GABAergic inhibition is essential for circuit development and function. In the developing visual system, GABAergic inhibition determines the onset and close of the critical period for ocular dominance plasticity. Furthermore, the development of GABAergic synaptic transmission is regulated by visual experience. These studies suggest that sensory input activity regulates the development of GABAergic neurons and their synaptic connections, and that, in turn, inhibitory GABAergic synaptic transmission regulates the development and plasticity of visual circuits. Despite this postulated pivotal role for inhibition in circuit development, the inability to visualize and perturb connectivity of GABAergic inhibitory neurons in an intact developing circuit has prevented a critical evaluation of this hypothesis. We have developed tools to address both of these limitations and now propose experiments to determine mechanisms controlling the development of GABAergic neurons and their synaptic connections in the intact Xenopus tadpole optic tectum and to determine how GABAergic inputs control the development of visual receptive fields and visually guided behavior. To visualize GABAergic neurons in the intact animal, we will use the VGAT promoter to drive expression of fluorescent proteins (FPs) and other genes of interest specifically in GABAergic neurons. We will decrease synaptic transmission onto GABAergic neurons by VGAT-driven expression of peptides which we have shown specifically inhibit GABAergic or glutamatergic synaptic transmission in a cell autonomous manner.
In Aim 1, we will collect time-lapse images of individual GABAergic tectal neurons expressing FP and FP-tagged synaptic proteins to identify mechanisms that control GABAergic neuronal development. We will test the role of glutamatergic and GABAergic synaptic transmission in GABAergic neuron development by expressing peptides to block synaptic transmission onto the imaged cell.
In Aim 2, we will combine in vivo time-lapse imaging, to identify stable and dynamic dendrites, and retrospective serial section electron microscopy (EM) to determine the synaptic rearrangements that occur during GABAergic neuronal development. Data from Aims 1 and 2 will demonstrate the activity-dependent mechanisms that control the morphological development and synaptic connectivity of GABAergic neurons in vivo.
In Aims 3 and 4 we will test the effect of blocking inhibitory and excitatory transmission on tectal visual receptive field properties, using cell-attached and whole recordings, and a visual avoidance assay, that will extend our studies into the behavioral arena. The aberrant development and function of inhibitory circuits is thought to underlie a variety of developmental neurological disorders, including autism, schizophrenia and depression, however the basic mechanisms governing the development of inhibitory circuits are relatively unknown. The experiments proposed here should illuminate activity-dependent mechanisms of GABAergic circuit development in vivo.

Public Health Relevance

This project will determine the mechanisms governing the development of GABAergic inhibitory neurons and their synaptic connections in the intact developing brain. We will determine the role of inhibitory neurons in perceiving visual information and in visually guided behavior. These studies will help us understanding developmental errors in brain circuit formation.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Central Visual Processing Study Section (CVP)
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Steinmetz, Michael A
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Scripps Research Institute
La Jolla
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McKeown, C R; Thompson, C K; Cline, H T (2017) Reversible developmental stasis in response to nutrient availability in the Xenopus laevis central nervous system. J Exp Biol 220:358-368
Lau, Melissa; Li, Jianli; Cline, Hollis T (2017) In Vivo Analysis of the Neurovascular Niche in the Developing Xenopus Brain. eNeuro 4:
Pratt, Kara G; Hiramoto, Masaki; Cline, Hollis T (2016) An Evolutionarily Conserved Mechanism for Activity-Dependent Visual Circuit Development. Front Neural Circuits 10:79
Thompson, Christopher K; Cline, Hollis T (2016) Thyroid Hormone Acts Locally to Increase Neurogenesis, Neuronal Differentiation, and Dendritic Arbor Elaboration in the Tadpole Visual System. J Neurosci 36:10356-10375
Liu, Han-Hsuan; Cline, Hollis T (2016) Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis. J Neurosci 36:7325-39
Truszkowski, Torrey L S; James, Eric J; Hasan, Mashfiq et al. (2016) Fragile X mental retardation protein knockdown in the developing Xenopus tadpole optic tectum results in enhanced feedforward inhibition and behavioral deficits. Neural Dev 11:14
Gambrill, Abigail C; Faulkner, Regina; Cline, Hollis T (2016) Experience-dependent plasticity of excitatory and inhibitory intertectal inputs in Xenopus tadpoles. J Neurophysiol :jn.00611.2016
Bestman, Jennifer E; Huang, Lin-Chien; Lee-Osbourne, Jane et al. (2015) An in vivo screen to identify candidate neurogenic genes in the developing Xenopus visual system. Dev Biol 408:269-91
Muñoz, Rosana; Edwards-Faret, Gabriela; Moreno, Mauricio et al. (2015) Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells. Dev Biol 408:229-43
Faulkner, Regina L; Wishard, Tyler J; Thompson, Christopher K et al. (2015) FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles. eNeuro 2:e0055

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