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Hollis Cline Lab
Research

Overview:

My research is focused on understanding the mechanisms by which experience controls the development of the brain. My lab addresses this fundamental question by examining the development of the visual system in Xenopus tadpoles. The visual system of Xenopus is well known for its experience-dependent plasticity. We have established this preparation as an excellent experimental system in which to conduct in vivo time lapse imaging studies of neuronal development and synaptogenesis, combined with both gene transfer and electrophysiological studies of visual system function. Over the past 15-20 years we have demonstrated a role for afferent coactivity, postsynaptic NMDA receptor activity, and downstream activation of calcium-dependent enzymes including CaMKII in controlling retinotectal synaptic maturation, optic tectal cell structural plasticity and topographic map formation (Wu et al., 1996; Zou and Cline, 1996; Wu and Cline, 1998; Zou and Cline, 1999; Ruthazer et al., 2003, 2006; Haas et al 2006). More recently, we have demonstrated that visual experience has multiple effects on visual system development. A relatively brief period, 4 hours, of visual experience enhances the growth rate of tectal cell dendritic arbors through a mechanism that requires glutamatergic synaptic maturation, and the RhoA GTPases (Li et al., 2000; Li et al., 2002; Sin et al., 2002). This same brief period of visual experience increases the excitability of tectal neurons, their sensitivity to visual stimuli, synaptogenesis and synaptic strength (Aizenman et al 2002, 2003; Aizenman & Cline 2007).

The finding that we can use visual stimulation to modify the development and properties of the retinotectal system has spurred our interest in determining the function of activity-induced genes on visual system plasticity. For instance, our studies of Homer, Arc and CPG15 demonstrate that each has distinct roles in controlling neuronal plasticity. Homer is a widely expressed scaffold protein, which affects calcium signaling and metabotropic glutamate receptor (mGluR) signaling. In addition to finding a role in axon guidance (Foa et al., 2001; Foa et al., 2005), our more recent work indicates that experience-dependent changes in postsynaptic Homer expression regulates mGluR-mediated plasticity of retinotectal transmission (Van Keuren-Jensen & Cline 2006). This is particularly interesting in light of recent work suggesting that mGluR-mediated plasticity of synaptic transmission may play a role in developmental neurological disorders such as Fragile X. CPG15, another activity-induced protein, is noteworthy because it a GPI-linked signaling molecule whose expression results in a large increase in dendritic arbor development, coupled with an increase in glutamatergic retinotectal synaptic maturation and a coordinated elaboration of presynaptic retinal axon arbors (Nedivi et al., 1998; Cantallops et al., 2000; Nedivi et al., 2001). CPG15 mediates these changes by promoting synapse formation, which in turn enhances axonal arbor growth (Javaherian and Cline, 2005). We have recently discovered another interesting property of CPG15: the protein is harbored in vesicles within growing axons and using the pH-sensitive GFP variant tagged to CPG15, we found that retinal ganglion cell activity in the intact tadpole increases surface expression of CPG15. These data suggest that CPG15 is akin to a membrane-tethered activity-induced growth factor. It now appears that many activity induced genes, including Arc (Rial Verde et al 2006) and Homer, function in a homeostatic manner to maintain synaptic strength within a functional operating range, despite experience-dependent increases or decreases in synaptic strength.

The goal of this body of work is to generate a comprehensive understanding of the role of experience in shaping brain development. We have taken a multidisciplinary approach to this question which has successfully revealed the complexity of brain development. Our experiments use a combination of molecular/genetic manipulation and quantitative observations of structural and functional plasticity in response to visual stimulation. Our experiments have demonstrated a diverse range of effects of visual activity on the development and plasticity of the visual system and have the potential to reveal both direct and homeostatic mechanisms of circuit development.

Research Projects

The Dynamic Connectome
A thorough understanding of circuit development and brain function requires knowledge of the connectivity of brain networks. In vivo time-lapse imaging of dendritic and axonal structures have shown that they are dynamic over the timecourse of hours and days. Importantly the structural dynamics are the physical substrate of synaptic dynamics and therefore dynamics in the connectivity map. We have extensive experience in using molecular genetic approaches combined with in vivo timelapse imaging and electrophysiology to identify activity-dependent mechanisms governing dynamic neuronal structure and connectivity in the developing visual system. We have also assessed the dynamics in the connectivity map of the optic tectum by combining in vivo time lapse imaging with serial section electron microscopy and three-dimensional reconstruction of optic tectal neuronal dendrites and axons. For these experiments we co-express GFP and a membrane-targeted horseradish peroxidase, mHRP. We use 2 photon time lapse imaging of GFP expressing neurons in living animals to identify dynamic branches within neurons and serial section electron microscopy (EM) of the same neuron, visualized by mHRP expression, to generate 3 dimensional reconstructions of labeled neurons and their synaptic partners. By comparing the live imaging data and the serial section EM data, we determine the synaptic connectivity and ultrastructural synaptic features of dynamics and stable dendritic and axonal branches. This type of analysis allows us to identify stable and dynamic components of the connectome and to determine how connectivity changes with experience and under conditions that model human neurodevelopemental diseases.

Regulation of Neurogenesis
The development of brain networks depends of the spatial and temporal control of cell proliferation and differentiation. We have recently begun several projects to investigate the control of neurogenesis in the tadpole brain. A significant advantage of studying neurogenesis in tadpoles is that the animals develop externally so the entire process from progenitor cell proliferation to neuronal differentiation and integration into brain circuits can be easily visualized and manipulated in the intact animal. We have found that visual experience controls neurogenesis and we have conducted microarray analysis to identify candidate molecular genetic pathways through which sensory experience controls neurogenesis.

Balanced inhibition in Visual circuit function and behavior
The retinotectal system processes and integrates visual and mechanosensory information and controls behavioral responses to sensory inputs. The interaction between excitatory and inhibitory synaptic inputs is essential for these brain functions. Excitatory synaptic activity is balanced by inhibitory synaptic input throughout the CNS. Despite the widespread expectation that balanced inhibition to excitation is essential for circuit function and information processing, the consequences of manipulating the ratio of inhibition to excitation on information processing and behavior have not been directly tested. We are using in vivo imaging, electrophysiological methods and behavior combined with molecular genetic manipulations to identify mechanisms controlling the development of inhibitory and excitatory neurons and explore the consequences of disrupting the balance of inhibition to excitation in the intact brain. Our studies suggest that disrupted the balance of inhibition to excitation in neurons in the optic tectum affects visual system function and visually-guided behaviors in ways that may be akin to deficits in information processing seen in neurodevelopmental disorders such as autism spectrum disorders.