Joshua Trachtenberg - US grants

laboratory mouse; long term potentiation

DESCRIPTION (provided by applicant): Previous studies suggested that the alpha-Calmodulin Kinase II (a-CaMKII) plays a critical role in hippocampal synaptic plasticity and in hippocampal-dependent learning and memory (L&M). Recent work from our laboratory also indicates that this kinase is required for memory consolidation in neocortical sites. These L&M studies, however, were limited by the fact that a-CaMKII was deleted in many brain regions. Using newly developed techniques, we have now derived mutant lines to generate sub-region (CA1, CA3 and dentate gyrus) and cell-type (excitatory neurons) restricted deletions of this kinase. Additionally, we have also used Cre -recombinase in Herpes Simplex Viral (HSV) vectors to specifically manipulate genes in a regional-specific manner. With these unique tools we plan to determine the role of this kinase in hippocampal pre- and post-synaptic plasticity, in learning and in memory consolidation. The specific aims of this proposal are: 1- To determine the role of a-CaMKII in either CAl, CA3 or dentate gyrus in learning and memory. Various models suggest specific roles for each hippocampal sub-region in L&M. We will use mice with post-natal and region restricted null mutations of a-CaMKII to test the role of this kinase in four forms of hippocampal-dependent learning: spatial learning in the Morris water maze, working memory in the 8-arm maze, contextual discrimination with fear conditioning, and social recognition. 2 - To test the role of pre- and post-synaptic a-CaMKII on the induction of long-term potentiation (LTP) in CAl, CA3 and dentate gymus. We will test the pre and post-synaptic role of this kinase in synaptic plasticity not only in CA1, but also in CA3 and dentate gyrus. These studies will also be essential to interpret the behavioral analysis of the mutant lines proposed in Specific Aim #1. 3 - To test the hypothesis that a-CaMKII-dependent plasticity in the hippocampus is critical for early stages of memory consolidation, but that later stages of consolidation require a-CaMKII-dependent plasticity in cortical sites. Recent findings in our laboratory suggest that a-CaMKII is critical for LTP and for memory consolidation in the neocortex. To directly test this hypothesis, we will use cortical and hippocampal transgenic and viral manipulations of a-CaMKII function. The studies proposed here will not only further our understanding of the role of a-CaMKII in synaptic plasticity and in L&M, but they will also be critical for insights into cognitive deficits, such as those associated with aging.

The experiments proposed in this application investigate the roles of dendritic spine growth and retraction in experience-dependent plasticity in the mouse visual cortex. Recent evidence indicate that dendritic spines are dynamic in the developing and adult cortex: new spines appear daily and grow towards axons to establish novel synapses while some existing spines retract, breaking their synaptic connection. These changes in synaptic connectivity may play important roles in rapidly rewiring cortical circuits during experience-dependent plasticity. The extent to which spine growth and retraction underlie functional changes in cortical circuits will be examined in transgenic mice expressing a green fluorescent protein transgene. 2-photon laser scanning microscopy and intrinsic signal optical imaging will used to repeatedly image spine dynamics and functional changes in cortical ocular dominance in vivo over periods of weeks in the same mice before and after monocular deprivation. By imaging changes in structure and function in the same preparation, it will be possible to determine whether synapse elimination underlies the functional loss of deprived eye inputs and novel synapse formation underlies the gradual strengthening of experienced eye inputs. In vivo electrophysiology and fixed tissue anatomy will be used to determine which cells in which layers are the first to alter their responsiveness and connectivity following monocular deprivation and to map the progression of these changes through the remainder of the cortical circuit. Taken together, these experiments should provide a detailed understanding of the onset and progression of experience-dependent changes in cortical structure and function at the level of receptive fields and synapses. Results from these experiments may aid in the provision of rationally-based therapeutic approaches to amblyopia, scotoma, and stroke.

computer program /software; molecular /cellular imaging

DESCRIPTION (provided by applicant): Autism is a severe neurodevelopmental disorder characterized by impairments in social interaction and communication. Rates of autism have exploded over the past decade and it is now estimated that 1 out of every 166 children exhibit some form of autism. The neuropathological cause of autism remains elusive, though a strong genetic contribution is evident from the 60%-92% concordance between monozygotic twins versus 0-10% in dizogtic twins. Research in human populations indicates that the number of loci associated with autism exceeds 15. Of specific relevance to this proposal is the phosphatase and tensin homologe (Pten). This oncogene regulates the growth of post-mitotic neurons. The PTEN signaling pathway is emerging as one of two major pathways that regulate the susceptibility to autism spectrum disorders. Mice with a cortical Pten deletion exhibit many of the characteristics associated with autism, including macrocephaly, deficits in social interactions, impaired social learning, hyperactivity, and increased anxiety-like behavior. Many of these deficits are reversible with chronic treatment of the mTOR inhibitor, rapamycin. In this proposal, we employ mice in which the Pten gene is deleted specifically from the cortex by CRE-mediated excision beginning some 6-8 weeks after birth. We recently demonstrated that cortical layer 2/3 pyramidal neurons experience a unique growth of their apical dendrites following PTEN deletion. This growth expands the apical dendritic tree by upwards of 1mm and adds hundreds of new dendritic spines. Our goal is to fully characterize any changes in basic physiology and sensory information processing in these growing neurons. To this end we use 2-photon laser scanning microscopy to guide the placement of in vivo whole cell patch recordings from growing neurons in the mouse primary visual cortex. We also use 2-photon imaging to image network activity in the cortex of knockout and wildtype mice. Results from these studies may provide targets for the development of rationally based therapeutics for autism spectrum disorders. PUBLIC HEALTH RELEVANCE: The PTEN signaling pathway is emerging as one of two major pathways that regulate the susceptibility to autism spectrum disorders. We earlier characterized robust changes in dendritic structure in conditional knockout mice. Here we measure changes in physiology and sensory processing in these growing neurons using in vivo whole cell patch recordings and 2-photon in vivo imaging of network activity.

Project Summary The proposed work addresses a problem highlighted by the NEI Audacious Goals Initiative as ?essential to resolve?: identifying ways to regenerate damaged neurons and promote their reconnection to the correct targets in the central nervous system. In mice, a crushed optic nerve can be regenerated by concurrent manipulation of growth-control pathways and neural activity. Yet these regenerating optic nerves may not form appropriate connections because they grow into an atrophied thalamus whose inputs to cortex are weakened. Thus, functional regeneration requires strengthening of thalamocortical inputs representing the damaged eye to re- establish binocular mapping of visual space onto cortical circuits. Similar challenges are faced in early postnatal development, when a weak incoming input from the ipsilateral eye must match the mapping laid down in a cortex already dominated by the contralateral eye. This proposal examines the circuit mechanisms in primary visual cortex necessary for successful regeneration and integration of weak inputs in primary visual cortex, using in- vivo two-photon microscopy of calcium activity in alert mice and whole-cell slice electrophysiology, and then tests the effectiveness of inducing similar conditions in adulthood. The overall hypothesis is that compartmentalized dendritic activity promotes large-scale integration of new inputs into primary visual cortex. Preliminary data suggest that direct cholinergic input to one class of inhibitory neurons, the regular-spiking, somatostatin- expressing interneurons that inhibit dendrites, is lost as the critical period closes, leading these neurons to shift from compartmentalized dendritic activity to more synchronous activity. Chemogenetic control of somatostatin interneurons will be used to promote dendritic compartmentalization in adult cortex and to test whether this enhances regeneration. These experiments are expected to reveal new mechanisms that explain how the closure of a critical period in visual development reduces the capacity for establishment and strengthening of synaptic connections in cortex. In the long term, this knowledge is likely to promote incorporation of weak inputs onto their appropriate targets during regeneration after injury or disease in adulthood, which would achieve a key goal of the NEI and improve treatment options for vision loss.

PROJECT SUMMARY/ABSTRACT The visual system exhibits a heightened sensitivity to the quality visual experience during an interval late in development termed the critical period. Discordant vision during the critical period is the cause of amblyopia, a prevalent visual disorder in children. Treatment of amblyopia is most effective in children before the close of the critical period. Subsequently, the flexibility with brain circuitry diminishes in adulthood and effective therapy is more difficult. In a mouse model of amblyopia, disrupting normal vision by closing one eye for only a few days (monocular deprivation, MD) during the critical period, but not thereafter, also perturbs the normal binocularity of neurons in visual cortex and decreases visual acuity. Yet how these adaptive changes, or plasticity, first emerge within neurons that form the circuits in visual cortex is poorly understood. Likewise, how plasticity propagates from the first neurons to adapt to other neurons connected to these neurons by synapses is unclear. The short duration of the critical period in mice is one factor impeding the study of how the greater plasticity confined to the critical period contributes to the induction as well as recovery from amblyopia. The nogo-66 receptor gene (ngr1) is required to close the critical period. In ngr1 mutant mice, plasticity during the critical period is normal, but it is retained in adult mice. Importantly, ngr1 mutant mice spontaneously recover visual acuity in this model of amblyopia. In the proposed research, we take advantage of this extended critical period in ngr1 mice to investigate what is unique about plasticity during the critical period that promotes recovery from amblyopia. We compare how MD alters the function and connectivity of populations of neurons in visual cortex with a combination of sophisticated repeated in vivo calcium imaging and laser-scanning photostimulation synaptic mapping. We will begin to unravel how plasticity within visual cortex proceeds during abnormal vision (MD), as well as how this plasticity is restricted to the critical period with these experiments. In addition to improving understanding of how experience-dependent plasticity changes the function of brain circuits, these studies may reveal new avenues for developing therapeutic approaches to treat amblyopia and perhaps other neurodevelopmental disorders that result from maladaptive developmental plasticity.