The human brain is a network of ninety billion neurons that allows for many of the behavioral adaptations considered unique to our species. One-fifth of these neurons are layered in an epithelial sheet known as the cerebral cortex, which is exquisitely folded into convolutions called gyri. Defects in neuronal number clinically present with microcephaly (Greek for “small head”), and in inherited cases these defects can be linked to mutations that identify genes essential for neural progenitor proliferation. Most microcephaly genes are characterized to play a role in the centrosome, however rarer presentations of microcephaly have identified different mechanisms. Charged multivesicular body protein/Chromatin modifying protein 1A (CHMP1A) is a member of the ESCRT-III endosomal sorting complex, but is also suggested to localize to the nuclear matrix and regulate chromatin. We show that loss-of-function mutations to human CHMP1A cause a rare microcephaly syndrome with reduced cerebellar volume. CHMP1A mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation, and loss of enrichment of INK4A promoter DNA in chromatin immunoprecipitations performed against BMI1, indicating a loss of the normal repression of INK4A by BMI1. Defects in zebrafish produced by morpholino-based knockdown of the CHMP1A orthologue resembled those seen after bmi1 knockdown, and were partially rescued by INK4A orthologue knockdown. Chmp1a is expressed in dividing cells in the developing cerebral cortex and cerebellar external germinal layer, and in vitro knockdown assays using short hairpin RNA implicate a role in Wnt- and Shh-pathway signal transduction. Altogether, this suggests that CHMP1A serves as a critical link between cytoplasmic and nuclear signals that regulate neural progenitor proliferation. Compared to microcephaly, polymicrogyria is a more heterogeneous brain malformation that has been suggested to implicate molecular mechanisms involved in pattern formation in the cortex. Many cases of polymicrogyria show an asymmetric distribution, and we demonstrate that these cases are strongly biased towards a right-predominant pattern. Using whole-exome sequencing in patients with polymicrogyria, we identify rare mutations in two primary microcephaly genes, ASPM and WDR62. Interestingly, some of these patients lack profound microcephaly, suggesting heretofore underappreciated pleiotropic effects of these centrosomal genes.

Visual perception can be improved by the intentional allocation of attention to specific visual components. This “top-down” attention can improve perception of specific locations in space, or of specific visual features at all locations in space. Both spatial and feature attention are thought to involve the feedback of attention signals from higher cortical areas to visual cortex, where it modulates the firing rates of specific sensory neurons. However, the mechanisms that determine how top-down attention signals modulate the firing rates of visual neurons are not fully understood. Recently, a sensory mechanism called normalization has been implicated in mediating neuronal modulations by attention. Normalization is a form of gain control that adjusts the dynamic range of neuronal responses, particularly when more than one stimulus lies within a neuron's receptive field. Models of attention propose that this sensory mechanism affects how attention signals modulate the firing rates of sensory neurons, but it remains unclear exactly how normalization is related to the different forms of top-down attention. Here we use single unit electrophysiological recordings from the middle temporal area (MT) of rhesus monkeys to measure the firing rates of sensory neurons. We ask the monkeys to perform a behavioral task that directs their attention to a particular location or feature, allowing us to independently measure modulations to firing rates due to normalization, spatial attention, or feature attention. We report that variations in the strength of normalization across neurons can be explained by an extension of conventional normalization: tuned normalization. Modulation by spatial attention depends greatly on the extent to which the normalization of a neuron is tuned, explaining a neuron-by-neuron correlation between spatial attention and normalization modulation strengths. Tuned normalization also explains a pronounced asymmetry in spatial attention modulations, in which neurons are more modulated by attention to their preferred, versus their non-preferred, stimulus. However, feature attention differs from spatial attention in its relationship to the normalization mechanism. We conclude that while spatial and feature attention appear to be mediated by a common top-down attention mechanism, they are differently influenced by the sensory mechanism of normalization.

The avoidance of light by fly larvae has been studied for over a century. Early 20th-century investigators found that larvae crawled away from light sources incident at an angle (e.g. a sunlit window). Contemporary studies project light from directly above or below and find that larvae accumulate in shadows and have stereotyped responses to sudden changes in light intensity. Now, as then, both the sensory and behavioral mechanisms for phototaxis remain controversial. Here, I unify the historic and modern approaches in the Drosophila larva using a novel apparatus and high-resolution behavioral analysis to allow for the precise quantification of larval movement in response to photosensory inputs. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, where the larva pauses and sweeps its head back and forth (head-sweeping) until it begins a new run in a new direction. My analysis reveals that the larva uses head-sweeps as spatiotemporal probes of local light information to determine the direction of successive runs. I find all forms of phototaxis are mediated by the same sensorimotor transformation and establish the necessity of the larval eye to decode the direction of incident light. This work provides the necessary foundation for the decryption of the neural circuits controlling phototaxis.

The complex circuitry of the visual system contains around one hundred functionally distinct neuronal types that become specified and connect with the appropriate synaptic partners during development. Previous studies have indicated that immature retinal ganglion cells already express subset-specific molecules that guide them to make precise synaptic choices. In the mammalian retina, members of the cadherin family of adhesion molecules are attractive candidates for this role. To test this idea I began by investigating the expression of cadherins 1-26 in the mouse retina and superior colliculus using in situ hybridization. I then studied the connectivity of cadherin-expressing neurons by analyzing mouse lines in which a marker was inserted after the start codon of each of six cadherin genes of interest. In this way, I identified functional circuits in the visual system that are marked by cadherins. One such circuit is formed of direction-selective retinal ganglion cells (DSGCs), which fire in response to objects moving in one (preferred) direction, and their synaptic partners, the starburst amacrine cells. There are four DSGC subsets, distinguished by their preference for dorsal, ventral, nasal, or temporal motion on the retina. I determined that cadherin 6 is selectively expressed by the two DSGCs subtypes that respond to dorsal or to ventral movement. In collaboration with other lab members I used in situ hybridization and gene expression profiling to identify other molecular markers that distinguish between the four DSGC subsets and that distinguish DSGCs from other retinal ganglion cells. Finally, I used birthdating and lineage tracing methods to ask when DSGCs become molecularly specified. I determined that at least two subsets of DSGCs are specified at or shortly after their birth. For cadherin 6-positive DSGCs, I went on to show that they are specified even before their birth, and that they arise from committed retinal progenitors. Globally, my experiments aimed not only to examine cadherin expression and function in the visual system, but also to demonstrate a method of using molecular signatures to probe the mechanisms of neural circuit assembly in the central nervous system.

The human brain is one of the most exquisite structures in nature, featuring extreme functional complexity and capacities that allow for advanced cognitive abilities. During the development of the human brain, neural progenitors undergo massive proliferation, which is known to inevitably result in spontaneous mutations; yet the degree of somatic mosaicism within the human brain is unexplored. Several hypotheses have been proposed that various types of somatic mosaicism may serve as an adaptive mechanism to diversify neurons and thereby promote the functional complexity of human brains. Previously proposed mechanisms to increase somatic mosaicism within the brain include elevated somatic LINE-1 element retrotransposition, and the creation of somatic aneuploidy during neurogenesis. On the other hand, genomic diversity needs to be balanced by genomic stability, in order to protect against deleterious mutations that reduce the fitness of the cells, or oncogenic mutations that might promote cancers. In fact, brain-specific somatic mutations have also been proposed to contribute to the unexplained burden of neurological diseases. To directly study genomic variability from cell-to-cell within the human brain, we developed a method to isolate and amplify single neuronal genomes from postmortem and surgically resected human brain tissues. We quantified the frequency of somatic LINE-1 retrotransposition events and aneuploidy in human cortical neurons, and found that the frequencies of both are low, with no sign of brain-specific elevation, arguing against the hypotheses that these two mutational sources are obligate generators of neuronal diversity. Additionally, aneuploidy analysis was performed on bulk and single cortical cells from a hemimegalencephaly brain. Hemimegalencephaly is an asymmetrical brain overgrowth syndrome caused by somatic mutations in brain. Single-cell analysis identified an unexpected mosaic tetrasomy of chromosome 1q, affecting both neuronal and glial populations, as a genetic cause of hemimegalencephaly. These results demonstrate that single-neuron sequencing allows systematic assessment of genomic diversity in the human brain and the identification and characterization of pathogenic somatic mutations underlying neurological disorders.

The transient receptor potential channels, ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), are non-selective cation-permeable channels that have retained their function as chemical sensors since their first appearance in metazoan species several hundred million years ago. In vertebrates, TRP channels have evolved multiple functions which make it difficult to understand exactly how they transmit signals to the brain that are interpreted very differently. For example, TRPA1 and TRPV1 are sensitive to various chemicals and activation of these channels produce sensations with opposing effects. Pain is felt when TRPV1 is activated by spider toxins, but activation by plant cannabidiol results in a pain-relieving sensation. Similarly, TRPA1 activation by delta-tetrahydrocannabinol is reported to relieve symptoms of pain, but TRPA1 activation by the active ingredient in wasabi results in a repulsive or noxious sensation. Much of what we know about TRPA1 and TRPV1 comes from the use of plant products or exposure to substances that cause or alleviate pain and inflammation. In this study, whole-cell voltage clamp recordings of heterologously expressed human TRPA1 and human TRPV1 were tested for sensitivity to a hallucinogenic plant compound, salvinorin A and an arthropod-defensive compound, para-benzoquinone. Neither compound has yet been reported to activate TRP channels but both are known to be involved in pain and inflammation signaling in humans. I show that the arthropod compound, para-benzoquinone, activates and desensitizes TRPA1 in a cysteine-dependent manner, but activation of TRPV1 is not dependent on cysteine reactivity. Although salvinorin A is known to be a potent agonist of the kappa-opioid and cannabinoid receptors, here I show that it also acts as a highly potent agonist of both TRPA1 and TRPV1. Its interaction with TRP channels may contribute to its antinociceptive effects in behavioral studies with animals that are reported to be independent of opioid signaling.

