABAergic. Populations of Reelin positive, non-GABAergic were found in the olfactory bulb and piriform cortex; the perforant pathway; the entorhinal cortex, stratum lacunosum-moleculare of the hippocampus proper, and the dentate gyrus; lastly a small population was found in layer V of the visual cortex. These results suggest Reelin signaling may directly modulate excitatory synaptic circuits in the postnatal brain. In heterozygous reeler multiple morphological abnormalities were identified compared to wild type littermates. Branched analysis revealed a marked decrease in basal dendrite nodes in layer V cells the heterozygous reeler motor cortex and hippocampus as well as a decrease in basal length in the hippocampus. A detailed Sholl analysis indicated abnormalities in both the cortex and hippocampus of the heterozygous reeler. In the cortex we found decreased basal nodes and number of intersections as well as length at specific compartments of neuronal dendritic structure. More significant differences were found in the hippocampus, which showed a decreased total number of intersections as well as decreased intersections length of CA1 neurons. Changes in both the cortex and the hippocampus of the heterozygous brains were comparable to the homozygous reeler mutant. These findings point to underlying neuronal morphological correlates for the electrophysiological changes found in homozygous reeler mice and the physiological abnormalities exhibited in heterozygous reeler mice.

Cortical spreading depression (CSD) is a depolarizing wave that travels through the cerebral cortex, and is followed by an inhibition of cortical activity. The propagation of CSD elicits metabolic challenges in tissue that may be irrecoverable in an ischemic brain, and thus has implications in neurological disease. Limiting the incidence of CSD may be instrumental in limiting the extent of neuronal damage following brain injury. Transcranial direct current stimulation (tDCS) is a form of brain stimulation that alters the level of cortical activity. Anodal tDCS, which increases cortical excitability, is used to treat a variety of neurological syndromes but may have the potential to exacerbate certain pathologies. This contention has never been evaluated using in vivo brain recordings. This study seeks to determine the effects of anodal tDCS on CSD, a phenomenon common to many neurological disorders. CSD was induced in the rat cortex by administration of potassium chloride. Animals were subjected to either anodal tDCS or sham stimulation. Cortical electrical activity was monitored using an intracortical multielectrode array, and data was analyzed to measure the effects of anodal tDCS versus sham on CSD incidence, velocity, amplitude, and several other characteristics of the wave. The hypothesis of the study was that anodal tDCS would increase the incidence, velocity, and amplitude of the CSD wave. No significant effects of anodal tDCS on CSD were observed in this study. Results indicate that anodal tDCS does not increase the velocity, amplitude, or frequency of the spreading depression wave, nor does it interrupt the wave. These data have implications for the use of anodal tDCS in the treatment of neurological disorders associated with spreading depression.

In this literary review, a possible mechanism used by propofol and the consequences of this mechanism are discussed. Propofol is able to bring about loss of consciousness by inhibiting the Ih current in hyperpolarization-activated cyclic nucleotide-gated type 2 (HCN2) channels. The inhibition leads to an increase in the hyperpolarization of the thalamocortical neurons, which results in temporally impaired delta oscillations (Ying et al., 2006). This leads to significant phase offsets which result in fragmentation and the isolation of neural networks. Propofol also leads to a breakdown in the basal ganglia-thalamo-cortical (BGTC) loop, which disrupts cortical and subcortical communication. This breakdown is a result of decreases seen in beta band coherence and the phase amplitude coupling (PAC) between subcortical and cortical regions of the loop. The reduction in coupling leads to interrupted communication which contributes to neural network fragmentation (Swann et al., 2016). Although fragmentation is seen, there are instances of increased global connectivity. The default mode network (DMN) increases its connections to structures outside its network during sedation. However, these connections are not representations of efficient global communication. Instead, they lead to a decrease local efficiency resulting in local network deterioration and an overall decrease in efficient global and local network interactions (Stamatakis et al., 2010). Certain characteristics of the transition from consciousness to loss of consciousness were identified. Delta oscillations are significantly more powerful during sedation, and the sharp increase that can be seen in their power is indicative of loss of consciousness (Lewis et al., 2012). Another indicator of loss of consciousness can be seen in frontal EEG channels by way of PAC analysis of alpha power and slow oscillations. Negative trough-max PAC exists at baseline, but switches to positive peak-max PAC during moderate sedation in those who are more sensitive to propofol. An increased propofol sensitivity can be detected at baseline and is represented by a weak alpha network that is not very small-worldly (Chennu et al., 2016). In summary, it is clear that the timing of our neural networks is crucial to consciousness and that it is through temporal modifications that propofol is able to induce its effects.

