Forebrain Acetylcholine Modulates Isoflurane and Ketamine Anesthesia in Adult Mice

All procedures were approved by the Animal Care Committee of the University of Western Ontario (London, Canada) and conducted according to the guidelines of the Canadian Council for Animal Care (Ottawa, Canada). The experiments were performed on male mice 3 to 9 months old. Two groups of mutant mice and a control group, referred to as wild-type, were used. The first group of mutants, the heterozygous knockdown mice, showed ~45% decreased vesicular acetylcholine transporter expression in the brain¹⁵ ; these mice were backcrossed with C57BL/6J animals for five generations.¹⁵  The second group of mice, named forebrain knockout mice, had homozygous vesicular acetylcholine transporter deletion restricted to the forebrain. Forebrain knockout mice were generated by crossing a Nkx2.1-Cre mouse line (C57BL/6J-Tg[Nkx2-1- cre]2Sand/J) with mice in which both alleles of the vesicular acetylcholine transporter gene were floxed and then backcrossed with C57BL/6J mice for five generations.¹⁶  Control mice had intact vesicular acetylcholine transporter genes. The mice were housed three or four per cage under climate-controlled conditions (21 to 23°C) with a 12-h light/dark cycle and lights on at 7:00 am and had access to water and regular mouse chow ad libitum.

Loss of the righting reflex was used as the behavioral endpoint to investigate the hypnotic properties of isoflurane (Forane, Baxter Corporation, Canada), following previously described methods with slight modifications.²⁴  To determine the isoflurane concentration that induced loss of the righting reflex, each mouse was placed in a small Plexiglas chamber (23 × 12 × 12 cm) connected to an isoflurane vaporizer with 1 l/min flow of 100% oxygen. The floor of the chamber was warmed to 36°C by a heating pad with a feedback control. The outflow from the chamber was connected to an infrared gas analyzer (RGM5250, Ohmeda, USA), with accuracy of 0.1% for isoflurane. Isoflurane was administered to the chamber starting at 0.5% concentration and was incremented by 0.125% until loss of the righting reflex occurred in the mouse. The concentration of isoflurane was shown to reach a plateau in less than 10 min after a change of dose, as verified by the infrared gas analyzer. Each concentration of anesthetic was maintained for an equilibration period of 15 min, after which the chamber was rotated to place the mouse on its back. An animal was considered to show loss of the righting reflex if it did not turn onto all four feet within 30 s, confirmed by a subsequent trial. The dose that induced loss of the righting reflex will be called the threshold loss-of-righting-reflex dose. The percentage of mice showing loss of the righting reflex at each dose of isoflurane was established for wild-type, heterozygous knockdown, and forebrain knockout mice, and ED₅₀ was estimated from the dose-response equation described under “Statistical Analysis.” Isoflurane administration started between 10:30 am and 3:30 pm.

Anesthetic responses to ketamine were studied using cumulative intraperitoneal doses, as described previously.²⁵  A mouse was given an initial dose of ketamine at 100 mg/kg intraperitoneally, and then increments of 20 mg/kg were injected intraperitoneally at 10-min intervals. At 8 to 10 min after each incremental dose, righting was tested by placing a mouse in a supine position, and loss of the righting reflex was defined if the mouse was unable to turn from the supine position to land on its feet in two trials separated by 30 s. Ketamine administration started between 10:30 am and 3:30 pm. Anesthetic (isoflurane and ketamine) sensitivity of each group of mice (wild-type, heterozygous knockdown, or forebrain knockout) was tested in different batches (two to eight mice per batch) on different days, interleaving with other groups in a quasirandom order. Each mouse was tested for isoflurane or ketamine only once. Most mice were given ketamine after isoflurane, separated by at least 4 days. The experimenter was not blinded to the identity of the mice. Based on previous literature, 10 to 20 mice per group was adequate for determination of ED₅₀ (loss of the righting reflex) of isoflurane²⁶  and ketamine²⁷  in mice.

Five control mice, seven forebrain knockout mice, and eight heterozygous knockdown mice were implanted with depth electrodes. Under ketamine–xylazine anesthesia, a pair of electrodes named H1 and H2 with ~0.6-mm separation was placed in both left and right dorsal hippocampus (3.8 mm posterior to bregma, 2.8 mm lateral to midline, and ~3 mm ventral to dura; coordinates were from the atlas of Franklin and Paxinos²⁸ ); the right and left sides were distinguished if needed. Distal hippocampus H1 electrode was targeted at the distal dendritic layers of CA1 (stratum lacunosum-moleculare or distal stratum radiatum), using electrophysiologic criteria,²⁹  which placed “hippocampal alveus” H2 electrode at the CA1 alveus or layer VI of the somatosensory cortex. A single electrode was placed in layer V of the right frontal cortex at 2 mm anterior to bregma, 2 mm lateral to midline, and 1.5 mm ventral to dura. Each electrode comprised a 125-µm stainless steel wire insulated with Teflon, except at the cut tip. A jeweler’s screw was placed epidurally over the cerebellum. All electrodes and screws were fixed onto the skull with dental cement. Field potential was recorded monopolarly from one electrode, with the cerebellar screw serving as both reference and ground.