RNA-binding proteins are key players in post-transcriptional regulation of gene expression by orchestrating RNA fate from synthesis to decay. Hundreds of proteins with RNA-binding capacity have been identified so far, yet only a small fraction has been functionally characterized and presumably many more RNA-binding proteins await discovery. The roles of RNA-binding proteins in the nervous system are of particular interest because accumulative evidence has linked RNA-based mechanisms to neural development, maintenance and repair. Here, the three RNA-binding proteins under study are IGF-II mRNA binding proteins IMP-1 and IMP-2, known to be involved in mRNA localization, translational control and stability, and adenomatous polyposis coli (APC), identified as a novel RNA-binding protein. To systematically identify their RNA binding profiles, a high-throughput approach combining protein-RNA crosslinking and immunoprecipitation with next-generation sequencing (HITS-CLIP) was applied in embryonic mouse brain. A nonparametric method was developed to computationally analyze the CLIP sequencing data, mapping transcriptome-wide protein-RNA interactions. The identified target mRNAs of IMP-1 and IMP-2 were highly enriched for functions related to neural development, especially neuron projection morphogenesis and axon guidance signaling. Moreover, these target mRNAs were associated with a variety of neurological diseases, including neurodevelopmental and neurodegenerative disorders. Supporting roles in axon development, knockdown of IMP-1 or IMP-2 caused aberrant trajectories of commissural axons in chicken spinal cord. APC mRNA targets were highly enriched for APC-related functions, including microtubule organization, cell and axon motility, Wnt signaling, cancer and neurological disease. Among the APC targets was Tubulin β-2B (Tubb2b), previously known to be required for neuronal migration. It was found that Tubb2b was synthesized in axons, and localized preferentially to dynamic microtubules in the peripheral domain of the growth cone. Blocking the APC binding site in the Tubb2b mRNA 3'UTR caused reduction in its expression in axons and loss of the growth cone peripheral area, and impaired cortical neuron migration in vivo. These findings offer an informative snapshot of the protein-RNA interactome, which can provide a basis to better understand the roles of RNA-binding proteins in the nervous system.

Visual cognition is notoriously limited: only a finite amount of information can be fully processed at a given instant. What is the source of these limitations? Here, we suggest that the organization of higher-level visual cortex into content-specific channels constrains information processing across the visual system. Each channel is primarily involved in representing one particular type of visual content (e.g. faces, cars, certain types of shapes, etc.). Furthermore, each channel has a finite processing capacity/bandwidth and is limited in the amount of information it can process. When multiple items are simultaneously presented across space, or quickly in time, the extent to which those items activate overlapping channels will constrain the amount of information that can be successfully processed. To examine this, we used brain/behavior correlations in which we directly compared behavioral performance on a perceptual task with the amount of overlap amongst the neural channels used to support the items from the behavioral task. In Chapter 1, we found that the amount of information that could be encoded on a change detection task was correlated with the amount of channel overlap within occipitotemporal cortex, but not early visual regions such as V1-V3. In Chapter 2, we extend this finding by showing that the amount of information that could reach visual awareness in a masking paradigm was also predicted by overlap amongst occipitotemporal, as well as occipitoparietal channels, but once again not in V1-V3. Finally, in Chapter 3, we sought to identify which particular channels were the most behaviorally relevant and found that virtually any part of higher-level visual cortex (e.g. across occipitotemporal cortex, within category selective regions, within the least active voxels, amongst a random sample of voxels, etc.) was significantly correlated with behavioral performance. Together, these results suggest that visual cognition is limited by a set of neural channels that extend across the majority of higher-level visual cortex. These findings have direct implications on many prominent models of visual cognition, specifically those focused on perceptual limitations, and help clarify the large-scale representational structure in higher-level visual cortex. / Psychology

Understanding how odors are encoded in the brain is of fundamental importance to neurobiology. The first two stages of olfactory information processing have been relatively well studied in both vertebrates and invertebrates. However, the organizational principles of higher order olfactory representations remain poorly understood. Neurons in the first relay of the olfactory system segregate into glomeruli, each corresponding to an odorant receptor. Higher-order neurons can receive input from multiple glomeruli, but it is not clear how they integrate their inputs and generate stimulus selectivity.

This thesis presents the analysis and design of biological interfacing technologies in light of a need for radical improvements in scalability. It focuses primarily on structural and functional neural data acquisition, but also extends to other problems including genomic editing and nanoscale spatial control. Its main contributions include analysis of the physical limits of large-scale neural recording, experimental development of a screening platform for ion-dependent molecular recording devices, characterization of the design space for molecularly-annotated neural connectomics, and new designs for high-speed genome engineering and bio-nano-fabrication. Articulating governing principles and roadmaps for these domains has contributed to the initiation of multi-institutional projects that are strategically targeted towards scalability.