BACKGROUND: Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative disorder that had been largely ignored for decades since its initial characterization in 1928 by Dr. H. Martland. Within the last several years, a dramatic increase of attention in the media has been given to the subject and is now a household term. As a consequence of repetitive concussive and subconcussive events, CTE has been clearly distinguished form other neurodegenerative disorders such as Alzheimer’s Disease and Parkinson’s. Its prominence in military personnel and professional athletes has established the importance for its characterization in order to develop preventative approaches and regulations. Recently, characterization and staging of CTE has been achieved and interventional approaches are already being implemented in response to this increased understanding. However, not everything is known about the pathology and its underlying mechanisms. No known studies have been done to quantify the connection between MLV pathology and CTE. OBJECTIVE: To determine if MLV pathology observed in CTE may serve as a potential biomarker. METHODS: The white and grey matter of brains from subjects with CTE and control subjects without CTE were analyzed for the presence and characteristics of multilumen vessels (MLVs) in the dorsolateral frontal cortex (DLFC). A total of 123 slides were analyzed, 88 from CTE cases and 35 from Non-CTE cases. The quantification of overrepresented MLVs and their features was the primary endpoint of the study. Associations were then made between the characteristics of multilumen vessels and controlling factors such as age at death and years of exposure to repetitive head injuries (RHIs). Finally, regression analysis was used to test for the predictive qualities of the density and average number of lumen for stage of CTE while controlling for age at death and years of exposure. RESULTS: It was found that MLVs are overrepresented in the CTE cohort in comparison to the Non-CTE cohort. There is also a possible connection between the presence of MLVs with their pathologic observable features to the progressive nature of CTE. In addition, the MLVs found in CTE have observable characteristics that are dissociable from MLVs in Non-CTE. Furthermore, for increased confidence of the findings, results remain significant even with the application of stricter parameters for defining MLVs. Finally, there was evidence of a connection between the development of MLVs to the progression of CTE. CONCLUSIONS: The findings suggest there is a strong relationship between age of death and the frequency of MLVs. Furthermore, the development of MLVs is likely positively impacted by the progression of CTE.

The anterior cingulate cortex is an important interface of cortical, motor, and limbic networks, and thus is a brain area uniquely situated to affect a wide variety of higher order functions. The aim of this study was to characterize the morphology of two distinct populations of anterior cingulate cortex (ACC) pyramidal neurons, a dorsal-caudal population projecting to the premotor cortex (PMC) and a ventral-rostral population projecting to the amygdala. Retrograde tracers injected into area 6DC of the “cognitive” premotor cortex, and into the basolateral nucleus of the “affective” amygdala were used to label distinct projection neurons in the ACC. Whole-cell patch clamp recording and intracellular filling techniques were used to fill the dendritic arbor of these labeled projection neurons. High resolution confocal microscopy and 3D neuronal reconstructions were used to quantify dendritic morphological parameters. Amygdala projecting neurons were more superficial than premotor projecting neurons, with an average soma-to-pia distance of 498 μm compared to 1,012 μm, respectively (amygdala projecting: 498 ± 139 μm vs. PMC projecting: 1012 ± 113 μm, p<.05). Overall, amygdala and PMC projection neurons had very similar average dendritic lengths, branch points, branch densities, and vertical and horizontal extensions in both apical and basal compartments. Amygdala projecting cells had greater apical tuft branch points than deep PMC projecting cells (8.25 vs. 3.3 apical tuft branch points, p<.05). Superficial PMC projecting cells had smaller total vertical and apical vertical extensions than deep PMC projecting cells (Total vertical: 304.98 vs. 750.96 μm, apical vertical: 241.78 vs 601.95 μm, p<.05). Sholl analyses revealed that the distribution of apical dendritic length as a function of distance from the soma of amygdala projections had bimodal peaks, while that of superficial and deep PMC cells had a single peak. Total spine number of amygdala projecting neurons was greater than PMC projecting cells (~17,000 spines vs. ~2,100 spines). Three major classes of morphology were visualized within the ACC neuron reconstructions dataset: regular-tufted, narrow-tufted, and untufted, with the regular-tufted cells containing more branch points than narrow tufted but less basal branch point density. The work in this study assessing cellular morphological properties of specific amygdala and PMC inputs and outputs within the ACC helps to characterize functional dynamics of both emotional and motor planning networks.

Alzheimer's disease is a neurodegenerative disease characterized by plaques of amyloid-beta protein (Aβ) and neurofibrillary tangles of the microtubule-associated protein tau. Plaques contain several variants of Aβ, including full-length Aβ1-40 and Aβ1-42, and N-terminally truncated and cyclized pyroglutamate-3 (pE3) Aβ. In particular, pE3 Aβ has been shown to be extremely toxic and resistant to degradation. Microglia are phagocytic cells in the brain that have been shown to take up and degrade Aβ. The purpose of this study is to compare the uptake and degradation of three different Aβ variants: Aβ1-40, Aβ3-40, and AβpE3-40 using both primary murine microglia and N9 microglia, a murine microglial cell line. The three techniques used to discern the differences in uptake and degradation are fluorescence-activated cell sorting, immunofluorescence, and Western blot. According to these techniques, primary microglia and N9 microglia take up and degrade the three Aβ variants differently. Overall, N9 cells appear to prefer to take up and degradeAβ1-40, whereas primary microglia prefer to take up AβpE3-40 and degrade Aβ1-40.