For all mice, habituation to the recording setup started 7 days after electrode implantation. At 1 to 4 weeks after surgery, loss of the righting reflex to increasing isoflurane doses was assessed while local field potentials were recorded from the hippocampus and frontal cortex. The signals were digitized as described under “Statistical Analysis.” Before anesthetic administration, each mouse was placed in a larger chamber (30 × 30 × 30 cm) and recorded for at least 2 min during awake immobility (no gross movements, head held up, and frontal local field potentials showed low-voltage fast activity) and during walking (when mouse walked, turned its body, and reared). For isoflurane anesthesia, the mouse was placed in a small Plexiglas chamber described above, and 0.5% isoflurane was delivered for 15 min, before incrementing by 0.125%, which was again maintained for 15 min, and the procedure was repeated until 1% isoflurane was reached. Local field potentials were recorded for 2 min, starting 13 min after each isoflurane concentration. The dose of isoflurane that induced loss of the righting reflex was recorded.

Ketamine dose response in the same group of implanted mice was done at least 4 days after isoflurane administration. Each mouse was placed in a large (30 × 30 × 30 cm) chamber, and local field potentials were first recorded during awake immobility and walking during baseline. An initial dose of ketamine of 100 mg/kg intraperitoneally was injected, and field potentials and behavior were recorded 8 to 10 min after. Incremental doses of 20 mg/kg intraperitoneal ketamine were then given, and recordings made 8 min after, until loss of the righting reflex was indicated. Because all mice received 120 mg/kg intraperitoneal ketamine (except one forebrain knockout mouse in which 100 mg/kg intraperitoneal induced loss of the righting reflex), the statistical analysis of local field potentials to ketamine dose was limited to 120 mg/kg intraperitoneal. Five to seven mice per group was an adequate sample size for spectral power analysis in previous studies of isoflurane on the hippocampus of rats.^9,29 

At the end of the experiments, the mice were deeply anesthetized with pentobarbital and perfused through the heart with 400 ml of cold saline followed by 500 ml of cold 4% formaldehyde solution in 0.1 M phosphate buffer (pH 7.4). The brain was removed and postfixed in the latter solution at 4°C. Using a freezing microtome, the brain was sectioned at 40 µm, and electrode placements in the hippocampus and frontal cortex were histologically verified in thionin-stained brain sections.

Power spectral analysis of field potentials was performed by custom software.³⁰  Hippocampal and frontal cortex signals from the rat were amplified and filtered between 0.3 Hz and 0.3 kHz (first-order bandpass filter) by a Grass 7P511 amplifier (Astro-Med, USA). Local field potentials were then fed into a Data Translation D303 analog-to-digital converter (Measurement Computing Corporation, USA) and sampled and stored at 1 kHz by custom-made scripts written in SciWorks 7 (DataWave Technologies, USA). Artifact-free signals were manually selected. Each field potential segment consisted of 4,096 points (4.096 s), and one tenth of the total segment length was tapered by a cosine function ½ [1 − cos (10 πt/T)], where t is the time from each end, and T is the segment length. After fast Fourier transform, the spectral values were smoothed by an elliptical window falling off at [1 − (f_i/f_m)²]^0.5, where f_i is the deviation from the center frequency, and f_m indicates half the smoothing bandwidth = 2. Effectively, the frequency resolution is 0.244 Hz (reciprocal of 4.096 s), and after smoothing (2 * f_m + 1 = 5 frequency bins), statistically independent values were 1.22 Hz apart. Each spectral value derived from one segment is estimated to have 10 degrees of freedom, and the average spectrum of each immobile or anesthetic condition consisted of more than 25 segments, i.e., more than 250 degrees of freedom, and walking baseline condition consisted of more than 6 segments (more than 60 degrees of freedom). Power was determined in logarithmic units, and 5.7 log units of power corresponded to a 1-mV peak-to-peak sinusoidal wave. Cross-spectral measures²⁹  were presented as coherence and phase between local field potentials derived from two electrodes.

For a group of animals (wild-type, heterozygous knockdown, and forebrain knockout) under one condition (walking or immobility baseline or after an anesthetic dose), an ensemble logarithmic power spectrum was averaged across mice and compared pairwise between two conditions.³⁰  This comparison allows frequency-dependent differences to be revealed by distribution-free statistics. A Wilcoxon test (paired or unpaired as appropriate) was used to assess whether the absolute power at each digital frequency was different between two conditions with probability of P < 0.05 (two-tailed). At each anesthetic dose, the power deviation (difference of power at each dose minus that at baseline immobility) at each digital frequency was compared between two conditions; this was used to compare power deviations between two groups of mice. Spectral measures between the two conditions were considered statistically different from each other within a frequency range in which five contiguous digital frequencies (1.22 Hz range) showed P < 0.05 difference individually; this compensates for multiple comparisons.

