Anesthetic effects on electrophysiological responses across the visual pathway

Abstract

Anesthetics are widely used in electrophysiological tests to assess retinal and visual system functions to avoid experimental errors caused by movement and stress in experimental animals. To determine the most suitable anesthetic for visual electrophysiological tests, excluding ketamine and chloral hydrate due to regulatory and side effect concerns, this study investigated the effects of ethyl carbamate (EC), avertin (AR), and pentobarbital sodium (PS) on visual signal conduction in the retina and primary visual cortex. Assessments included flash electroretinogram (FERG), pattern electroretinogram (PERG), pattern visual evoked potentials (PVEP), and flash visual evoked potentials (FVEP), FERG and FVEP were used to evaluate the responses of the retina and visual cortex to flash stimuli, respectively, while PERG and PVEP assessed responses to pattern stimuli. The research showed that AR demonstrates the least disruption to the visual signal pathway, as evidenced by consistently high characteristic peaks in the AR group across various tests. In contrast, mice given EC exhibited the lowest peak values in both FERG and FVEP, while subjects anesthetized with PS showed suppressed oscillatory potentials and PERG responses. Notably, substantial PVEP characteristic peaks were observed only in mice anesthetized with AR. Consequently, among the three anesthetics tested, AR is the most suitable for visual electrophysiological studies.

Fig. 1

Scotopic ERG and a-wave Fitting. (A) Representative scotopic and photopic ERG responses in mice treated with different anesthetics. (B) The scotopic and photopic ERG waveforms from different groups were processed with a band-pass filter of 0.3–30 Hz to remove the influence of oscillatory potentials. (C–E) A-wave fitting waveforms for EC (C), AR (D), and PS (E). (F–H) Quantitative statistics of the a-wave related parameters, R_max (F), S (G), and T_d (H), derived from fitting with the Lamb and Pugh model. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 2

Fitting of scotopic ERG b-waves. (A–C) Representative scotopic ERG b-waves of EC (A), AR (B) and PS (C). ERGs were recorded in dark-adapted mice with the light intensities increasing from − 3.6 to 2.15 log cd s m⁻². (D–F) B-wave fitting waveforms of EC (D), AR (E) and PS (F). (G–I) Statistical analysis of the b-wave parameters, R_max (G), N (H), and K (I), obtained by applying the Naka-Rushton equation. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple com-parisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 3

Record of the oscillatory potentials. (A) Representative waveforms of oscillatory potentials (OPs) in three groups were recorded under dark adaptation, in response to a flash with a luminance of 1.4 log cd s m⁻². (B) The frequency-domain waveforms of oscillatory potentials (OPs) for three groups: EC (gray), AR (blue), and PS (green). (C, D) Quantitative statistics of the implicit time (C) and amplitude (D) of OPs across three groups. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 4

Analysis of photopic ERG b-waves. (A–C) Exemplary photopic ERG b-waves were recorded from EC (A), AR (B), and PS (C) mice, with the light intensities progressively increasing from − 0.6 to 1.4 log cd s m⁻² against a background illumination of 1.48 log cd s m⁻². (D–F) Curves depicting the fitted b-wave responses for EC (D), AR (E), and PS (F). (G–I) Quantitative evaluation of b-wave parameters, such as R (G), N (H), and K (I), derived through the application of the Naka-Rushton equation. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple com-parisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 5

Characterization of PERG under varied anesthetic conditions in mice. (A) Schematic of the PERG experiment. (B) The waveforms of PERG results in mice under the influence of three different anesthetics: EC, AR, and PS, are illustrated. (C–D) The implicit times (C) and amplitude (D) for N1, P1, and N2 waves among the groups are quantified. (E–M) Statistical analysis of parameters following frequency-domain analysis, including magnitude (E–G), phase (H–J), and power spectral density (PSD) (K–M). Compared to the other two groups, the PS group mice (G, J, M) demonstrated insensitivity to graphic stimuli when compared with the EC (E, H, K) and AR (F, I, L) groups. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple com-parisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 6

The effects of different anesthetics on FVEP differ between anesthetics. (A) Representative FVEP waveforms from distinct groups: EC (bottom), AR (middle), and PS (top), elicited under a luminance of − 0.3 log cd s m⁻². (B, C) The density plots show the density distribution of N1 (B) and P2 (C) amplitudes across various groups in their FVEP responses. (D–F) Latencies of P1 (D), N1 (E), and P2 (F) in different groups. (G–I) Peak amplitudes for P1 (G), N1 (H), and P2 (I) in different groups. Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple com-parisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.

Fig. 7

Evaluation of PVEP Outcomes. (A) Schematic representation of PVEP waveforms in EC (left), AR (center), and PS (right) mice. (B–C) Statistical analysis of the N1 (B) and P2 amplitudes (C) in the PVEP of AR mice. (D–I) Frequency domain analysis of the PVEP results for the AR group, including magnitude (D, E), phase (F, G), and power spectral density (PSD) (H, I). Frequency domain analysis results are statistically analyzed for recordings at a depth of 800 μm (D, F, H) and across all depths (E, G, I). Representative data are presented as mean ± SEM; n = 6 independent biological replicates. Two-way ANOVA tests with Tukey’s multiple com-parisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were independently performed at least three times to ensure repeatable results.