Intracellular calcium (Ca2+) signaling can regulate synaptic plasticity via gene expression in neurons. One pathway that links Ca2+ to gene expression is the G-protein coupled receptor - inositol triphosphate receptor (GPCR-IP3R) pathway. Ca2+ signaling dynamics could be affected spatiotemporally by subcellular ultrastructure, such as the endoplasmic reticulum (ER) and distributions of Ca2+-regulating proteins in the GPCR-IP3R pathway. Computational models have been built to better understand intracellular Ca2+ signaling but have yet to consider accurate neuronal ultrastructure and protein localizations. In order to make a precise model simulating neuronal Ca2+ dynamics, we aimed to analyze realistic somatic ultrastructure and distributions of signaling proteins in the GPCR-IP3R pathway within cerebellar Purkinje neurons. Here, using electron microscopy, we found out that the sub-surface ERs are denser and mostly parallel to the plasma membrane (PM). We performed immunohistochemistry for multiple proteins in the GPCR (coupled to Gαq subunit) signaling pathway, but none of these proteins showed prominent asymmetries except for IP3R. In the initial model with idealized geometry, we found out that specific ER membrane structure organization near the PM may facilitate local Ca2+ concentration increases. In addition, heterogeneous IP3R distribution could lead to various degrees of Ca2+ release from the internal ER Ca2+ store. Based on the results above, we concluded that the unique ER organization and heterogeneous Ca2+-signaling protein distributions may shape Ca2+ signaling dynamics within the Purkinje somas.

In order to form a memory, transient experiences are captured by neurons in the brain and transformed into long-lasting changes in cell circuitry and connectivity. To elucidate the mechanisms underlying memory formation it is necessary to determine how pyramidal neuron (PN) activity is transformed into changes in the future output of the neuron. NPAS4, an immediate early gene that is expressed transiently following PN activity, has been linked to changes in inhibitory synaptic connectivity. Specifically, NPAS4 leads to recruitment of somatic CCK basket cell synapses and destabilization of CCK dendritic inhibitory synapses. This cell-autonomous regulation of inhibitory synapses indicates that NPAS4 plays a role in shaping CA1 PN activity in vivo but, to date, there are no studies investigating this. Here we use an optotagging approach to compare the in vivo activity of simultaneously recorded, intermingled, NPAS4 wild type and knockout (KO) CA1 PNs from freely moving mice. We find that NPAS4 KO neurons have impaired spatial tuning and that this is accompanied by deficits in the stability of their firing across the session. Furthermore, NPAS4 KO neurons are less bursty within the place field. This reduction in bursting has implications for other aspects of spike timing including theta-coupling and phase precession. Specifically, we find that NPAS4 KO neurons are less theta-coupled within the place field and that their phase precession slopes are more shallow. Taken together our results demonstrate that NPAS4, through the reorganization of inhibitory synapses, is important for both the tuning of place fields in CA1 and for the refinement of sequences.

Glutamatergic excitatory synapses are one of the main currencies of the mammalian central nervous system. Excitatory synapses have been most well-characterized in terms of dendritic spines, which are small membranous protrusions. Dendritic spines influence synapse function by boosting synaptic potentials and sequestering synaptically-generated second messengers. Spines have been extensively studied in densely spiny principal neurons, but little is known about how they expand the information-gathering capabilities of sparsely spiny interneurons (INs). We find in the mouse primary visual cortex, parvalbumin-positive INs have a low density of spines that enclose functional glutamatergic synapses. Both spine and dendritic synapses contain calcium-permeable AMPA and NMDA receptors (CP-AMPARs, NMDARs), but NMDARs are enriched at spine synapses. Despite these similarities, spine synapses are embued with distinct sensitivities to the ongoing activity of the neuron. Glutamate receptor-mediated calcium (Ca) influx at proximal dendritic sites is bi-directionally modulated by the timing of action potentials (APs). Surprisingly, spine synapses are largely insensitive to APs but coincident activity originating in the adjacent dendrite strongly influences spine NMDAR-mediated Ca influx. Thus, while glutamate receptors on spines and dendrites are modulated by the activity of the neuron, they are distinctive in the type of coincident activity detected.

