Grayson O. Sipe - US grants

DESCRIPTION (provided by applicant): Synaptic plasticity is critical for normal neurodevelopment and adult circuit function. Recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in developmental synaptic plasticity. However, the mechanisms driving these roles are poorly understood. Purinergic signaling has been implicated in many neurodevelopmental processes, but studies on purinergic signaling in microglia have focused primarily on neuroinflammatory roles. It is unknown whether purinergic signaling contributes to the motility of non-inflamed microglia that underlies roles in synapse surveillance and plasticity. P2Y12 is a purinergic receptor associated with microglial motility and is highly expressed in resting, ramified microglia. However, P2Y12 is rapidly downregulated following inflammation, suggesting that it serves functions under healthy neurophysiological conditions. We propose that P2Y12 may be critical for basal microglial motility, synaptic surveillance, and synaptic plasticity. Using a mouse model, in Aim 1, we will determine whether genetic or pharmacological disruption of P2Y12 affects basal microglial morphology or motility in vivo. In Aim 2, we will test whether P2Y12 disruption affects microglial synaptic surveillance and experience-dependent synaptic plasticity in the mouse visual system. In addition, P2Y12 has been implicated in a purinergic autocrine system in peripheral macrophage chemotaxis, suggesting that a similar mechanism may exist in microglia. Therefore in Aim 3, we will test whether microglial release of purines in an autocrine manner is necessary for efficient chemotaxis towards chemokines implicated in synaptic plasticity. Through these studies, we will explore novel roles for purinergic signaling in non-inflamed microglia.

Emerging evidence suggests that astrocytes are not passive support cells in the central nervous system, but rather play active roles in modulating neuronal activity through ionic buffering, metabolic support, and neurotransmitter uptake. Specifically, astrocytes show selective expression of a subset of neurotransmitter transporters, including GLT1 (glutamate transporter) and GAT3 (GABA transporter), which raises the possibility that astrocytes can influence neuronal activity by changing neurotransmitter uptake. Studies investigating astrocytic calcium transients have largely characterized responses using slice or in vitro preparations rather than in vivo; likewise studies into the role of neurotransmitter uptake have almost exclusively been conducted in slices in situ. Previous work has shown that large-scale astrocytic somal calcium transients can be elicited in vivo by diffuse release of neuromodulators such as acetylcholine and norepinephrine. Yet, it remains unclear how small calcium transients in the distal processes of astrocytes relate to neuronal activity and how astrocytes influence cortical sensory processing. Our lab has developed a novel visual stimulation paradigm using natural movies that reliably drives both neuronal activity and astrocytic process calcium transients in the mouse primary visual cortex. In Aim 1, I will correlate astrocytic process calcium transients and local neuronal calcium activity simultaneously using dual-calcium imaging in vivo. Using transgenic Cre lines to restrict neuronal calcium imaging to excitatory and inhibitory neuron subpopulations, I will be able to dissect how calcium activity in astrocytes relates to specific components of the cortical circuit. In Aim 2, I will use pharmacological and genetic manipulations of GLT1 to explore how astrocytic glutamate uptake contributes to excitatory synaptic activity during visual processing. In Aim 3, I will use pharmacological and genetic manipulations of GAT3 to determine how astrocytic GABA uptake contributes to inhibitory synapse activity during visual processing in the cortex. By exploring astrocytic calcium activity and neurotransmitter uptake during sensory processing in the mouse visual cortex, this work will increase our understanding of astrocytic roles in cortical processing and may provide insight into disorders characterized by imbalances of excitation and inhibition such as epilepsy and schizophrenia.

My career goal is to lead an independent research program studying astrocyte-neuron dynamics, arousal, and alcohol use disorder (AUD). I have benefitted from experimental training in numerous techniques including two-photon microscopy, neurophysiology, circuit anatomy, and behavior. During the mentored phase of this grant (K99), I will continue to work closely with my co-mentors, Drs. Mriganka Sur and Elena Vazey. Mriganka is an expert in cortical information processing, neuron- astrocyte circuits, and optical techniques. Elena Vazey is an expert in noradrenergic signaling, stress, alcohol-related behaviors, and chemogenetics. In addition, I will receive advice from my mentoring team consisting of Drs. Kerry Ressler, Heather Richardson, and Thomas Kash. Their combined expertise ranges across stress pathophysiology, alcohol-related processing, limbic and reward circuits, neuromodulation, and anxiety behaviors. The additional training from my mentoring team will equip me with the conceptual and technical acumen to become a significant contributor to the fields of alcohol behavior and stress. This training will be done within the Brain and Cognitive Sciences department at MIT, which provides both a vibrant intellectual research community and expansive research infrastructure support. During my postdoctoral fellowship, I developed novel methods of simultaneously imaging astrocyte-neuron networks to study astrocyte roles in information processing. This work led me to study how astrocytes affect neuromodulation of cortical circuits by norepinephrine (NE). I have found an intriguing astrocyte-neuron calcium signature that reflects a shift in cortical processing during periods of high arousal. Furthermore, the drugs that are currently being tested in arousal disorders and AUD block these signatures, suggesting that astrocyte-neuron interactions may provide crucial insight into the pathophysiology of stress and AUD. My immediate goals are to understand how these events relate to arousal and alcohol drinking behavior, and to determine the relationship with abnormal NE release. (Aim 1) I will study the astrocyte-neuron processing in the prefrontal cortex (PFC) while mice actively drink alcohol on using in vivo two-photon imaging. (Aim 2) I will determine the role of NE in affecting astrocyte-neuron physiology through pharmacological and chemogenetic manipulations of NE release. (Aim 3) Finally, during R00 phase, I will manipulate astrocyte-specific mechanisms to determine the role of astrocytes in arousal-mediated alcohol consumption. These experiments will clarify how astrocyte-neuron dysregulation in the PFC is related to NE release and AUD.