At each recording electrode, the average integrated power of the local field potentials was determined by averaging the logarithmic power across bins within standardized frequency ranges: delta (0.9 to 4 Hz), theta (6 to 11 Hz), beta (13 to 30 Hz), low gamma (gamma 1, 30 to 57 Hz), high gamma (gamma 2, 63 to 100 Hz), and ripples (100 to 200 Hz). The theta band was operationally defined to exclude the immobility-associated hippocampal theta rhythm of 3 to 6 Hz that could occur after less than 0.75% isoflurane or 100 mg/kg ketamine. For evaluation of the dose response to isoflurane/ketamine of each mouse, the difference in average logarithmic power from baseline immobility (power deviation) was calculated for each frequency band (delta, theta, beta, low and high gamma, and ripples) at each anesthetic dose. The selection of a pair of H1–H2 electrodes depended on placement of the H1 electrode at the distal dendritic layer of CA1, as confirmed histologically, and by a walking theta H1–H2 phase of more than 90°. For gamma coherence and phase measures, H1–H2 electrode pairs with more than 90° gamma phase during baseline walking were selected. When measures (power, coherence, or phase) were available from both sides, which occurred in ~40% of the mice, the left and right measures were averaged to give one measure for each mouse. The average power deviation, coherence, or phase per mouse for different frequency bands at each electrode (H1, H2, and frontal cortex) or electrode pair (H1–H2 and H2–frontal cortex) was subjected to two-factor repeated-measures ANOVA (GB Stat, Dynamic Microsystems Inc., USA) with Greenhouse–Geisser adjustment (SAS 9.1.3; SAS, USA); the factors were group (wild-type, forebrain knockout, or heterozygous knockdown) and dose. Post hoc Newman–Keuls test was applied if the group or group × dose effect was statistically significant at P < 0.05 (two-tailed). For comparison of H1–H2 versus right H2–frontal cortex coherence, a three-factor repeated-measures ANOVA was used, with the additional factor being the electrode pair (intrahippocampal H1–H2 pair vs. “extrahippocampal” H2–frontal cortex pair), and only mice with both H1–H2 and H2–frontal cortex coherence measures were selected.

Prism software version 8.0 (GraphPad Prism Inc., USA) was used for curve fitting. ED₅₀ was determined by nonlinear regression of the population percent loss of righting versus anesthetic dose curve with the equation Y = Y_min + (Y_max − Y_min) /[1 + 10 ^log(ED₅₀^{– X) * m}], where Y is the percentage of the population showing loss of the righting reflex; Y_min and Y_max are the minimal and maximal values of Y, respectively; ED₅₀ is the drug dose at half (Y_max − Y_min); X is the logarithmic drug dose; and m is the Hill’s slope constant. Group measures of anesthesia sensitivity (age of mouse, anesthetic administration times, and threshold loss-of-righting-reflex dose) were also compared using nonparametric statistics Kruskal–Wallis one-way ANOVA or paired Wilcoxon (statistical criterion adjusted to P = 0.017 for three planned paired comparisons among three groups). The data were expressed as mean ± SD, with 95% CI. Number (n) refers to number of animals unless otherwise specified. P < 0.05 (two-tailed) was considered statistically significant.

The threshold dose of isoflurane that induced loss of the righting reflex was assessed in two different groups of mutant mice and compared to control wild-type mice (mice from Groups 1 and 2 in table 1 were combined). The isoflurane concentrations that induced loss of the righting reflex were 0.76 ± 0.08% (mean ± SD, n = 18), 0.78 ± 0.07% (n = 24), and 0.83 ± 0.07% (n = 23) for the homozygous forebrain knockout, heterozygous knockdown, and wild-type groups, respectively. The loss-of-righting-reflex dose was significantly lower for the forebrain knockout group (P = 0.005, unpaired Wilcoxon) but not the heterozygous knockdown group (P = 0.021, unpaired Wilcoxon, not meeting criterion of P = 0.017 for planned comparisons), as compared with the wild-type group (fig. 1A; table 2). Nonlinear curve fit using a sigmoidal dose response graph gave ED₅₀ of 0.76 (95% CI, 0.76 to 0.77; n = 23) for the wild-type group, which did not overlap with the CI estimate of ED₅₀ for the forebrain knockout group (mean ED₅₀, 0.69; 95% CI, 0.69 to 0.70; n = 18; table 2) or that for the heterozygous knockdown group (mean ED₅₀, 0.72; 95% CI, 0.72 to 0.72; n = 24; table 2; fig. 1B). The estimate of the Hill’s slope was not statistically different among mouse groups (table 2).

