Mark T. Harnett, Ph.D. - US grants

PROJECT SUMMARY This proposal is submitted in response to the NIH Exploratory Bioengineering Research grants program. The proposal develops a technology platform that will enable the parallel measurement of intracellular dynamics from ensembles of neurons in cortical circuits in awake, behaving rodents in a fully automated fashion. We will develop a novel, high-density, multichannel whole-cell patch-clamping platform that is guided by recently developed algorithms that enable automated intracellular recordings in vivo. The three Specific Aims provide for a systematic development of the proposed technologies and application to an urgent systems neuroscience question. AIM 1 develops the surgical methodology, electrode localization strategies and custom skeletal implants for to distribute robotic patch clamping electrodes to the requisite targets in the cortex. AIM 2 develops the parallel patch-clamp robot itself as well as the algorithms to control dense arrays of intracellular recording electrodes. AIM 3 will utilize the functionally characterized platform to obtain parallel whole-cell patch-clamp recordings in behaving mice within and across different lamina and circuits in a cortical association area. This project is well suited to the goals of bioengineering research program ? we are using a multidisciplinary approach to develop a robotic platform that removes a critical barrier for studying neuronal circuit functioning with a high degree of cell and circuit specificity during behavior. The successful development of this technology platform will empower neuroscientists to map the activities of neurons in specific circuits throughout the nervous system, enabling a mechanistic understanding of how circuits function in behaviors, and reveal how cells and circuits go awry in pathological states.

Acetylcholine (ACh) exerts diverse and powerful effects on animal behavior and underlying cortical neural dynamics. However, identifying the cellular and circuit substrates mediating these processes has proved challenging due to the many targets of ACh action and the lack of specific tools. ACh is thought to act on local cortical circuit components, specifically interneurons, to indirectly influence pyramidal neuron dynamics. We hypothesize that direct cholinergic neuromodulation of pyramidal neurons dendrites is an important new locus for the effects of ACh on cortical dynamics and behavior. By leveraging a new genetically targeted pharmacological tool with unprecedented specificity, we will causally test the contribution of AChR-dependent dendritic mechanisms to a cortical sensorimotor computation.!Our preliminary evidence shows that muscarinic acetylcholine receptors (mAChRs) potently modulate the excitability of distal apical trunk dendrites in layer 5 cortical pyramidal neurons (L5 PNs). These dendrites exhibit an active supralinear mechanism that can drive high frequency somatic spiking during coincident ?bottom-up? and ?top-down? cortical input. L5 PN trunk dendrites are therefore well positioned to implement a canonical cortical computation for combining multiple inputs. In the mouse barrel cortex, bottom-up sensory information is combined with top-down motor input via this subcellular coincidence detection mechanism to produce a whisker object localization signal. We will use this system to test a novel role for ACh in cortical function by characterizing the effect of mAChR activation on L5 PN dendritic integration (Aim 1) and identifying its ion channel mechanism (Aim 2). We will then employ a novel genetically-targeted pharmacology strategy with unprecedented specificity to causally test the contributions of mAChR-dependent dendritic mechanisms to a cortical sensorimotor computation during behavior (Aim 3). These experiments will establish a new pathway linking a single neuromodulator ? and its ion channel target(s) in a genetically defined L5 PN cell type ? to cellular processing, circuit computation, and behavior, providing critical insight into how ACh modulates brain function. !

PROJECT SUMMARY This proposal is submitted in response to the NIH Exploratory Bioengineering Research grants program. The proposal develops a technology platform that will enable the parallel measurement of intracellular dynamics from ensembles of neurons in cortical circuits in awake, behaving rodents in a fully automated fashion. We will develop a novel, high-density, multichannel whole-cell patch-clamping platform that is guided by recently developed algorithms that enable automated intracellular recordings in vivo. The three Specific Aims provide for a systematic development of the proposed technologies and application to an urgent systems neuroscience question. AIM 1 develops the surgical methodology, electrode localization strategies and custom skeletal implants for to distribute robotic patch clamping electrodes to the requisite targets in the cortex. AIM 2 develops the parallel patch-clamp robot itself as well as the algorithms to control dense arrays of intracellular recording electrodes. AIM 3 will utilize the functionally characterized platform to obtain parallel whole-cell patch-clamp recordings in behaving mice within and across different lamina and circuits in a cortical association area. This project is well suited to the goals of bioengineering research program ? we are using a multidisciplinary approach to develop a robotic platform that removes a critical barrier for studying neuronal circuit functioning with a high degree of cell and circuit specificity during behavior. The successful development of this technology platform will empower neuroscientists to map the activities of neurons in specific circuits throughout the nervous system, enabling a mechanistic understanding of how circuits function in behaviors, and reveal how cells and circuits go awry in pathological states.

The mammalian cortex plays a critical role in integrating multiple streams of information to guide adaptive behavior. For example, head direction (HD) information is combined with visual and spatial input in the mouse retrosplenial cortex (RSC). Accurate integration of these signals is a necessary component of navigation: recognizing a distant landmark while facing north vs. facing south has very different interpretations for one's position and future actions. However, the mechanisms by which any cortical association area integrates different inputs at the level of individual neurons during behavior is unknown. RSC is therefore a compelling model system in which to test general associative computations during a complex behavior: the combination of visual and HD information during navigation. Anatomical evidence suggests that HD inputs computed in the anterior thalamus make their synapses at distal apical dendrites in RSC, while visual and motor synapses are located closer to the somas of RSC principal neurons. This arrangement suggests that nonlinear dendritic integration may be used by RSC to combine HD with other inputs. Active dendritic integration is theorized to allow single neurons to respond flexibly to different combinations of input, where the state of one input nonlinearly influences the impact of another input. Our overarching hypothesis is that such mechanisms could work in concert with neural circuit computations to implement context-dependent cortical computations. Congruent with this idea, RSC neurons in navigating rats exhibit complex conjunctive receptive fields, a feature that is lacking from commonly studied primary sensory cortices. RSC is therefore an ideal area to evaluate the role of dendrites in associative computations during navigation. However, current methods are not well-suited to this level of investigation: they either allow mice to behave freely or they achieve sub-cellular resolution. This has led to a critical gap in our understanding of navigation, and by extension, associative cortex function. We have recently developed technology that bridges this gap: an animal-actuated rotating headpost that allows mice to engage in 2-D navigation by freely turning their head during conventional 2-photon imaging. We will use this new approach to test the hypothesis that neurons in RSC use sub-cellular processing to flexibly combine HD and visual information during navigation behavior. These experiments will provide new insights into cellular- and circuit-level mechanisms of navigation, and of associative cortical function in general. Results from this project will be valuable for understanding brain disease states as well as for building biologically-inspired artificial neural networks.