The brain converts fleeting experiences into long lasting changes in circuit connectivity and function. Activity induced immediate early genes(IEGs) are rapidly expressed in response to cellular stimuli. When a mouse is placed in an enriched environment, the inducible transcription factor(ITF) NPAS4 is expressed in hippocampal CA1 pyramidal neurons in response to depolarization and not other types of stimuli (Lin et al., 2008; Bloodgood et al., 2013). Expression of NPAS4 is further known to mediate inhibitory circuit rewiring onto pyramidal neurons through an increase in somatic inhibition by CCK basket cells and a decrease in dendritic inhibition (Bloodgood et al., 2013; Hartzell et al., 2018). Inhibitory circuit reorganization in CA1 through depolarization-dependent NPAS4 expression is likely to play an important functional role in encoding pyramidal cell place fields (Del Pino et al., 2017; Wilson et al., 2001; Danielson et al., 2016). However, the role of neuromodulatory inputs on NPAS4 expression an animal experiences in vivo is unknown. Neuromodulators such as dopamine, acetylcholine and noradrenaline are known to be released during periods of attention, novelty, and salience which an animal experiences as it travels through an enriched environment (Avery et al., 2017). With bath application of D1 dopamine receptor agonist SKF81297 on mouse acute slices, we show diminished NPAS4 expression in hippocampal CA1 pyramidal neurons. This result describes potential for dopamine to modulate NPAS4 mediated changes in inhibitory circuit connectivity in vivo and place field formation as the animal explores an enriched environment.

The mechanisms linking inducible transcription factor (ITF) activity with neuronal connectivity and receptive field properties are of great interest, but poorly understood. Of note, the immediate early gene transcription factor (IEG-TF), neuronal PAS domain 4 (NPAS4), is an ITF that is expressed exclusively in response to neuronal activity or depolarization. In the mouse visual cortex, NPAS4 is expressed at high levels during the critical period, a time during development in which neural plasticity is increased. To study NPAS4 in the visual cortex, we have elected to use in vivo calcium imaging. However, it is not known if GCaMP expression affects neuronal development or the induction of NPAS4. By utilizing in utero electroporation, we aimed to introduce GCaMP into the pyramidal neurons of the mouse visual cortex and analyze the function of the cells that were electroporated. We first found that the cells electroporated with GCaMP in early development are neurons. In addition, expression of GCaMP does not impact basal or stimulus-dependent induction of NPAS4 in the visual cortex of mice. Thus, GCaMP can be used to study the impact of NPAS4 expression on neurons in vivo without disrupting endogenous NPAS4 induction.

Experience-dependent expression of immediate-early gene transcription factors can transiently change the transcriptome of active neurons and initiate persistent changes in cellular function. However, the impact of inducible transcription factors (ITFs) on circuit connectivity and function is poorly understood. We investigate the specificity with which the ITF NPAS4 governs experience-dependent changes in inhibitory synaptic input onto CA1 pyramidal neurons (PNs). We show that novel sensory experience selectively enhances somatic inhibition mediated by cholecystokinin-expressing basket cells (CCKBCs) in an NPAS4-dependent manner. NPAS4 specifically increases the number of synapses made onto PNs by individual CCKBCs without altering synaptic properties. Additionally, we find that sensory experience-driven NPAS4 expression enhances the amount of PN inhibition that can be suppressed by depolarization-induced suppression of inhibition (DSI), a short-term form of cannabinoid-mediated plasticity expressed at CCKBC synapses. Our results indicate that CCKBC inputs are a major target of the NPAS4-dependent transcriptional program in PNs, and consequently that NPAS4 is an important regulator of cannabinoid-sensitive inhibition in the hippocampus.