The ages (range, 3 to 9 months) of the mice (table 3) were not significantly different among the three mouse groups tested for isoflurane sensitivity (Kruskal–Wallis one-way ANOVA, degrees of freedom = 2, isoflurane chi-square test = 2.66, P = 0.264). The start time of isoflurane (0.5%) administration each day (range, 10:30 am to 3:30 pm) was not strictly controlled, and it turned out to be statistically different among the three groups (Kruskal–Wallis one-way ANOVA, chi-square test = 10.8, degrees of freedom = 2, P = 0.004), with earlier times for the heterozygous knockdown group than either the wild-type (P = 0.006, unpaired Wilcoxon) or the forebrain knockout group (P = 0.006; table 3). However, when the isoflurane administration times were matched (range, 10:30 am to 1:00 pm each day), the isoflurane loss-of-righting-reflex dose of wild-type mice (n = 13 in subgroup) was still higher than that of heterozygous knockdown mice (n = 17 in subgroup, unpaired Wilcoxon, P = 0.027), as in the complete groups of wild-type and heterozygous knockdown mice. Thus, isoflurane administration time was likely not a critical factor in determining the difference in loss-of-righting-reflex dose between heterozygous knockdown and wild-type mice.

The cumulative doses of ketamine (intraperitoneal) that just induced loss of the righting reflex were 144.3 ± 39.4 mg/kg (n = 14), 162 ± 31.7 mg/kg (n = 20), and 167.6 ± 24.1 mg/kg (n = 21) for the forebrain knockout, heterozygous knockdown, and wild-type groups, respectively (fig. 1C). The threshold ketamine dose for loss of the righting reflex was significantly lower for the forebrain knockout group as compared to the wild-type group (P = 0.006, unpaired Wilcoxon), but not significantly different between heterozygous knockdown and wild-type groups (P = 0.602, unpaired Wilcoxon). Sigmoidal curve-fit gave a mean ED₅₀ of 160 mg/kg intraperitoneal (n = 21) for the wild-type group, significantly different from the mean ED₅₀ of the forebrain knockout group (124 mg/kg intraperitoneal, n = 14) or the mean ED₅₀ of the heterozygous knockdown group (150 mg/kg intraperitoneal, n = 20; table 2; fig. 1D). ED₅₀ differences suggest that the population of forebrain knockout mice or heterozygous knockdown mice was significantly different from wild-type mice in their loss of the righting reflex response to ketamine (table 2). The estimate of the Hill’s slope was not statistically different among mouse groups (table 2).

The ages (range, 3 to 9 months) of the mice (table 3) were not significantly different among the three mouse groups tested for ketamine sensitivity (Kruskal–Wallis one-way ANOVA, degrees of freedom = 2, chi-square test = 4.77, P = 0.092). The time of day ketamine was administered was also not significantly different among the mouse groups (Kruskal–Wallis one-way ANOVA, chi-square test = 0.99, degrees of freedom = 2, P = 0.608; table 3).

The spontaneous hippocampal local field potentials in response to increasing isoflurane dose were analyzed by power spectral analysis (Supplemental Digital Content 1, https://links.lww.com/ALN/C538, illustrates a spectrogram of the electrical activity at H1 electrode after different doses of isoflurane). An ensemble average for a particular dose was defined to be the average power spectrum of all animals in each group: wild-type (n = 5 mice), forebrain knockout (n = 6), and heterozygous knockdown (n = 6), as shown for the field potentials at distal hippocampal (H1) electrode for the three groups of mice (fig. 2A). For the wild-type group, a no-change zone in the ensemble average spectra associated with various isoflurane doses could be identified at a frequency range of 25 to 30 Hz (arrow in fig. 2A1). For the forebrain knockout or heterozygous knockdown group, a no-change zone at 25 to 30 Hz could be observed with a dose of up to 0.88% isoflurane (fig. 2, A2 and A3). Power at lower (less than 25 Hz) or higher (more than 30 Hz) frequencies than the no-change zone both decreased with increasing isoflurane dose, with the 30 to 100 Hz gamma power decrease qualitatively larger for forebrain knockout and heterozygous knockdown mice than wild-type mice (fig. 2A).

To elucidate the differences between doses or mouse groups, the local field potential absolute power or the power deviation from baseline were statistically compared between two conditions at each digital frequency (“Materials and Methods”). At the H1 electrode, comparison of the ensemble power spectra was made between the wild-type (n = 5) and forebrain knockout (n = 6) groups (fig. 2B) and between the wild-type and heterozygous knockdown (n = 6) groups (fig. 2C). During baseline immobility, there was no statistically significant difference in the absolute power between the wild-type and forebrain knockout groups (fig. 2B1) or between the wild-type and heterozygous knockdown groups (fig. 2C1). For responses to isoflurane, power deviation from baseline immobility was estimated for each animal of one group and compared between groups. For comparison between the wild-type and forebrain knockout groups, statistically significant differences in the power deviation from baseline were found at 0.5 to 0.63% isoflurane, mainly at 50 to 150 Hz, but also at a beta (13 to 30 Hz) range (fig. 2, B2 and B3). For comparison between wild-type and heterozygous knockdown groups, statistically significant differences in power deviation were found in a limited range around 30 Hz at 0.63% isoflurane (fig. 2, C2 and C3). At 0.75% isoflurane, no statistically significant difference was found in the power deviations between the wild-type with forebrain knockout or heterozygous knockdown groups (fig. 2, B4 and C4).

