Cellular and molecular analysis of startle modulation

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Abstract A remarkable feature of the nervous system is its ability to adjust stereotyped behavioral responses in a context dependent manner. In vertebrates, sudden and intense acoustic stimuli evoke an evolutionarily conserved startle response. While the execution of the acoustic startle response is extremely stereotyped, response probability is modulated in a context-dependent manner. For example, repeated presentation of a startling stimulus suppresses a behavioral response, representing a simple form of learning known as habituation. In humans, modulation of startle behavior is impaired in several neuropsychiatric disorders, including in Attention Deficit- Hyperactivity Disorder and autism spectrum disorders. Despite its importance, the molecular mechanisms underlying startle modulation not well understood. Zebrafish show a remarkable behavioral plasticity, and we have previously shown that larvae exhibit modulation of the acoustic startle response- including prepulse inhibition and habituation- with behavioral and pharmacological characteristics similar to those in mammals. We previously conducted the first forward genetic screen in vertebrates to isolate mutants defective in startle modulation, and identified 14 mutants with defects in habituation behavior. None of these 14 mutants exhibit morphological defects or overt defects in startle performance. Importantly, five of the six mutants we cloned so far encode genes previously not implicated in vertebrate habituation. Here we propose to build on our success in using a molecular genetics, phenotype based strategy to decipher the molecular and circuits mechanisms that drive vertebrate habituation behavior. Specifically, rather than focusing on a single habituation gene, our strategy is to continue to use whole genome sequencing to clone six additional mutants from our screen. Combined with the six mutants we have already cloned, this provides an unparalleled toolbox critical to attain a comprehensive model of the molecular-genetic and circuit mechanisms underlying habituation. Simultaneously, we focus on select genes as entry points to further link genetic mutants to behavioral phenotypes and to decipher the molecular and circuit mechanisms that regulate behavior. The experiments in this proposal will: (1) use a molecular genetic approach including transgenic behavioral rescue to identify the neuronal populations in which three genes critical for habituation function; (2) to use molecular and pharmacogenetic approaches in conjunction with a behavioral assay to determine the signaling pathways through which the adaptor protein-2 sigma subunit (AP2s1) critical for receptor endocytosis and the huntingtin interacting gene hip14 promote habituation; and 3) to use an established whole genome sequencing/bioinformatics pipeline to identify the causative gene mutations for six additional habituation mutants isolated from our genetic screen, generate CRISPR/Cas9 alleles to confirm their identity and determine their expression in the brain. Combined this will provide both breadth and depth both at the molecular and at the circuit level critical to comprehensively address fundamental molecular genetic questions in vertebrate startle modulation, also relevant to human disease conditions.