Local Versus Global Effects of Isoflurane Anesthesia on Visual Processing in the Fly Brain

SSVEP recordings before anesthesia (0% isoflurane). a, Schema of an experiment showing the electrode inserted laterally from the left. The left LED panel is shown flickering at 13 Hz, corresponding to the [13 off] flicker configuration. b, Exemplar mean bipolar rereferenced SSVEP, averaged over 10 trials in the [13 off] condition. The same data from one fly are presented in c and d in different formats. c, Exemplar baseline-corrected SSVEP power spectrum, averaged over the same 10 trials in b (S_EB(f); see Local field potential analysis). The blue and the red lines mark the first (f₁ = 13 Hz) and second (f₂ = 26 Hz) harmonic, respectively. d, Baseline-corrected SSVEP power at f₁ (blue) and f₂ (red) for the 10 trials of the [13 off] condition (S_EB(f_1/2)). Note that the narrow shaded areas represent the SD across 10 trials, showing the robust and repeatable nature of the SSVEP paradigm. The grouping into peripheral and central channels is depicted at the bottom. The channels are consistently aligned on the x-axis, b–d.

Baseline-corrected SSVEP power (S_EB) and coherence (C_EB) before anesthesia (0% isoflurane). a, b, Grouping trials according to ipsilateral and contralateral flicker configurations. A trial consisted of a presentation of one of the following eight flicker configurations: [off 13], [off 17], [13 off], [17 off], [13 13], [13 17], [17 13], and [17 17]. Trials were classified as either ipsilateral or contralateral according to the location of the flicker, with respect to electrode insertion site. a, When analyzing SSVEP power at f₁ = 13 Hz or f₂ = 26 Hz, trials in which a single flicker was shown ([13 0] and [0 13], row 1) were classified according to the location of the flicker. Trials in which different flickers are shown at each panel ([13 17], [17 13], row 2) are classified according to the location of the 13 Hz flicker. Trials in which both panels show the same flicker ([13 13], row 3) are classified as ipsilateral, as this component dominated the response (see c–f). Trials in which only a 17 Hz flicker is shown ([17 0], [0 17], and [17 17]) are excluded from the grouping. b, Corresponding classification scheme when analyzing SSVEP power at f₁ = 17 Hz or f₂ = 34 Hz. c–f, Group average (N = 13) baseline-corrected SSVEP power (S_EB; see Local field potential analysis) for f₁ = 13 (c), f₁ = 17 (d), f₂ = 26 (e), and f₂ = 34 (f) Hz for each of the eight flicker configurations. Grouping flicker configurations as ipsilateral or contralateral accounted for much of the variance, as indicated by the color code (see legend). Error bars represent the SEM across flies (N = 13). g, Group average (N = 13) baseline-corrected SSVEP power at f₁ and f₂ for ipsilateral and contralateral flicker configurations. Shaded area represents the SEM across flies. h, Group average (N = 13) baseline-corrected SSVEP coherence (C_EB) for P, CP, and C channel pairs. Schematics of the fly brain with superimposed examples of channel pairs from each grouping are shown at the bottom. SSVEP coherence followed a similar trend to SSVEP power: higher coherence at f₁ than f₂ and a decrease toward the center. Contralateral flickers evoked coherence predominantly at f₁. Error bars represent SEM across flies (N = 13).

