Calibration of Sub-Threshold Evoked EPSPs in Dendritic Spines using Voltage-Sensitive Dyes

Abstract

Dendritic spines are specialized structures in the brain that receive excitatory synaptic inputs. Activation of synaptic receptors on spines leads to current influx that depolarizes the activated spine, the parent dendrite and the neuron's somatic and axonal compartments. Local membrane potential fluctuations affect voltage-dependent channels producing calcium influx in an interplay of electrical and biochemical signaling. We combine 2-photon glutamate uncaging with 2-photon voltage-sensitive dye (VSD) imaging to study changes in membrane potential in individual spines. We compare two methods of calibrating these signals in spines that may be spatially distant from the somatic compartment. Subthreshold calibration signals have limited amplitude, and actual amplitudes can fall short of the expected values due to patch clamp limitations and space clamp effects, both leading to over-estimated EPSP amplitudes. Backpropagating action potentials have large amplitude but an average attenuation decay rate must be incorporated leading to some uncertainty. We measure the small but significant nonlinearity in dye response that would lead to under-estimated EPSP amplitudes if not accounted for. Calibrated evoked spine EPSP (spEPSP) amplitudes, along with somatic EPSP (soEPSP) amplitudes are input to a compartmental model (NEURON) to predict spine neck resistance (Rneck) values that are consistent with our measurements. While highly variable, predicted Rneck is centered on 200MOhm and is consistent with independent estimates based on FRAP experiments using a cytosolic dye in the absence of the VSD.