The average power deviation as a function of isoflurane dose was estimated for standardized frequency bands at each electrode (H1, H2, or frontal cortex) and statistically evaluated by two-way (group × dose) repeated-measures ANOVA. The average power within a frequency band generally decreased with increasing isoflurane dose (fig. 3). For a given isoflurane dose, a significantly lower power in forebrain knockout (or heterozygous knockdown), compared to wild-type group, indicates a stronger anesthetic effect on forebrain knockout (or heterozygous knockdown) group versus wild-type group. At the distal hippocampal (H1) electrode, post hoc tests (after a statistically significant group × dose interaction effect) revealed that at 0.5 to 0.63% isoflurane, the forebrain knockout group power was significantly more depressed than wild-type group at theta, beta, and gamma 2 power bands (Supplemental Digital Content 1, https://links.lww.com/ALN/C538); at 1% isoflurane, the power of the heterozygous knockdown group was less than that of the wild-type group at the gamma 1 and gamma 2 power bands, whereas the power of the forebrain knockout group was less than that of the wild-type group at the gamma 2 power band (table 4; fig. 3A1). Post hoc tests also indicated some differences between forebrain knockout and heterozygous knockdown groups at delta, theta, and beta power bands at various isoflurane doses (table 4; fig. 3A1).

At the alveus hippocampus (H2) electrode, post hoc tests after a statistically significant group × dose interaction effect indicated that at 0.63% isoflurane, forebrain knockout was found to be less than wild-type for delta and theta power bands (fig. 3B; table 4). At 1% isoflurane, heterozygous knockdown was found to be less than wild-type, which was equal to forebrain knockout for beta, gamma 1, and gamma 2 power bands (fig. 3B; table 4), whereas forebrain knockout was found to be less than heterozygous knockdown for delta power at 0.88% isoflurane and for theta power at 0.5 to 0.63% isoflurane. At the frontal cortex electrode, none of the average power at any frequency band was significantly different among groups (fig. 3C; table 4). There were no statistically significant group-related effects shown by the average ripples (100 to 200 Hz) power at H1, H2, or frontal cortex electrode (table 4).

Spontaneous hippocampal local field potentials in response to increasing ketamine dose were analyzed by power spectral analysis (Supplemental Digital Content 2, https://links.lww.com/ALN/C539, illustrates spectrogram of the electrical activity at distal hippocampus H1 electrode after different doses of ketamine). Ensemble power spectra at H1 electrode are shown for wild-type (n = 5), forebrain knockout (n = 5), and heterozygous knockdown (n = 6) groups of mice (fig. 4A). All doses of ketamine tested, including the dose that induced loss of the righting reflex, increased 30 to 120 Hz power in each of the three groups (fig. 4A). All ketamine doses also decreased 6 to 25 Hz power, leaving power at 25 to 30 Hz relatively unchanged in each group of mice (arrow in fig. 4A).

Comparison of the ensemble averages between groups shows no difference in the power spectra at H1 electrode during baseline (rows 1 and 2 of fig. 4, B and C). Power spectra during walking were not statistically different among groups (data not shown), but gamma power was higher during walking than immobility in each group (arrow in fig. 4, B1 and C1). The ensemble average power spectra during baseline immobility were not statistically different between wild-type and forebrain knockout groups (fig. 4B2) or between wild-type and heterozygous knockdown groups (fig. 4C2). After 100 mg/kg intraperitoneal ketamine, the increase in 50 to 100 Hz gamma power was statistically higher in wild-type than forebrain knockout mice (fig. 4B3; Supplemental Digital Content 2, https://links.lww.com/ALN/C539) and in wild-type than heterozygous knockdown mice (fig. 4C3). The same was observed after 120 mg/kg intraperitoneal ketamine (fig. 4, B4 and C4). Interestingly, at a ketamine dose that induced a loss of the righting reflex, there was no statistically significant difference between wild-type and forebrain knockout groups, or between wild-type and heterozygous knockdown groups (fig. 4, B5 and C5). The mean doses for loss of the righting reflex, evaluated for mice with field potential recordings, were 168, 160, and 140 mg/kg intraperitoneal for the wild-type (n = 5), forebrain knockout (n = 6), and heterozygous knockdown (n = 6) groups, respectively.

The average power of standardized frequency bands at each electrode was compared between mouse groups. Ketamine increased the average power of most frequency bands in all mice, except theta- and beta-band power may decrease or increase with ketamine, dependent on mouse group and electrode. Delta power was increased by ketamine at all electrodes, but this was not statistically significant among groups (row 1 of fig. 5; table 5). Theta power change with ketamine was not significantly different among groups at hippocampal electrodes H1 and H2 but was statistically higher in the forebrain knockout group than other groups at the frontal cortex electrode (row 2 of fig. 5; table 5). A significantly higher (P < 0.05) increase in beta power was found in the forebrain knockout group than in other groups of mice at all electrodes (row 3 of fig. 5; table 5). A significantly smaller increase in gamma 1 power at the hippocampal alveus (H2) electrode was found in the heterozygous knockdown group compared to wild-type or forebrain knockout group (row 4 of fig. 5; table 5). Ketamine induced a large increase in gamma 2 power at H1 and H2 electrodes, but this increase was significantly lower (P < 0.05) in forebrain knockout or heterozygous knockdown group than wild-type mice (fig. 5, A5 and B5; table 5), with the heterozygous knockdown group often showing the lowest increase (fig. 5, A5 and B5). The increase in gamma 1 and gamma 2 power in the frontal cortex was smaller than in the hippocampus and was not group-dependent (fig. 5, C4 and C5; table 5). The ketamine-induced increase in average ripples (100 to 200 Hz) power was also not significantly group-dependent at any electrode (table 5).

