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Neuroscience in Anesthesiology and Perioperative Medicine: Research Reports

Inhibition of α5 γ-Aminobutyric Acid Type A Receptors Restores Recognition Memory After General Anesthesia

Zurek, Agnieszka A. BSc*; Bridgwater, Erica M. BSc*; Orser, Beverley A. MD, PhD, FRCPC†,‡

Author Information
doi: 10.1213/ANE.0b013e31824720da

General anesthetics and benzodiazepines are routinely administered to millions of patients each year to allow them to tolerate surgery. Unfortunately, these neurodepressive drugs may cause cognitive deficits that persist much longer than would be expected on the basis of their pharmacokinetic properties. For example, up to 47% of elderly patients who have undergone anesthesia for noncardiac surgery exhibit cognitive deficits at the time of hospital discharge.1 The duration of anesthesia has been shown to be an independent predictor of cognitive dysfunction in the early postoperative period.2 Similarly, the risk of severe brain dysfunction, including delirium, increases in critically ill patients who receive benzodiazepines.3 Such cognitive deficits are associated with poor long-term outcome, yet no specific treatments have been developed.4,5 A current research priority is to understand the neurobiological basis of postanesthetic cognitive deficits, including the brain structures and types of memory that are susceptible to disturbance by anesthetics and the molecular mechanisms underlying these deficits.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and GABA subtype A (GABAA) receptors are principal targets for most inhalation anesthetics.6,7 The prototypic volatile anesthetic isoflurane interacts with a putative binding cavity on the GABAA receptor, allosterically increasing receptor function.6 In particular, increased activity of GABAA receptors containing the α5 subunit (α5GABAA receptors) is thought to contribute to acute, desirable memory blockade during anesthesia.8,9

At the cellular level, both onset of and recovery from isoflurane modulation of GABAA receptors occur on a time scale of milliseconds to seconds.7 In humans, the rate of uptake of isoflurane is rapid (onset 3 to 5 minutes).10 The elimination of isoflurane also occurs within minutes with an initial, fast, 5-minute component and a slower 15-minute component.11 In laboratory animals, 97% of the isoflurane is eliminated from the brain within 270 minutes.12 Surprisingly, despite relatively rapid elimination, isoflurane has been shown to cause anterograde and retrograde memory deficits that persist for days to months in laboratory animals.1315

Using a mouse model, we previously showed that isoflurane administered at 1 minimum alveolar concentration (MAC) for 1 hour caused deficits in fear-associated learning and memory that lasted for at least 24 to 48 hours.16 These postanesthetic memory deficits were prevented by pretreating the mice with the drug L-655,708 30 minutes before administration of isoflurane.16 L-655,708 is an imidazobenzodiazepine that acts at the benzodiazepine site of the GABAA receptor to reduce GABA affinity and to reduce the opening of the integral chloride channel.17,18 The affinity of L-655,708 is 50-fold greater for α5GABAA receptors than for other receptor subtypes.17 Thus, our previous results were interpreted as showing that preventing the activation of α5GABAA receptors during isoflurane anesthesia prevents memory deficits that persist for 24 to 48 hours.

The above results raise the following critical question: can memory impairment that occurs after isoflurane has been eliminated be reversed by inhibiting α5GABAA receptors? The main aims of the current study were to determine whether isoflurane causes deficits in anterograde recognition memory and whether L-655,708, administered after isoflurane anesthesia, restores memory back to baseline levels. Additionally, we examined whether working memory and short-term memory were equally impaired after isoflurane and measured the time required for spontaneous recovery of recognition memory. Furthermore, to determine whether the expression of α5GABAA receptors is necessary for the development of memory deficits after isoflurane, we studied genetically modified mice lacking α5GABAA receptors (Gabra5–/– mice). Finally, to determine whether other volatile anesthetics also impair recognition memory, learning and memory were examined 24 hours after exposure to sevoflurane.

METHODS

Animal Model

Experiments were approved by the Animal Care Committee of the University of Toronto and complied with the guidelines of the Canadian Council on Animal Care. The Gabra5–/– mice were generated using a C57BL/6J and Sv129Ev background, as described previously.19Gabra5–/– mice breed normally, have a normal lifespan, and do not display an overt behavioral phenotype.19 They exhibit normal motor coordination, with no evidence of compensatory changes in the expression of other GABAA receptor subunits.19 Mice were housed in groups under standard conditions and were supplied with food and water ad libitum. A circadian cycle of 14 hours light, 10 hours dark was maintained in the housing room, and all experiments were performed during the light phase. For all behavioral tests, age-matched 3- to 4-month-old male wild-type and Gabra5-/- mice were studied. To reduce variability in learning and memory performance caused by acute stress during the conditioning and testing phases of the study, we handled each mouse for at least 10 minutes daily for 5 days before the start of the behavioral experiments. Mice were randomly assigned to treatment groups, and the experimenter was blinded to the drug treatment of individual mice.

