Kurt Haas

During learning, neurons in the brain are remodelled in both their structure and function in ways that can dramatically shape how they process incoming information and compute an output. This is especially the case in early development when neurons are extremely plastic. The spatial arrangement of a neuron’s synapses determines how inputs interact to perform computations, such as through recruitment of nonlinear conductances by spatially clustered activity. However, it remains poorly understood how such functional arrangements arise. Here, I generate the capabilities that allow me to record and analyze sensory-evoked activity in an in vivo model system and to analyze how sensory inputs shape neural activity. I develop and validate the genetic and optical tools and protocols that allow me to record neural activity across the neuron, being able to capture both synaptic input across the dendritic arbor and the action potential output. Leveraging the albino Xenopus laevis visual system as an accessible vertebrate model of early brain circuit formation I then use these tools to image growing neurons during plasticity-inducing visual training while recording and manipulating neuronal firing, I show that dendrite growth and pruning are correlated to neurons’ evoked calcium responses. Lastly, I use the functional data from these experiments to build and validate a mathematical model to predict locations of synaptic input on dendritic arbors, using fluorescence-based calcium data.

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ASD is a prevalent neurodevelopmental disorder characterized by social impairments and repetitive behaviors. Repeated studies indicate a role of genetics in the etiology of the disorder. In the search for genetic causes, whole exome sequencing has identified de novo likely gene disrupting and missense mutations in multiple, unrelated probands in many genes. The research community is now faced with the task of determining how dysfunction in genes caused by point mutations contribute to the pathophysiology of the disorder. From the high-confidence genes associated with ASD, we chose phosphatase and tensin homolog, PTEN, as our gene of interest. We first utilized an unbiased approach to identify novel genetic interactions of wildtype PTEN using a synthetic dosage lethality (SDL) screen in the Saccharomyces cerevisiae model system. Our screen yielded eight strong genetic interactions, ‘sentinels’ specific for PTEN catalytic function. We then tested for disruptions to these genetic interactions by designing a novel mini-SDL to screen a total of 97 PTEN point mutations. Functional scores obtained from this screen indicated the level of dysfunction caused by each point mutation. Interestingly, we found the dysfunction of variants to lie on a continuum with many variants displaying a range of partial loss of function (LoF). By testing the stability of all PTEN variants in both yeast and HEK293 cells we determined that many variants were dysfunctional due to a partial or complete loss of protein stability. Further, we also tested the ability of PTEN point mutations to negatively regulate the AKT pathway similar to wildtype PTEN. From this assay, we identified variants that were fully functional, partial LoF, complete LoF, and dominant negative. Through the creation of a biallelic PTEN knockout cell line we were able to deduce that the main mechanism of dominant negativity in this assay was likely due to interference of mutant PTEN with endogenous PTEN. Combined, our data indicates that point mutations of PTEN disrupt protein function via several mechanisms. Understanding how each mutation alters function will hopefully advance our understanding of the disorder and lead to precision therapeutics in the future.

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Precise wiring between dendrites and axons during brain development is a critical requirement for forming proper neuronal connectivity, a prerequisite to generate correct brain function. Establishing this highly complex physical network entails forming precise patterns of dendritic and axonal arborization as well as correct targeting of these processes to appropriate brain regions. As compared to our current understanding of axonal development, relatively little is known regarding the structural organization and of dendritic arbor growth during dendritogenesis. Two major obstacles in studying dendritogenesis in vivo are technical challenges in observing the dynamic behavior of these structures in the developing brain, and post-imaging analyses of their complex growth patterns. Here, we used in vivo rapid time-lapse imaging in the intact and awake vertebrate brain to observe dynamic dendritogenesis and analyzed components of growing dendritic arbors of individual neurons to elucidate how short-term growth behaviors culminate to produce the dendritic arbor patterning of mature neurons. Of particular interest, this work establishes that dendritic growth cones exist on all growing dendrites, but due to their dynamic nature, they have been grossly under-reported in previous in vivo studies. In this study, I find that dendritic growth cone morphology correlates with branch behavior, report differences in two different dendritic filopodial populations in vivo, and describe how dendritic growth behavior changes over neuronal maturation. Further, we have developed a novel analysis tool called Dynamo to accurately track and analyze dendritic components. I have used this tool to screen three candidate guidance cue molecules, including ephrin-A1, ephrin-B1, and slit2, for their potential role in regulating dynamic behavior of growing dendrites, and found that slit2 exposure decreases branch motility and increases branchtip filopodial motility in vivo. I also find that neurons located in the caudomedial tectum project their dendrites in a biased rostral orientation to reach the tectal neuropil, and that interfering with the Slit receptor Robo3, prevents this biased dendrite growth. These findings provide novel insights into how dendrites develop in vivo in the awake vertebrate brain.

