Erin K. Purcell, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Neuronal loss is responsible for the profoundly devastating effects of neurological injury and disease for millions of patients worldwide, and the central nervous system has little capacity for self-repair. Regeneration of damaged neural circuitry with stem cell-derived neurons is a promising approach to the problem, particularly given the discovery that pluripotent stem cells can be derived by reprogramming a patient's own skin cells (induced pluripotent stem cells, iPSCs). However, functional integration of stem cell-derived neurons with host tissue continues to be a challenge met with few successes, and the field requires new and better tools to control stem cell fate and connectivity. We propose new optical methods to enable the construction of defined neural networks, where light is used to pattern specific neuronal subtypes and selectively connect them with target cell types. The approach uses a recently-described photosensitive bacterial transcription factor to drive gene expression as well as optogenetic control of neuronal spiking to selectively strengthen or weaken connections between specific populations of neurons. To demonstrate proof-of-concept, the project begins with a functional characterization of rat iPSC-derived neurons and subsequently generates pilot data to demonstrate the feasibility of using spatiotemporal patterns of light-activated gene expression and channel gating to build neural networks. For optical control of connectivity, the frequencies and patterns of stimulation are adopted from literature demonstrating either elimination or stabilization of synapses in mature neurons following optogenetic stimulation. For light-driven gene expression, the photosensitive transcription factor is delivered via recombinant replication-defective retroviruses with broad-spectrum neural promoters. Results are validated through a combination of whole-cell electrophysiology, identification of synaptic markers with immunohistochemistry, and spatially patterned optical induction of fluorescent reporter gene expression. These proof-of-concept studies will establish new protocols to control connectivity between specific neuronal populations with light, with the future potential to use a modified light-activated transcription factor to determine subtype specification. This will lay the foundation to use optical stimulation to define the identity and connectivity of neurons derived from stem cells, giving new tools to construct and reconstruct neural circuitry in vitro and in vivo in future work.

ABSTRACT Neuroprostheses are devices implanted in the brain that record or stimulate the electrical activity of nearby neurons. These devices hold tremendous potential for breakthroughs in advanced research and clinical applications. Despite the rapid growth in the use of these technologies, certain aspects of their influence on surrounding neurons, and the reasons underlying their often unpredictable function over time, remain unclear. In this study, we explore the effects of implanted neuroprostheses on the excitability of neurons at the device interface. We hypothesize that the insertion trauma and inflammatory response initiated by neuroprosthesis implantation will impact the ion channel expression and intrinsic excitability of local neurons. This is expected to result in an initial period of hyperexcitability followed by a period of relative hypoexcitability, where effects will be evident in whole-cell recordings and quantitative immunohistochemistry of ion channel expression. A novel approach for assaying the intrinsic excitability of neurons surrounding devices is developed, where whole-cell voltage- and current-clamp recordings are taken from neurons surrounding silicon devices contained in brain tissue slices collected following implantation in rats. This preparation will provide detailed information on the biophysical characteristics of neurons at the device interface, clear visual targeting of specific cell types for recordings, and correlating outcomes to local glial densities in subsequent histology. In separate subjects, ion channel expression will be quantitatively assessed at time points spanning twelve weeks surrounding devices implanted in the rat brain, where specific classes of voltage-gated sodium and potassium channels are surveyed. These studies will open up a new understanding of plasticity changes induced by the presence of devices implanted in the brain, focused on impacts to the intrinsic excitability of the cells that neuroprostheses electrically interface. The understanding gained will inform a variety of phenomena related to neuroprosthesis function, including observations of instability and variability in recording quality, responsiveness to stimulation, and placebo effects after device implantation. The research impacts a broad range of implanted devices which read-out or write-in neural activity, and future work will leverage the knowledge gained to improve long-term device function.

PROJECT SUMMARY The development of implantable devices capable of recording or stimulating electrical activity in the brain has created unprecedented opportunities to treat and study neurological diseases and injuries. However, a reactive tissue response typically occurs following implantation which is widely believed to interfere with long-term device performance. Inflammatory microglia and astrocytes encapsulate and isolate devices from neurons, while neuronal signal sources are lost within the recordable radius of the electrode surface. While these observations may contribute to signal instability and recording loss over time, the mechanistic link between specific inflammatory events and changes in signal quality remains unclear. Our group is expanding upon the current basic science understanding of device-tissue integration and recently published a study which showed shifts in subtype-specific markers of synaptic transmission surrounding implanted electrode arrays. Our data indicated an early elevation of markers of excitatory transmission (vesicular glutamate transporter-1, VGLUT1) three days post-implantation that was followed by a subsequent shift to increased expression of labeling for inhibitory neurotransmission (vesicular GABA transporter, VGAT). We hypothesize that structural and functional plasticity of synaptic inputs surrounding devices could contribute to loss of recorded signals. We further hypothesize that the timed elevation of glutamate and GABA release may act as ?go? and ?stop? cues which mediate the reactive tissue response. In this proposal, we will build upon our initial observations, further investigating the underlying mechanisms and functional consequences of synaptic plasticity on device performance. In Specific Aim 1, we will define the functional impacts of glutamatergic synaptic remodeling at the electrode interface on recorded signal quality and reactive gliosis. We will correlate transporter expression with signal quality and assess the effects of VGLUT1 knockdown on signal quality and tissue response. In Specific Aim 2, we similarly will define the functional impacts of GABAergic synaptic remodeling at the electrode interface on recorded signal quality and reactive gliosis. We hypothesize that while early glutamate release may incite neurotoxicity and reactive gliosis, subsequent GABA release acts in an anti-inflammatory capacity to preserve neuronal viability and mitigate further glial reactivity. In Specific Aim 3, we will reveal structural plasticity in the dendritic arbors of neurons at the electrode interface. For this aim, we will use two photon imaging to assess changes in dendritic spine density and morphology surrounding devices captured in ex vivo brain tissue slices. For all aims, we will test both silicon and polyimide-based arrays to compare results between device designs commonly used in the field. We will inspect impedance measurements and post-mortem scanning electron microscopy images for signs of device failure to strengthen the interpretation of our results. By exploring novel mechanisms of synaptic plasticity surrounding implanted electrode arrays, we expect to open new opportunities to both understand, and improve upon, long-term device function and biocompatibility.