The trace amine–associated receptor 1 (TAAR1) is an intracellular G–protein coupled receptor whose activation by trace amines, catecholamines and amphetamines leads to elevation of cyclic–AMP and activation of protein kinase A (PKA). Recently, the Amara lab discovered that TAAR1 also mediates the activation of the small GTPase, RhoA. TAAR1 is expressed in midbrain dopamine (DA) neurons, including those in the substantia nigra and ventral tegmental area, and thus is positioned to modulate both motor activity and addiction–related plasticity. Due to antibody limitations, however, neither the intracellular membrane localization of TAAR1 nor the site of signaling by this receptor has been clearly demonstrated in neurons. Dopaminergic neurotransmission is a coordinated process which requires synthesis, packaging, exocytosis, and reuptake of DA. Amphetamine (AMPH) can stimulate TAAR1, which has been shown to downregulate the surface expression of the dopamine transporter, thus decreasing DA reuptake and increasing extracellular DA concentrations. In addition, AMPH and elevation of cAMP decreases the activity of the vesicular monoamine transporter, VMAT2 in neurosecretory pheochromocytoma (PC12) cells, although the mechanism of this regulation remains undefined. The co–expression of TAAR1 and VMAT2 in the DA neuron and PC12 cells suggests that TAAR1 activation may mediate the effects of AMPH/cAMP on VMAT2. Towards understanding the role of TAAR1 in transporter trafficking and function in the DA neuron, this thesis seeks to define the mechanism of AMPH action on TAAR1 signaling and examine the intracellular membrane localization and pathways downstream of TAAR1 activation. In Chapter I, we used compartment–specific FRET–based sensors to determine the functional subcellular localization of TAAR1. Novel endomembrane targeting constructs were designed and targeting, and functionality was confirmed using standard biochemical techniques and confocal microscopy. Targeted FRET–based sensors for PKA and RhoA activation enabled us to assess TAAR1–mediated responses to AMPH treatment in discrete subcellular compartments. AMPH increased PKA activation in the synaptic vesicle compartment. However, TAAR1–mediated effects of AMPH on RhoA signaling was differentially localized to the Golgi and ER membrane compartments. In Chapter II, it was hypothesized that PKA activation of TAAR1 may negatively regulate VMAT2. We used midbrain DA neuron cultures and SK–N–SH neuroblastoma cells that express TAAR1 and VMAT2 and release catecholamines as model systems. With CRISPR–Cas9 technology the Amara lab generated TAAR1 knockout SK–N–SH cells that were used to examine the role of TAAR1 in VMAT2 regulation. VMAT2–mediated uptake of radiolabeled DA and serotonin was measured in the presence or absence of drugs that modulate VMAT2 activity. Inhibition of the Gα stimulatory (GαS) G–proteins upstream of PKA activation increased VMAT2 uptake; conversely, stimulation of cAMP decreased VMAT2 activity. Compared to wildtype cells, we found no difference in VMAT2 uptake in TAAR1 knockout cells treated with PKA agonists like dibutryl cAMP and forskolin. These data suggest that VMAT2 uptake is modulated by GαS signaling, cAMP and PKA activation, but does not require TAAR1. Taken together our results show that the cAMP–dependent inhibition of VMAT2 uptake by PKA is not mediated by the TAAR1 receptor.

Glioblastoma is the most common and most deadly form of brain cancer. With treatment, expected survival time after diagnosis is 15 months and the disease presents with universal morbidity. Current therapies include surgery, radiation, and chemotherapy. One of the current fields of interest for glioblastoma research is in immunotherapy and specifically tumor-associated macrophages (TAMs). Normally, macrophages in an infection or disease state work to degrade and digest pathogens and cancer cells. However, in glioblastoma, current evidence points to TAMs as active tumor-supporters and immunosuppressants. While the field is still relatively in its infancy, much is known about TAMs and their interactions with other immune cells and with cancer cells. This paper elucidates all the different aspects in which TAMs work to support GBM cancer cells and how they encourage tumor growth, progression, and migration. This review consolidates the pertinent information known on how TAMs are recruited, how they contribute to angiogenesis, how they promote tumor migration, how they interact with T cells, and how they become polarized to become tumor-supportive macrophages and suggests approaches for future research given this knowledge.