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During learning, and particularly during development, neurons in the brain undergo structural and functional changes that are intricately interrelated. This plasticity is guided by patterns of activity that encode information about the environment, allowing the brain to adapt to an organism's specific experiences. Here I developed optical methods and analysis tools to measure and analyze sensory-evoked activity patterns in the awake brain, and track how sensory information guides plasticity. Several different methods and their applications are presented. I described models and analysis tools for nonlinear decoding of somatic activity patterns in populations of neurons, and used them to track functional reorganization of neural circuits during training. I identified a group of ultrabright and stable organic dyes that enable two-photon imaging deep within living tissue, and applied them to produce a sensitive intracellular label for excitatory synapses. I developed a random access microscope capable of tracking activity at all excitatory synapses on a neuron simultaneously, enabling the first comprehensive measurements of a single neuron's dendritic input and firing output within the awake brain. I used this microscope to track neurons' comprehensive activity and structural changes across plasticity-inducing training, and identified rules by which somatic and dendritic activity direct the detailed growth patterns of dendrites, producing spatially clustered input patterns along neurons' dendritic arbor. Throughout this work, I've taken advantage of the Xenopus laevis model system to observe rapid experience-dependent plasticity in the awake, developing brain. These results demonstrate ways in which specific experiences direct the detailed connectivity of developing neural circuits.

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During early brain development, formation of functional neural circuits requires correct neuronal morphological growth and formation of appropriate synaptic connections. In addition, sensory experience and neural activity impart lasting effects on morphological and functional complexity by directing synapse formation and synaptic plasticity. Errors in these events may result in the creation of dysfunctional circuits underlying common neurodevelopmental disorders, including Autism Spectrum Disorder (ASDs), schizophrenia, and epilepsy. Therefore, to understand the normal development and the pathophysiology of these disorders, we must decipher the molecular mechanisms regulating developmental neural circuit structural and functional plasticity. This dissertation discusses work on the molecular mechanisms underlying structural and functional plasticity in the developing brain, ranging from cell adhesion molecules involved with initial synapse formation to transcription factors regulating sensory experience-driven functional plasticity. In the first half of the dissertation, using two-photon time-lapse imaging of individual growing neurons within intact and awake embryonic Xenopus brains, I found that the cell adhesion molecules, neurexin (NRX) and neuroligin-1 (NLG1), confer stabilization to labile dendritic filopodia, supporting their transition into longer and persistent branches through an activity-dependent multistep process. Disrupting NRX-NLG1 function destabilizes filopodia and culminates in reduced dendritic arbor complexity as neurons mature over days. These findings suggest that abnormalities in brain neuron structural development may contribute to ASDs. In the second half of the dissertation, I used in vivo two-photon calcium imaging of visual network activity and rapid time-lapse imaging of individual growing brain neurons to identify morphological correlates of experience-driven functional potentiation and depression during critical periods of neural circuit formation. Further, I identified the transcription factor MEF2A/2D as a major regulator of neuronal response to plasticity-inducing stimuli directing both structural and functional changes. Unpatterned sensory stimuli that change plasticity thresholds induce rapid degradation of MEF2A/2D through a classical apoptotic pathway requiring NMDA receptors and caspases-9, 3 and 7, demonstrating natural sensory experience fine-tunes the plasticity thresholds of neurons during neural circuit formation. Together, work in this dissertation provides new insights into the molecular and cellular mechanisms of how sensory experience and synapse formation direct structural and functional plasticity in the embryonic developing brain.

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The period of early brain development involves an exceptional amount of neuronal morphological growth and refinement to form functional brain circuits. Although it is known that neural activity influences dendrite morphogenesis, the molecular pathways which convert a neural activity input to changes in morphology are not well understood. Here I show that activation of the adenylyl cyclase pathway promotes growth of developing brain neurons in vivo, in a neuron maturation-dependent manner. Rapid time-lapse two-photon imaging of single neuron growth within the developing vertebrate brain and pharmacological manipulations reveal a synergistic role for PKA and Epac in growth downstream of β-adrenergic receptors and adenylyl cyclase. Inhibition of the protease calpain increases axonal and dendritic filopodial density, but only in axons is this effect downstream of PKA. Furthermore, experiments indicate that PKA localization by AKAPs may be important in its regulation of dendritogenesis. Together, the results presented here outline multiple steps of a signaling pathway important in dynamic dendritogenesis and axogenesis in vivo.

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PKMz (Protein Kinase M zeta) is a recently identified isoform of Protein Kinase C. It is persistently active upon synthesis because its sequence resembles the catalytic domain of PKC zeta but lacks the auto-inhibitory regulatory domain. Previous studies found that PKMz is critical for LTP maintenance, as well as learning and memory in the adult rat brain. However, it is not known whether and how it functions in developing neural systems. I have identified endogenous PKMz in Xenopus laevis tadpoles brain and found that its expression pattern is temporally and spatially correlated with synaptogenesis and dendritogenesis within tadpole retino-tectal system. By in vivo rapid time-lapse imaging and three-dimensional analysis of dynamic dendritic growth, I find that exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with ZIP (zeta-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Consistent with its established roles in regulating adult glutamatergic synaptic transmission, I also examined role of PKMz in regulating developing synapses, using both immunohistochemistry and in vivo patch clamp recording. Specifically, I find that knocking down endogenous PKMz using a morpholino impairs both transmission and maturation of glutamatergic synapses, and consistently induces promoted dendritic expansion as seen in ZIP treated neurons. The model that PKMz regulates dendritogenesis by regulating glutamatergic synaptic transmission was further investigated using a novel seizure model based on Xenopus tadpoles. I find that PTZ induced seizure activity increases normalized expression level of brain PKMz, which is required for over-stabilization of dendritic filopodia dynamics induced by seizure activity. Based on these findings, together with previous results from other related studies, I have constructed a discreet and stochastic computational model to simulate synaptotropic dendritic growth mechanism. I show that as formation of nascent synapses promotes dendritic expansion into region of synaptic partners by promoting maintenance of dendritic filopodia, synapse maturation drives further dendritic refinement and stabilization of appropriate dendritic structure.

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The effects of highly prevalent early-life seizures on neuronal activity-dependent developmental programs within the immature brain remain unclear. To address this issue, the present work examined the acute and persistent effects of early-life seizures on neuronal dendritogenesis, a key activity-dependent component of neural circuit development. A novel experimental model system of early-life seizures, based on the albino Xenopus laevis tadpole, was developed for these studies. The transparency of this organism allows in vivo imaging of neuronal growth and activity within the intact developing brain. Additionally, immobilization of tadpoles using reversible paralytics and immersion in agar, for electrophysiological or imaging experiments, allows examination of seizure activity and seizure-induced effects on neuronal growth for the first time within the unanaesthetized and awake brain. Chemoconvulsant-induced seizures in tadpoles were extensively characterized using behavioural assessment, measures of cell death, and in vivo examination of neural activity during seizures through electrophysiological recordings and imaging of intracellular calcium dynamics. Rapid and long-interval time-lapse in vivo two-photon imaging of individual fluorescently labelled growing optic tectal neurons within the intact tadpole brain revealed that seizures inhibit dendritic arbor growth, that these effects are mediated cell-autonomously by excessive AMPA-receptor mediated excitatory activity, and that a single seizure episode persistently stunts subsequent arbor growth. Reduced dendritic growth is a result of decreased branch elongation, increased branch elimination, and loss of dendritic filopodia. Seizures also persistently reduced the density of immunostained excitatory synaptic markers within the tectal neuropil. Rapid time lapse imaging at 5 minute intervals for 5 hours reveals selective effects on filopodial growth dynamics, characterized by rapid increase in the rate of elimination of pre-existing filopodia within minutes of seizure onset, followed by hyper-stabilization of filopodia generated during seizures. These data suggest that seizures interfere with neural circuit development by acutely destabilizing filopodia present prior to seizure induction and hyper-stabilizing filopodia formed during seizures, leading to a persistent inhibition of continued arbor elaboration and growth. This is the first examination of the effects of common early-life seizures on dendritic morphogenesis within the intact and awake brain, and these findings identify a potential morphological correlate of persistent seizure-induced neural dysfunction.

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During embryonic activity‐dependent brain circuit refinement, neurons receiving the same natural sensory input may undergo either long‐term potentiation (LTP) or depression (LTD). While the origin of variable plasticity in vivo is unknown, the type of plasticity induced plays a key role in shaping dynamic neural circuit synaptogenesis and growth. Here, we investigate the effects of natural visual stimuli on functional neuronal firing within the intact and awake developing brain using calcium imaging of 100s of central neurons in the Xenopus retinotectal system. We find that specific patterns of visual stimuli shift population responses towards either potentiation or depression in an N‐methyl‐D‐aspartate receptor (NMDAR)‐dependent manner. In agreement with the Bienenstock‐Cooper‐Munro (BCM) theory, our results show that functional potentiation or depression in individual neurons can be predicted by their specific receptive field properties and endogenous firing rates prior to plasticity induction. Enhancing pre‐training activity shifts plasticity outcomes as predicted by BCM, and this induced metaplasticity is also NMDAR dependent. Furthermore, network analysis reveals an increase in correlated firing of neurons that undergo potentiation. These findings implicate metaplasticity as a natural property governing experience‐dependent refinement of nascent embryonic brain circuits.

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