The spatiotemporal organization of intracellular features is fundamental to understanding the intercommunication of neurons in the brain. Neuronal communication is regulated by receptor activation, which is influenced by signaling messenger availability and internal calcium (Ca2+) concentrations. An excess or deficiency of cytoplasmic Ca2+ can lead to changes in secondary messenger diffusion, synaptic plasticity, gene expression, and even cause cell apoptosis. Existing models of intracellular calcium movement have used image-based recording methods to approximate a uniform distribution within the cytosolic space, but it is still unclear whether or how somatic features shape calcium mobility. A methodic analysis of the relationship between somatic arrangement and the Ca2+ signaling pathway does not currently exist. In this study, we used fine-scale electron tomogram reconstructions of cerebellar Purkinje neurons to create a precise model of the interior of the cell. We established structural motifs of endoplasmic reticulum (ER) and noticed a heterogeneous arrangement of organelles within the soma. In addition, we used fluorescent immunohistochemistry to analyze localization patterns of signaling proteins and observed how two receptors that mediate Ca2+ efflux from internal ER stores: inositol 1,4,5-trisphosphate receptor 1 (IP3R1) and ryanodine receptor 1 (RyR1) have distinct angular and radial domains. Overall, we found that ER serves as a diffusion barrier and can cause nonuniform diffusion of Ca2+. This thesis demonstrates how the localization organelles and calcium modulating proteins significantly contribute to intracellular communication and provides an architectural reference for emerging research on somatic neuronal signaling.

The blood-brain barrier (BBB) is a set of properties belonging to endothelial cells that make up blood vessels in the central nervous system (CNS). The BBB’s function is to reduce the transport of nonspecific ions and molecules between the blood and brain and import specific molecules to maintain brain homeostasis. The cerebral vasculature is also known to be tightly coupled to neural activity and dynamically changes blood flow to satisfy the energy demand of active brain regions. Although the cerebral vascular blood flow is tightly coupled to neural activity, the question remains whether BBB properties can dynamically respond to neural activity, as well. Through manipulating glutamatergic neurons in mice, we have shown that neural activity can dynamically regulate both the expression and function of BBB genes in the cerebral vasculature, namely, ATP- binding cassette (ABC) efflux transporters. Additionally, we found that neural activity dynamically regulates circadian clock-related genes in endothelial cells. This led us to ask the question of what is the functional role of the circadian clock in endothelial cells of the cerebral vasculature? Through knocking out a core clock gene, Bmal1, specifically in endothelial cells, we found that the vascular clock regulates ABC efflux transport and animal behavior. We propose the idea that neural activity entrains the vascular-specific circadian clock, which regulates ABC transporter activity in a circadian manner, and deletion of the clock in endothelial cells causes dysregulation of ABC transport, thus disrupting the chemical microenvironment of the brain, resulting in complex behavioral phenotypes.

As the number one cause of death in the world, and the leading cause of death in the United States, heart disease claims more lives than all forms of cancer combined (Go et al., 2013). Though several therapies are available to

alleviate its symptoms and delay the progression of cardiac deterioration, there is currently no cure for heart failure. The discovery of new proteins and pathways, and understanding their mechanisms, create targets for potential therapeutic approaches.

Calcium and β-adrenergic receptor (β-AR) signaling are both important factors in cardiac function. Here, we examined CREB Regulated Transcriptional Co-activator 2 (CRTC2), a known converging point between β-adrenergic receptor and calcium in glucose metabolism. Little is known about the function of CRTC2 in cardiomyocytes. Upon comparison of survival and cardiac function of wildtype and transgenic nuclear targeted CRTC2 (ntCRTC2) mice in the setting of chronic heart failure (CHF), we provide evidence of trends favoring mice with upregulated ntCRTC2. Understanding of a β-AR and calcium point of convergence will enable development of de novo treatments that modulate both systems, both of which are fundamental to cardiac function.