In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording

Abstract

In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V(m) can accurately represent population V(m) changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.

Figure 1

VSD setup, validation and calibration of whole-cell current-clamp recordings (I-clamp recording) with VSD response. The epifluorescent-based imaging setup is illustrated in (a). The slice is placed in an interface chamber under a long working distance objective, allowing access to the slice for stimulating and recording electrodes, including whole-cell patch electrodes. The slice is illuminated by an intense (200 W xenon) light source that is directed through a dichroic filter cube. The emitted fluorescence signal is then projected onto the CCD chip for acquisition. To limit bleaching and phototoxicity, a shutter is placed at the light source (not shown) and triggered to open 200 ms before acquisition. Images are typically acquired into memory and then transferred to a disc drive for analysis (though direct writing to disk is possible with some cameras, including the NeuroCCD by RedShirtImaging). Validating that the VSD response is predominantly measuring voltage change in principal cells is demonstrated in (b), where an evoked response recorded by VSD (red trace) is overlaid on a dendritic I-clamp recording from a CA1 pyramidal cell. The VSD response is averaged over a small region of interest adjacent to the tip of the electrode, which is patched onto the dendrite. Similar kinetics of the scaled VSD image and the simultaneous I-clamp recording (black trace) is evident in B. With the simultaneously acquired data, we can plot change in fluorescence with respect to change in voltage for each point acquired by the camera (c). The resultant linear relationship, over a physiologically relevant scale (−5 to +30 ΔV_m), demonstrates that whole-cell recordings can be used to calibrate the relationship of fluorescent change to transmembrane voltage deviations. Over a population of cells, this relationship clusters around approximately 0.1% df/f_i for each 10 mV voltage change (d); thus, even in areas or slices without direct whole-cell recordings, we have a measure of the magnitude of the voltage response (similar results in a larger number of cells is published for the dye RH795) (see ref. 6). *Neuro CCD-SM, RedShirtImaging, Decatur, GA.

Figure 2

Analysis and display of VSD data. The gray scale image displayed in (a) shows a single frame from the RedShirtImaging camera. As shown in this image, anatomical features produce relatively large differences in intensity. To identify changes in voltage, analysis begins by normalizing the data by subtracting a reference frame (f_i) from all other frames (delta f, or df), followed by division by the reference frame (df/f_i). Because of the small signal size presently inherent in VSD imaging, noise and dye bleaching during recording produce a strong nonspecific component of the raw signal (b). Averaged response of pixel intensities over time from a region of interest (ROI) encompassing a portion of the dentate blade (raw DG) as well as a trace from the inactive, unstimulated neuropil outside of the hippocampus (Raw bkg) shows a strong exponential decay of fluorescence, and, in this case, the presence of mechanical oscillations in the first quarter of the trace, probably due to residual vibrations from the shutter opening. Because both the bleaching and the effects of vibrations scale with intensity, the most effective way to remove them is to subtract a background signal from an area unaffected by the stimulus (b; bkg). When this is not possible, subtraction of an exponential fit to the baseline period (b; Exp fit; in red) of the recording, or subtraction of an interleaved non-stimulated control recording will also correct for bleaching. To match I-clamp conventions (c), signals are usually inverted so that a depolarization is upward (dye signal intensity decreases as membrane potential depolarizes). Because the imaging data are collected as a set of 2D images over time, enhancing characterization of various spatial, voltage or temporal aspects of the recording requires that data display be reduced from 4 dimensions (space (x and y), time and voltage) to 2 or 3 dimensions. 2D traces (voltage change over time) as shown in (b) and (d) are familiar to electrophysiologists and, by averaging, many pixels can show a strong signal to noise. However, similar changes in amplitude over time can also be represented in 3D raster scans as shown in (e). In a raster scan, changes in VSD signal along a line, for each time point, reveal changes in voltage (high intensities are red, and a color look-up table provides the actual intensity), with the addition of an axis coding for space. Using this display, where activity occurs along that line over time and how it propagates can be easily visualized. To show the spatial extent of a response, changes in fluorescence from baseline at a specific time point can also be displayed in the same orientation as the gray scale image (f). For this type of a 3D snapshot display, at the peak of the first response, activity is clearly limited to the dentate gyrus.

Figure 3

Unlike other electrophysiological measures of neuronal population activity, VSD imaging can directly measure the spatiotemporal dynamics of inhibitory responses by reporting hyperpolarization. This is dramatically demonstrated by an alveus stimulation that activates output axons arising from area CA1 pyramidal cells, which in turn activate feedback interneurons and generate a strong feedback IPSP. Action potentials generated by this stimulation propagate back to the neurons (producing a depolarization; red in (a)), but also via axon collaterals to excitatory synapses that impinge onto local inhibitory neurons. Activating these neurons produces the equivalent of feedback inhibition throughout area CA1, producing a hyperpolarization (blue in (b)). The spatiotemporal dynamics of this response can be described purely spatially as in (a) and (b), but can also be portrayed by comparing traces averaged over ROIs from specific anatomical areas (c). These inhibitory responses from intact neurons can show different kinetics than records from I-clamp recordings (dendritic I-clamp; (c)), as IPSPs are very sensitive to internal anion concentrations (perturbed in whole-cell patch) and holding potential. As many signal transduction pathways underlying synaptic plasticity are also lost during whole-cell patch recordings, the potential for efficiently studying neuronal circuitry with the neuron’s intracellular milieu and therefore these transduction pathways intact is another important feature of VSD imaging with great promise.