Cross-spectral measures (coherence and phase) were estimated for H1–H2 (intrahippocampal) electrode pairs and for H2–frontal cortex (called extrahippocampal) electrode pairs, as illustrated in figure 6C1. Ensemble coherence for delta to gamma 2 frequency bands were plotted in figure 6 for the three groups of mice (four or five mice for each group). H1–H2 coherence at the theta or gamma (gamma 1 or gamma 2) band was higher during walking than awake immobility, as shown in previous studies.³⁰  Ketamine (at least 100 mg/kg intraperitoneal) suppressed theta and beta power and enhanced gamma power in the hippocampus (fig. 5 and fig. 6, C2 and C3), corresponding to decreased theta and beta coherence and increased gamma H1–H2 coherence (fig. 6A). By contrast, H2–frontal cortex coherence was increased at theta and decreased at a gamma range after ketamine (fig. 6B). A three-way (pair × group × dose) repeated-measures ANOVA yielded a statistically significant dose effect for theta, beta, gamma 1, and gamma 2 frequency bands, with a statistically significant pair × dose interaction effect, indicating opposite dose effects for H1–H2 as compared to H2–frontal cortex pairs (compare fig. 6, A and B; table 6). Delta coherence for both electrode pairs (H1–H2 and H2–frontal cortex) increased with ketamine dose (fig. 6, A1 and B1), and ANOVA revealed a statistically significant dose effect without pair × dose interaction (table 6). Analysis of left H1 versus right H1 electrodes or left H2 versus right H2 electrodes (with nearly zero phase between the right and left sides) also showed delta coherence increase and gamma coherence decrease after ketamine (data not shown). In summary, ketamine increased delta coherence globally (left and right hippocampus and frontal cortex), whereas gamma coherence increase was confined locally to one side of the hippocampus.

Walking compared to immobility during baseline also increased gamma 1 and gamma 2 power, but this was found to be about twofold (~0.3 log units) as compared to fourfold to eightfold increase after 100 mg/kg ketamine (fig. 6, C2 and C3). The gamma 1 (50-Hz) phase shift across H1–H2 electrodes was found to be different between forebrain knockout (n = 5) or heterozygous knockdown (n = 4) group compared to wild-type group (n = 5) after ketamine (fig. 6C4). Two-factor (groups × dose) repeated-measures ANOVA of H1–H2 phase at 50 Hz gave no main group effects (group F[2,11] = 3.57, P = 0.064; dose F[3,33] =1.97, P = 0.138; the four doses were 0 dose walk, 0 dose immobility, ketamine 100 and 120 mg/kg intraperitoneally), but a statistically significant group × dose interaction (F[6,33] = 4.70, P = 0.002), with statistically significant (Newman–Keuls) post hoc differences illustrated in figure 6C4. There was no statistically significant difference in the 50-Hz phase among the three groups during baseline walking; the average phase was ~170° (fig. 6C4), and average coherence was ~0.2 in wild-type and forebrain knockout groups, with higher coherence (~0.3) in the heterozygous knockdown group (fig. 6A4). After ketamine, the H1–H2 phase was significantly higher in the forebrain knockout or heterozygous knockdown group as compared to the wild-type group (fig. 6C4). The 50-Hz phase between H2–frontal cortex electrodes showed no statistically significant differences among the three groups of mice (P > 0.05, two-way ANOVA). H2–frontal cortex phase averaged ~0° during baseline and decreased significantly with ketamine (fig. 6C5); the high phase variability was expected because of a low H2–frontal cortex coherence (~0.1 in fig. 6B4).

We showed that mice with ablation of the vesicular acetylcholine transporter gene from the basal forebrain (forebrain knockout group) and mice with heterozygous depletion of brain vesicular acetylcholine transporter (heterozygous knockdown group) had a lower ED₅₀ (loss of the righting reflex) for isoflurane anesthesia compared to control (wild-type) mice. The ED₅₀ (loss of the righting reflex) for ketamine was also lower in forebrain knockout and heterozygous knockdown mice than wild-type mice when estimated by sigmoidal dose-response curve-fitting. At 0.5 to 0.63% isoflurane, near the loss-of-righting-reflex dose, hippocampal theta, beta, or gamma 2 power was lower in the forebrain knockout mice than the respective measure in the wild-type mice. Similarly, hippocampal gamma 2 power was lower in the forebrain knockout and heterozygous knockdown mice than the wild-type mice at pre–loss-of-righting-reflex doses of 100 to 120 mg/kg intraperitoneal ketamine.