Anesthesia

Mice were randomly assigned to treatment with isoflurane (1.3%; 1 MAC) or vehicle gas (70% air, 30% O2) for 1 hour. For treatment, each mouse was placed in an airtight acrylic chamber (27 cm wide × 10 cm deep × 10 cm high) that had been preflushed with the anesthetic gas mixture or the vehicle gas, delivered at 1 L/min. The concentrations of isoflurane, O2, and expired CO2 in the chamber were continuously analyzed with a commercial gas analyzer (Datex Ohmeda, Mississauga, Ontario, Canada). To prevent hypothermia, we maintained the temperature of the chamber at 35°C with a heating blanket, as previously described.16 After exposure to isoflurane or the vehicle gas, the mouse was removed from the chamber and was allowed to recover for 1 hour under a heat lamp before being returned to its home cage. This anesthesia regimen does not cause hypoxia or hypothermia.16 Behavioral testing was performed 24 or 72 hours after discontinuation of treatment. At that point, motor function had fully recovered, and the sedative and analgesic actions of isoflurane had dissipated.16 We have previously shown that the concentration of isoflurane in the brain at 24 hours after anesthesia, as measured with gas chromatography, is undetectable or at trace levels (0.0095%).16 For experiments with sevoflurane, mice were treated with sevoflurane (2.3%; 1 MAC) or vehicle gas (70% air, 30% O2) for 1 hour under the conditions described above.

Novel Object Recognition

Memory performance was studied after exposure to an inhaled anesthetic with the novel object recognition task. The novel object recognition assay relies on the natural preference of rodents to explore novel rather than familiar objects.20 The test involves the training or sample phase, a retention delay, and the choice phase.20 During the training phase, the mouse is allowed to explore 2 identical “sample” objects in a familiar context.20 The mouse is returned to its home cage for a retention period. For the choice phase, the mouse is reintroduced to the training context and presented with 1 familiar sample object and 1 novel object.20 Usually, mice will explore the novel object significantly more than the familiar object.20,21 This bias toward novelty is interpreted as indicating “recognition” or recall of the familiar object. The length of the retention delay (the time between the sample and choice phases) can be varied to study the influence of increasing mnemonic demands on performance of the task.22,23 Memory deficits after a short retention delay (1 minute) suggest impairment of encoding, whereas memory deficits after a longer delay (1 hour) implicate the processes of memory consolidation, retention, or both.

Object recognition was assessed in a 20 cm × 20 cm × 30 cm opaque chamber in a dimly lit room. Movement and interaction with the test objects (interlocking building blocks or toy cars) were recorded with a video camera mounted above the chamber. Each mouse was habituated to the chamber for 15 minutes on the day before testing. Mice were randomly assigned to be trained with 1 pair of sample objects. Pilot studies were performed to confirm that there was no inherent preference for either of the objects. Additionally, the set of objects and the position of the familiar and novel objects in the test chamber were counterbalanced and randomized throughout the experiments. No external motivational factors, such as food deprivation or appetitive or aversive stimuli, were used.

The timelines and general experimental protocols are outlined in Figure 1. Mice were trained on the object recognition memory paradigm 24 or 72 hours after exposure to isoflurane or vehicle gas (Fig. 1, A–C). During the training phase, each mouse was placed in the chamber and allowed to explore the 2 identical sample objects for 10 minutes. After either 1 minute (to test working memory; Fig. 1A) or 1 hour (to test short-term memory; Fig. 1B), the mouse was reintroduced to the same context and was exposed to 1 familiar sample object and 1 novel object. All of the mouse's movements were video-recorded, and the time spent exploring each object was scored manually. Exploratory behavior was defined as sniffing, licking, or touching the object while facing it.21 Learning was deemed to have taken place if the time spent with the novel object was longer than the time spent with the familiar object. Additionally, memory was assessed by measuring the proportion of total exploration time that was spent exploring the novel object and calculating a discrimination ratio, where the discrimination ratio was the time spent exploring the novel object divided by the total time spent exploring both objects.21 Mice that spent a larger proportion of time with the novel object, as evidenced by a discrimination ratio more than the chance value of 0.5, were deemed to have remembered the familiar object (i.e., the object to which they had previously been exposed). Typical discrimination ratios that indicate learning range from 0.61 to 0.72.21,2426

F1-26
Figure 1:
The timelines and general experimental protocols are shown. A, Wild-type mice that were exposed to isoflurane anesthesia (1 hour, 1 minimum alveolar concentration [MAC]) or vehicle gas received an injection of a vehicle 23.5 hours later. To assess working memory performance, we trained these mice on the object recognition paradigm 24 hours after isoflurane and tested 1 minute after training. B, To determine whether short-term memory was impaired by isoflurane and whether memory deficits could be reversed, we exposed wild-type mice to isoflurane anesthesia and mice received an injection of either vehicle or L-655,708 (0.35 mg/kg or 0.7 mg/kg) 23.5 hours after isoflurane. To assess short-term recognition memory, we trained this group on the object recognition paradigm 24 hours after isoflurane and tested 1 hour after training. C, Timeline of experimental treatment in which wild-type mice were tested 72 hours after isoflurane (1 hour, 1 MAC). Mice were treated with vehicle by injection 71.5 hours after exposure to isoflurane or vehicle gas and 30 minutes before behavioral training. Short-term memory was assessed 1 hour after training. D, Timeline of experimental treatment in which short-term memory was tested in Gabra5–/– mice. Mice received injections of L-655,708 (0.7 mg/kg) or vehicle 10 minutes before isoflurane anesthesia. Mice were trained on the object recognition paradigm 24 hours after isoflurane, and short-term memory was tested 1 hour after training. E, Timeline of experimental treatment in which prevention of isoflurane-induced memory deficits was assessed in wild-type mice. Mice received injections of L-655,708 (0.7 mg/kg) or vehicle 10 minutes before isoflurane anesthesia. Mice were trained on the object recognition paradigm 24 hours after isoflurane, and short-term memory was tested 1 hour after training. F, Wild-type mice were exposed to sevoflurane and received a vehicle injection 23.5 hours later. Sevoflurane-treated mice were trained on the object recognition task 24 hours after anesthesia and tested 1 hour after training on the task.

