Optogenetic Interrogation of Hippocampal Circuit Stabilization

Understanding the response of excitatory and inhibitory populations to varying input is vital to understanding how a brain region transforms information. Optogenetics - the combined use of optics and genetics to control the activity of proteins, provides neuroscientists with a tool to interrogate neuronal circuits with high spatio-temporal resolution and targeted cell specificity. This thesis examines the effects of optogenetic manipulations on hippocampal circuit responses. The hippocampus is a structure required for the formation and retention of episodic memories and is comprised of anatomically distinct subregions including cornu ammonis 3 (CA3) and cornu ammonis 1 (CA1). Both regions, despite differences in local circuitry, contain excitatory cells that fire in a spatially selective manner as an animal explores an environment. Based on these differences in circuitry, studies have proposed different computational roles of each region. In order to gain insight into how distinct hippocampal networks respond to light-induced external drive we measured the responses of neurons in CA1 and CA3 to optogenetic perturbation. To date, no work has explored the differences in CA3 and CA1 network responses to acute optogenetic manipulation of the circuits. This thesis uses a combined approach of optogenetic perturbation with simultaneous high-density electrophysiological recordings to answer two fundamental questions related to the computational roles of region CA3 and CA1. The first question asks, what role does region CA3 play in shaping spiking activity in downstream CA1? To address this question, electrophysiological recordings of CA1 were combined with optogenetic silencing of CA3 using the light-driven proton pump ArchT in both freely moving and urethane-anesthetized rodents. Since the major projection from CA3 to CA1 is excitatory, our initial hypothesis predicted an overall decrease in CA1 activity due to the expected decrease in excitatory drive from CA3. Surprisingly, suppression of CA3 resulted in a robust and consistent increase in interneuron firing in CA1 (awake: 68\% increase, 10\% decrease, 22\% no response n = 87, anesthetized: 59\% increase, 26\% decrease, 15\% no response, n = 96). The second question asks, how do excitatory and inhibitory populations in CA3 and CA1 differentially respond to incoming signals? To address this question, integrated opto-electrode devices were used to simultaneously manipulate and measure the responses of CA3 and CA1 circuits to perturbations. We found that focal suppression of CA3 driven by both ArchT and the light-driven chloride channel stGtACR2 resulted in a paradoxical increase in firing of both inhibitory and excitatory cell at all distances from the site of photoinhibition. In contrast, CA1 cells responded to focal photoinhibition by showing nearly 100\% decrease in cell response at the site of illumination. Paradoxical increases in firing in response to external inhibitory input to interneurons can be a feature of networks with highly-recurrent excitatory connections that are unstable in the absence of inhibition (ISNs: inhibitory-stabilized networks. Broad (600 $\mu$m diameter) photoinhibition was applied and network responses were measured over a range of laser intensities to test whether differences in responses between CA3 and CA1 can be attributed to CA3 operating in an ISN-regime. Paradoxical increases in pyramidal cell or interneuron firing were not observed when inhibitory opsins were expressed in both pyramidal cells and interneurons. When external input was restricted to interneurons, CA1, and to a smaller extent, CA3 showed increased firing in response to varying intensities of photoinhibition, suggesting both CA1 and CA3 operate as ISNs. Taken together, these results indicate that perturbations of neuronal activity can produce paradoxical effects that affect both local and connected regions. The emerging patterns depend on the detailed interactions between excitatory and inhibitory subpopulations within a region, and can be broadly explained by network models of global stabilization through inhibition. Our results further highlight the need for simultaneous monitoring of cellular responses when using optogenetics or other manipulations that alter neuronal activities

Uncovering temporal structure in hippocampal output patterns.

Place cell activity of hippocampal pyramidal cells has been described as the cognitive substrate of spatial memory. Replay is observed during hippocampal sharp-wave-ripple-associated population burst events (PBEs) and is critical for consolidation and recall-guided behaviors. PBE activity has historically been analyzed as a phenomenon subordinate to the place code. Here, we use hidden Markov models to study PBEs observed in rats during exploration of both linear mazes and open fields. We demonstrate that estimated models are consistent with a spatial map of the environment, and can even decode animals' positions during behavior. Moreover, we demonstrate the model can be used to identify hippocampal replay without recourse to the place code, using only PBE model congruence. These results suggest that downstream regions may rely on PBEs to provide a substrate for memory. Additionally, by forming models independent of animal behavior, we lay the groundwork for studies of non-spatial memory

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity