Methods for voltage-sensitive dye imaging of rat cortical activity with high signal-to-noise ratio

Abstract

We describe methods to achieve high sensitivity in voltage-sensitive dye (VSD) imaging from rat barrel and visual cortices in vivo with the use of a blue dye RH1691 and a high dynamic range imaging device (photodiode array). With an improved staining protocol and an off-line procedure to remove pulsation artifact, the sensitivity of VSD recording is comparable with that of local field potential recording from the same location. With this sensitivity, one can record from approximately 500 individual detectors, each covering an area of cortical tissue 160 microm in diameter (total imaging field approximately 4 mm in diameter) and a temporal resolution of 1,600 frames/s, without multiple-trial averaging. We can record 80-100 trials of intermittent 10-s trials from each imaging field before the VSD signal reduces to one half of its initial amplitude because of bleaching and wash-out. Taken together, the methods described in this report provide a useful tool for visualizing evoked and spontaneous waves from rodent cortex.

Figure 1. Schematic drawing of the recording apparatus

Light from the tungsten lamp was filtered with a 630 ± 15 nm band pass filter and reflected via a 655 nm dichroic mirror for illumination. The filament of the lamp is focused onto the back focal plane of the macroscope, to achieve Köhler illumination. Fluorescent light from the cortex is collected with the macroscope, passed through the 695 nm long-pass filter, and forms a real image onto the aperture of the diode array. The field of view on the cortex is approximately 4 mm in diameter. The numerical aperture of the macroscope is about 0.45, with optical magnification of 5X and working distance approximately 20 mm.

Figure 2. Sensitivity of VSD recordings

The top two traces in each panel are local field potential and VSD recordings from the same location in the visual cortex. Another VSD recording (bottom trace in each panel) was 2 mm away from this location. A. Under 1.5% isoflurane, there are spontaneous spindle-like bursts. Dots mark events in which VSD has higher amplitude than local field potential. Triangles mark events in which local field potential has higher amplitude than VSD. Diamonds mark events seen in local field potential but not in VSD. B. Recordings from the same animal as in A, about 5 min later, with isoflurane anesthesia lowered to 1.1%. Most of the peaks in local field potential are also seen in VSD, but the correlation between the two signals is lower. The baseline fluctuations cannot be attributed to noise, since the analogous recordings had much less fluctuation when anesthesia was elevated (A).

Figure 3. Staining depth

A. Left, Visible light image of the stained cortex. Middle, fluorescence image from the same cortical section showing distribution of RH1691 staining. Right, fluorescence intensity (red) and integrated fluorescence intensity (blue). Broken lines mark the tissue depth with fluorescent intensity higher than 2/3 of the maximum intensity. B. Photograph of a stained cortical section. Arrows indicate boundary of the cranial window. Broken line indicates the border between gray and white matter. Staining of RH1691 can be seen by eye as a light blue hue.

Figure 4. Comparison of RH795 and RH1691

Recordings from two separate animals stained with RH795 and RH1691 (A and B, respectively). Epileptiform spikes induced by bicuculline had large signals in both VSD and local field potential recordings (top and middle traces on each panel). Pulsation artifact was time locked to the electrocardiogram (ECG, bottom traces on each panel). VSD signal from RH795-stained cortex had large pulsation artifact, comparable in amplitude to the epileptiform spike (A), while the artifact was much smaller in the VSD signal from RH1691-stained cortex.

Figure 5. Pulsation artifact of RH1691

VSD signals from three locations of visual cortex are shown (Optical 1-3, black traces on the top). Visually evoked cortical activity was seen at all three locations when the contralateral eye was stimulated by a light flash (stim). The gray trace under each raw black trace is the pulsation artifact, reconstructed with an ECG triggered averaging procedure (see text). Note that in one location (Optical 1), there was virtually no pulsation artifact. The pulsation artifact is time-locked to the ECG and contains virtually no components from the recorded activity. After subtraction, the signals from the three locations had similar waveform and contained almost no artifact (bottom three traces).

Figure 6. Total recording time

A. Amplitude of VSD signal (bicuculline induced spikes) is plotted against light exposure time. Data from four animals were normalized to the amplitude of the first recording trial. In three animals (diamonds, triangles, squares), the signal amplitude remained stable (or slightly increased) for a period (flat period). After the flat period, the signal had a higher rate of decline with light exposure (declining period). Note that one animal did not have optimum staining, and the stable time was much shorter (stars). B. Data from another animal, in which part of the cortex was shielded from light exposure. In the shielded area, the signal amplitude remained unchanged (solid diamonds), while signal in unshielded area declined (empty diamonds), suggesting that the signal decline was related to light exposure. The shielded area did not have a flat period after the shielding was removed, suggesting that the flat period may be related to dye wash-out.

Figure 7. Sensory evoked response

A. Signal from a single detector viewing a cortical area of 160 μm in diameter near the principal barrel in the barrel cortex. Ten trials are displayed from 105 trials with identical whisker stimuli. B. Signal from a single detector viewing a cortical area of 160 μm in diameter in the V1 area. Ten trials are displayed from ~30 trials recorded with identical light stimuli, to the contralateral eye.

Fig. 8. Propagating waves in barrel and visual cortices

The top traces in panels A and B are signals from a single detector in the recording field. Bars under each trace mark the time of a spontaneous event (spt) and an evoked response (evk) during the recording trial. The vertical broken lines mark the time of stimulation. The bottom images in each panel are frames showing the spatiotemporal patterns of the evoked (top row) and spontaneous events (bottom row). The imaging field is ~4 mm in diameter. Each image is a 0.625 ms snap shot, with every 8th frame displayed (5 ms frame intervals). The bars under the top trace mark the duration of the images. A. In barrel cortex, the evoked response started from the principal barrel and spread as a propagating wave to the entire imaging field within 15 ms. The spontaneous event started from the bottom of the field and propagated as a slow wave across the field. B. In visual cortex, the sensory evoked wave is slower than that in the barrel cortex. Note that the spontaneous wave in visual cortex was initiated from the bottom of the field, propagated upward and reflected near the top to propagate downward.

Fig. 9. Trial-to-trial variations in barrel cortex

Image rows 1-10: Ten consecutive trials with identical whisker deflection. The bottom images (AVG) are averaged from 105 trials from the same animal. Bottom row left: schematic diagram of the barrel pattern and the imaging field. Bottom row right: Isochromatic contour lines were superimposed from trials 5 (red), 6 (orange), 8 (green) and 10 (blue), along with contour lines from the averaged data (gray).