The current study shows that depletion of basal forebrain acetylcholine increased the sensitivity to isoflurane anesthesia in mice, using loss of the righting reflex as a hypnotic measure. ED₅₀ (loss of the righting reflex) was shifted significantly from 0.76% in control (wild-type) mice to 0.69% in forebrain knockout mice, which had no acetylcholine in the basal forebrain. Heterozygous knockdown mice with decreased acetylcholine release from forebrain also showed a statistically significant decrease in isoflurane ED₅₀ (loss of the righting reflex). The current results corroborate the increase in hypnosis sensitivity in rats after 192 immunoglobulin G–saporin lesion of medial septum-diagonal band cholinergic neurons, which resulted in a shift of isoflurane ED₅₀ (loss of the righting reflex) from 0.74% in control rats to 0.62% in lesioned rats.⁹ 

Both hippocampal and frontal cortical gamma activity decreased with isoflurane dose. At the H1 electrode, forebrain knockout compared to wild-type mice showed a statistically significant decrease in hippocampal gamma 2 activity at 0.5 and 1% isoflurane and a statistically significant decrease of theta and beta power at 0.63% isoflurane (fig. 3). On the other hand, heterozygous knockdown compared with wild-type mice showed a statistically significant decrease of hippocampal gamma 1 and gamma 2 power at 1% isoflurane, above the dose that induced loss of righting (labeled by # in fig. 3). Direct comparison of ensemble power spectra between forebrain knockout and wild-type groups or heterozygous knockdown and wild-type groups corroborated the main effects (fig. 2). We suggest that the decrease of the hippocampal gamma 2–, beta-, and theta-band power at a pre–loss-of-righting-reflex dose of 0.5 to 0.63% isoflurane was commensurate with the higher sensitivity to righting loss in forebrain knockout than wild-type mice. By contrast, whereas power within all frequency bands, except beta, recorded in frontal cortex decreased with isoflurane dose, there was no statistically significant group effect (fig. 3C), suggesting that basal forebrain acetylcholine did not contribute critically to the field potentials in the frontal cortex.

A decrease in high-frequency hippocampal gamma (gamma 2) during deep anesthesia was consistent with previous studies that used different anesthetics (isoflurane, halothane, propofol, and pentobarbital) in rodents^10,11,13,18  and with temporal lobe gamma activity after sevoflurane anesthesia in humans.^31,32 

We showed several original results of ketamine anesthesia on forebrain acetylcholine-depleted mice. First, although ketamine increased hippocampal gamma power in all mice, the increase was smaller in the forebrain knockout and heterozygous knockdown mice compared to the wild-type mice. Second, the forebrain knockout or heterozygous knockdown mice showed statistically lower ED₅₀ (loss of righting) to ketamine compared to the wild-type mice.

The hippocampal gamma increase after ketamine (~100 mg/kg intraperitoneal) administration was likely caused by an increase in hippocampal acetylcholine release,²⁰  and the gamma increase was blocked by systemic administration of muscarinic antagonist scopolamine.^23,33  Thus, we suggest that the lower gamma power induced by ketamine in forebrain knockout compared to wild-type mice was caused by a lack of acetylcholine release from the basal forebrain. Gamma oscillations in the hippocampus are generated by a network of pyramidal cells and local inhibitory interneurons,^34–36  and muscarinic excitation³⁷  of CA3 by septohippocampal cholinergic afferents may contribute to the proximal gamma oscillations maximal at the midapical dendrites of CA1.^35,36  Distal gamma oscillations in CA1 are proposed to be driven by the entorhinal cortex, either directly or via an interneuronal circuit.^38–40  Gamma is further enhanced by ketamine’s action in blocking N-methyl-d-aspartate receptors on inhibitory interneurons,⁴¹  which would disinhibit pyramidal cells. In this study, we showed that gamma was increased at both distal (H1) and alveus (H2) electrodes in CA1. An increase in distal gamma may be caused, in part, by ketamine’s action in enhancing neuronal activity in entorhinal cortex layer III neurons that project to the distal dendritic layer of CA1,⁴²  likely contributing to the high glucose utilization at this layer.⁴³  At the frontal cortex, gamma power increase after ketamine was not significantly dependent on mouse groups (fig. 5C).

Other than gamma power, ketamine increased beta power in the hippocampus and frontal cortex, with the forebrain knockout group showing a statistically higher increase than the wild-type or heterozygous knockdown group (fig. 5C). Beta activity in the motor cortex, associated with abnormal sensorimotor functions,⁴⁴  may have contributed to the group-dependent beta activity.