Animals that did not interact with each object (interaction time of 0 seconds) during the test period were excluded, as described previously.27,28 Mice meeting this exclusion criteria included several treated with isoflurane + vehicle injection (n = 2), vehicle gas + L-655,708 (0.7 mg/kg; n = 2), and mice treated with isoflurane + L-655,708 (0.7 mg/kg; n = 3). In addition, animals for which the discrimination ratio deviated from the mean discrimination ratio by 2 standard deviations or more were also excluded from the analysis.15 Two animals were excluded during the analysis phase, one from the group that received vehicle gas plus vehicle injection and the other from the group that received isoflurane plus L-655,708 (0.7 mg/kg).

To determine whether the treatments affected locomotor activity or exploration, we measured the total time spent exploring both objects during the training phase. This analysis was undertaken in response to a reviewer's comments and several of the 247 potential videos were not available because of file corruption or the files were unavailable. Videos were not analyzed for mice in the following groups: wild-type control in the treatment experiment (n = 1), isoflurane-treated in the treatment experiment (n = 2), Gabra5–/– control (n = 1), Gabra5–/– isoflurane-treated (n = 1), Gabra5–/– treated with vehicle gas and L-655,708 (n = 2), Gabra5–/– mice treated with isoflurane and L-655,708 (n = 1), control group in the sevoflurane experiment (n = 3), and sevoflurane-treated group (n = 2).

Drug Treatment

Selective antagonists for α5GABAA receptors are currently not available; however, the inverse agonist L-655,708 preferentially decreases the activity of α5GABAA receptors. Careful dose selection is important to ascribe to L-655,708 selective actions on α5GABA receptors.17,29 The doses of L-655,708 used in this study were selected on the basis of in vivo binding data, pharmacokinetic analyses, and previous memory studies.30,31 In experiments to study reversal of memory deficits by L-655,708, doses of this agent (0.35 mg/kg or 0.7 mg/kg, i.p.) or vehicle (90% saline, 10% dimethylsulfoxide [DMSO], i.p.) were administered 23.5 hours after exposure to isoflurane or vehicle gas and 30 minutes before training in the object recognition paradigm (Fig. 1B). This time schedule was selected so that all mice in the treatment cohort would be studied 24 hours after isoflurane anesthesia. In the prevention experiments, wild-type and Gabra5−/− mice were treated with L-655,708 (0.7 mg/kg) or vehicle (90% saline, 10% DMSO, i.p.) injection 10 minutes before administration of isoflurane, sevoflurane, or vehicle gas (Fig. 1, D and E). The 0.7 mg/kg dose administered 10 minutes before anesthesia has been studied previously, with no apparent effect on the potency of the anesthetic, as measured by the tail pinch assay,16 and no generalized effects on fear-associated learning.32 In previous studies, L-655,708 (0.7 mg/kg) caused 60% to 70% occupancy of α5GABAA receptors in vivo at 30 minutes after i.p. injection, with limited binding to other GABAA receptors.29,31 To control for the effect of injection, we gave control mice that were tested 1 minute after training (working memory) or 72 hours after anesthesia an injection of vehicle (90% saline, 10% DMSO, i.p.) 30 minutes before behavioral training (Fig. 1, A and C).

Statistical Analysis

Results are presented as means ± SE of the mean (SEM). To determine whether subjects within a group remembered the familiar object, the mean time spent with the 2 objects was compared using a 1-tailed Student t test, which assumed equal variances. To determine whether the memory performance between treatment groups differed, we compared data with an unpaired, 2-tailed Student t test for equal variances or a 2-way analysis of variance (ANOVA) (L-655,708 dose × gas), as appropriate. Post hoc analyses, when required, were conducted with Tukey Honestly Significant Difference (HSD) test. The Shapiro–Wilk test was used to validate the assumption of normality (all P > 0.09). The discrimination ratios of all but 2 groups were normally distributed; the discrimination ratios of the control group in the Gabra5–/– experiment as well as the control group in the sevoflurane experiment had non-normal distributions. In these cases, the Kruskal–Wallis and Mann–Whitney U tests were used to corroborate the findings of the parametric statistics. The homogeneity of variances was verified for each group with the Levene test, which indicated that the variance of discrimination ratios in each experiment was comparable between groups (P > 0.13). