Automated Remote Focusing, Drift Correction, and Photostimulation to Evaluate Structural Plasticity in Dendritic Spines

Abstract

Long-term structural plasticity of dendritic spines plays a key role in synaptic plasticity, the cellular basis for learning and memory. The biochemical step is mediated by a complex network of signaling proteins in spines. Two-photon imaging techniques combined with two-photon glutamate uncaging allows researchers to induce and quantify structural plasticity in single dendritic spines. However, this method is laborious and slow, making it unsuitable for high throughput screening of factors necessary for structural plasticity. Here we introduce a MATLAB-based module built for Scanimage to automatically track, image, and stimulate multiple dendritic spines. We implemented an electrically tunable lens in combination with a drift correction algorithm to rapidly and continuously track targeted spines and correct sample movements. With a straightforward user interface to design custom multi-position experiments, we were able to adequately image and produce targeted plasticity in multiple dendritic spines using glutamate uncaging. Our methods are inexpensive, open source, and provides up to a five-fold increase in throughput for quantifying structural plasticity of dendritic spines.

RGS14 Restricts Plasticity in Hippocampal CA2 by Limiting Postsynaptic Calcium Signaling

“…3 ). To optimize data collection from each neuron during long experiments, we used an automated method to image multiple dendritic spine positions simultaneously ( Smirnov et al, 2017 ). This interface tracks coordinates of multiple dendritic segments on the same neuron and employs autofocus and drift-correction algorithms during the experiment to maintain optimal focus.…”

“…For sLTP experiments, images were acquired as a Z stack of five slices with 1-µm separation, averaging five frames/slice. Using multiposition imaging of spines with high-throughput automation, dendritic spines at four positions on separate secondary/tertiary dendrites were imaged simultaneously, employing algorithms for autofocusing and drift correction to maintain position and optimal focus during long imaging experiments as recently described ( Smirnov et al, 2017 ). Baseline images were acquired over 5 min before two-photon glutamate uncaging (1 min, 30 pulses at 0.5 Hz) followed by 30 min of imaging after uncaging.…”

RGS14 restricts plasticity in hippocampal CA2 by limiting postsynaptic calcium signaling

“…For sLTP experiments images were acquired as a Z stack of five slices with 1 µm separation, averaging five frames/slice. Using multi-position imaging of spines with high-throughput automation, dendritic spines at four positions on separate secondary/tertiary dendrites were imaged simultaneously employing algorithms for autofocusing and drift correction to maintain position and optimal focus during long imaging experiments as recently described (Smirnov et al, 2017). Baseline images were acquired over 5 mins prior to two-photon glutamate uncaging…”

“…We then performed two-photon fluorescence microscopy during two-photon glutamate uncaging to induce spine structural plasticity on secondary/tertiary apical dendrites of CA2 and CA1 pyramidal neurons (Figure 3). In order to optimize data collection from each neuron during long experiments, we utilized an automated method to image multiple dendritic spine positions simultaneously (Smirnov et al, 2017). This interface tracks coordinates of multiple dendritic segments on the same neuron and employs autofocus and drift correction algorithms during the experiment to maintain optimal focus.…”

Design and implementation of multi-signal and time-varying neural reconstructions

“…The positions of synapses have been detected with high spatial accuracy using mGRASP 37 . Integrated systems for two-photon imaging and photo-stimulation are well suited for systematic interrogations of structural plasticity 38 . Low-intensity live-imaging of fluorophore-tagged subcellular protein complexes allow long-term tracking of mitochondrial trafficking in neurites 39 .…”