Ketamine increased intrahippocampal coherence at the gamma (30 to 100 Hz) frequency, indicating increased coherent gamma activities across proximal and distal layers of hippocampal CA1. By contrast, alveus hippocampus (H2)–frontal cortex coherence at the gamma frequency was decreased by ketamine (fig. 6), indicating a lack of gamma-band synchrony between local field potentials at H2 and frontal cortex, and a disruption of corticocortical information transfer.⁴⁵  Other studies reported that ketamine anesthesia disrupted corticocortical information transfer⁴⁶  and decreased wide-band coherence between neocortical areas.²¹ 

Ketamine-induced loss of the righting reflex, an animal behavioral measure of loss of consciousness,¹⁸  appears to be associated with hippocampal gamma activity in forebrain knockout and wild-type mice. A relation between the hippocampus and behavior induced by ketamine or its analog phencyclidine has been known for some time. Stereotypic rotational behavior in rats induced by low-dose phencyclidine was shown to depend on an asymmetrical 2-deoxyglucose metabolic activity induced in the hippocampus.⁴⁷  Low-dose ketamine induced behavioral hyperactivity and high-amplitude hippocampal gamma in rodents,²³  and inactivation of the medial septum suppressed both the behavioral changes and hippocampal gamma induced by ketamine.²³ 

Knockdown mice provided qualified support of a relation between ketamine anesthetic sensitivity and hippocampal gamma activity. Heterozygous knockdown mice showed the lowest hippocampal gamma compared to forebrain knockout and wild-type mice in response to ketamine, but their ketamine ED₅₀ (loss of the righting reflex) was intermediate between wild-type and forebrain knockout mice. This suggests that hippocampal gamma activity may not be the only indicator of behavioral sensitivity to ketamine. A reduced acetylcholine release in heterozygous knockdown mice could increase glutamate release of basal forebrain neurons by reduction of cholinergic presynaptic inhibition,⁴⁸  but because forebrain knockout mice did not show the same change, the glutamatergic transmission enhancement may occur in brainstem cholinergic neurons that project to the basal forebrain.¹  An enhanced distal (glutamatergic) input in heterozygous knockdown mice could result in high H1–H2 gamma coherence (fig. 6, A4 and A5) and ~180^o gamma phase (fig. 6C4) under many conditions, based on a model of overlapping rhythmic dipole fields.⁷ 

We suggest that basal forebrain activation of the hippocampus opposes anesthesia, and this activation is deficient in forebrain knockout mice that have no basal forebrain acetylcholine. The forebrain knockout mice showed a greater isoflurane dose–dependent decrease of hippocampal gamma power than control mice, in correspondence with a lower isoflurane ED₅₀ (loss of righting) in knockout as compared to control mice. Furthermore, despite an enhancement of hippocampal gamma power by ketamine, the reduced hippocampal gamma power in forebrain knockout compared to control mice also corresponded to a higher ketamine anesthesia sensitivity of forebrain knockout compared to control mice.

Previous studies have emphasized different actions of ketamine and isoflurane on the brain. Ketamine increases, whereas isoflurane decreases, cortical excitability and neuronal metabolism.^11,22,49  In contrast, the current study suggests a common aspect that cholinergic neurons in the basal forebrain provide resistance to general anesthesia induced by either isoflurane or ketamine.

Our study has several limitations. First, the number of animals was small, and a quantitative relation between hippocampal gamma-band (or other) power and behavioral anesthesia across individuals could not be established. Second, the lack of correlation between neocortical gamma and behavioral anesthesia was based on recordings from only the frontal cortex. Third, the distribution and metabolism of isoflurane and ketamine concentrations in different parts of the brain were not measured, and cumulative doses of ketamine intraperitoneal could give complex pharmacokinetics. Fourth, compensatory mechanisms could arise in mutant mice that may complicate an interpretation of solely a loss in cholinergic tone. Fifth, only male mice were used, and anesthetic sensitivity in female mice, which may be affected by estrous states, remains to be studied.

In summary, the current results suggest that forebrain cholinergic neurons are involved in the modulation of anesthesia of an inhalational anesthetic, isoflurane, and an injectable anesthetic, ketamine. The results also suggest that a reduction of hippocampal gamma activity is an indicator of the general anesthesia effect and that the hippocampus participates in the modulation of anesthesia sensitivity.

Supported by Canadian Institutes of Health Research (Ottawa, Canada) grant No. MOP-15685 and Natural Science and Engineering Research Council of Canada (Ottawa, Canada) grant No. 1037-2008 (to Dr. Leung), and Canadian Institutes of Health Research grant Nos. PJT 162431, PJT 159781, MOP 136930, and MOP 89919 and National Science and Engineering Research Council of Canada grant Nos. 06106-2015 and 06577-2018 RGPIN (to Dr. M. A. M. Prado and Dr. V. F. Prado).

Dr. M. A. M. Prado has received financial compensation from Wiley (Bridgewater, New Jersey) and the International Society for Neurochemistry for service as the Deputy Chief Editor of the Journal of Neurochemistry. Drs. M. A. M. Prado and V. F. Prado have received grants from the Alzheimer’s Society of Canada (Toronto, Canada), ALS Society of Canada (Toronto, Canada), Canadian Open Neuroscience Platform (Montreal, Canada), Tanenbaum Open Science Institute (Montreal, Canada), Canadian Institutes of Health Research (Ottawa, Canada), BrainsCAN-Western (London, Ontario, Canada), and Natural Science and Engineering Research Council of Canada (Ottawa, Canada) to support research in their laboratory. The other authors declare no competing interests.