Isoflurane anesthesia has region- and harmonic-dependent effects on SSVEP power. a, Experimental protocol. An experiment consisted of multiple blocks, each at a different concentration of isoflurane (top black line). Each block proceeded with (1) air puffs (light blue rectangles); (2) 30 s of rest; (3) 80 trials of flicker presentation, corresponding to 10 presentations for each of the eight flicker configurations (gray rectangles); (4) 30 s of rest; (5) air puff; (6) isoflurane concentration change; and (7) 180 s of rest for adjustment to the new isoflurane concentration. b, Isoflurane abolishes behavioral responses. Consecutive video frames (first and second row) in response to an air puff before any anesthesia was administered (0%, left column) and before 0.6% isoflurane exposure (right column). In 0% isoflurane, flies respond to the air puff by moving, which is seen as the large difference in pixel intensity between consecutive frames (left, third row). After 0.6% isoflurane exposure, flies do not respond to the air puff, and there are only small differences between consecutive frames (right, third row). c, Quantifying behavioral responses. Group average (N = 13) movement index (see Movement analysis) was reduced during exposure to 0.6% isoflurane and rebounded after isoflurane levels were reset to 0%. Error bars represent the SEM across flies. d, Isoflurane reduces spontaneous brain activity (ΔS_s), measured over four segments of 2.3 s before the start of the presentation of the visual stimuli. Group average (N = 13) effect of 0.6% isoflurane on spontaneous power (ΔS^0.6_S; see Local field potential analysis). Power is averaged across all channels. The average power for 20–30 and 80–90 Hz is significantly reduced. Error bars represent the SEM across flies. e, Isoflurane reduces SSVEP power (ΔS_E) at f₁ but increases power at f₂ in a concentration-dependent manner. SSVEP power at f₁ = 13 Hz (blue) and f₂ = 26 Hz (red) for the [13 off] flicker configuration (indicated by the schematic above), at increasing concentrations of isoflurane. For each fly, the SSVEP is first averaged over peripheral channels (triangles, channels 1–6) or central channels (circles, channels 9–14). The channel average is further averaged across flies (N = 3). Error bars reflect the SEM across flies. f, Isoflurane increases SSVEP power at f₂ for ipsilateral but not for contralateral flicker configurations. Spatial profile of SSVEP power at f₁ (blue) and f₂ (red) for contralateral (dark) and ipsilateral (light) flicker configurations in 0.6% isoflurane (ΔS^0.6_E). SSVEP power is averaged across ipsilateral or contralateral flicker configurations (Fig. 3a,b) first, then is averaged across flies (N = 13). Shaded areas represent the SEM across flies. The SSVEP power at f₁ is reduced in central channels for all flicker configurations, indicating an effect on global neural processing. In contrast, SSVEP power at f₂ is increased at the periphery, but only for ipsilateral flicker configurations, indicating an effect on local neural processing. The peripheral and central channels over which the average was taken in e are depicted at the bottom of f. ***p < 0.001 and **p < 0.01 in c and d.

A minimal model explains the unexpected increase in SSVEP power at f₂ due to isoflurane. a, Modeling the SSVEPs. The input (depicted as a blue square wave) is (linearly) differentiated to extract points of luminance increments and decrements before splitting into two streams corresponding to the On (pink) and Off (orange) pathways. Each pathway is modeled as a linear operation determined by the impulse response of the pathway. The responses of the two pathways are summed to give the recorded SSVEP. b, The impulse responses of the On and Off pathways were estimated from the response to a 1 Hz flickering stimulus (blue). An example from one channel is shown. No other parameters are fitted from the data. c, Exemplar On (pink) and Off (orange) impulse responses obtained from the 1 Hz flicker presented in both panels [1 1] in 0% isoflurane (air). Note that the negative of the On impulse response (gray) is not identical to the Off impulse response. d, The power spectra of the model output. When the negative of the On impulse response is the same as the Off impulse response, there is no power at f₂ (gray). When the empirical On and Off impulse responses are used, the power spectrum has a sharp peak at f₂ (black). e, Comparison between the model output (black) and the recorded SSVEP (blue, average across 10 trials) to a [13 0] stimulus in 0% (left) and 0.6% isoflurane (right) in the time domain. An example from one channel is shown. f, Corresponding comparison to e in the frequency domain. Spectra of model output (left) and recorded data (right, averaged across 10 trials of the [13 0] flicker configuration) in 0% (green) and 0.6% (blue) isoflurane show that the model correctly predicts that isoflurane increases SSVEP power at f_2. g, The SSVEP model predictions are in excellent agreement with the observed effects of isoflurane. The model correctly predicts the reduction in power at f₁ (blue) and the increase in power at f₂ (red) for each of three flies (marked by a cross, square, or asterisk), across all channels (14) and both flicker configuration ([13 13] or [17 17]; n = 168, ρ = 0.76). The empirical line of best fit (dashed black) closely resembles the line of perfect fit (solid black).