UC Berkeley

Synaptic scaling is a mechanism that neurons use to maintain homeostatic levels of excitability, allowing them to increase their sensitivity when excitatory inputs are weak and to decrease sensitivity at times of elevated excitation. In this way individual neurons and whole neural circuits are thought to maintain a balance between excitation and inhibition, and to operate in a part of their dynamic range that allows for their output to increase and decrease in response to altered inputs. So far, scaling has been studied as cell-wide responses to long-term genetic and pharamcological manipulations of excitability in an entire cell, group of cells, or in whole brain regions. However, under physiological conditions scaling will be elicited not by these kind of global changes in input or excitability, but rather by changes in synaptic drive at a subset of inputs. This thesis sets out to determine whether local excitation elicits scaling responses and, if so, whether the changes would be confined to the site of excitation or would trigger a global response throughout the cell.

Our lab has developed LiGluR, a light-controlled glutamate receptor that rapidly and reliably depolarizes neurons, resulting in calcium influx and inducing action potential firing. In chapter 2 of this thesis I describe the optimization of subcellular LiGluR activation in cultured hippocampal neurons, discussing critical parameters for spatially restricting the illumination. Establishing a targeted illumination system and verifying the spatial resolution of light activation is essential for the experiments later described in chapter 3. The conditions for long-term light activation are also set to take advantage of the noninvasive nature of LiGluR, thus enabling the study of processes that require prolonged, chronic manipulation. The light-activation protocol considers both the light for activation and the light for measuring output, determining maximum power levels for robust photostimuluation while maintaining good spatially resolution and cell health. Light activation is carried out on a high resolution scanning confocal microscope so that small features (such as synaptic receptors) can be properly visualized. I also discuss the expression pattern of LiGluR in neurons and illustrate a co-expression system for driving channel localization to postsynaptic sites.

In chapter 3, I describe LiGluR activation in combination with time-lapse imaging of AMPA receptors (AMPARs), a well established expression locus of different modes of synaptic plasticity. LiGluR is an ideal tool to build upon previous studies for its spatial and temporal properties. With targeted illumination I am able to activate LiGluR within a single neuron with sub-cellular resolution. By combining this approach with bath application of TTX, which blocks action potentials without affecting light-induced depolarization, the effect of LiGluR activation is further restricted. Synaptic scaling occurs on the time scale of hours; LiGluR is able to provide the reliable, long-term stimulation necessary to evoke these changes. The non-invasive nature of LiGluR is also ideal for coupling with time-lapse imaging of other fluorescently-tagged proteins that can be expressed within the same cell.

To measure synaptic scaling I monitored the surface expression of AMPA-type glutamate receptors using an ecliptic-tagged GluR2 subunit, a highly pH-sensitive reporter that is fluorescent when the receptor is surface expressed and quenched when it is exposed to the lumen of a vesicle. Expressing this receptor in cultured hippocampal neurons results in punctate surface expression concentrated at spines along the dendritic shaft. The fluorescence intensity increases in hippocampal neurons when culture-wide activity is blocked with TTX, verifying that the system is a faithful reporter of AMPAR accumulation under synaptic scaling conditions. We found that local excitation in a distal sub-region of the dendritic tree induced a global reduction in the surface population of AMPARs at dendritic spines. The loss of AMPARs began within 20-30 minutes and leveled off within 1 hour. This scaling response was blocked by chelating intracellular calcium with BAPTA-AM, blocking voltage dependent calcium channels with cadmium, or inhibiting the activity of calcium/calmodulin-dependent protein kinases with KN-93. Imaging of internal calcium with a fluorescent calcium indicator dye showed that the optical excitation of LiGluR in the distal dendrite does not lead to a calcium rise outside of the illuminated area, indicating that the spread of the homeostatic effect propagates via another means. We consider the possibility that phosphorylated CaMKII, which has been shown to be highly mobile, spreads throughout the dendrite to give a global homeostatic reduction in AMPA receptor surface levels.

Our method employed here for spatially-controlled neuronal stimulation coupled with time-lapse imaging can further be extended to examine other important questions underlying neuronal circuit function.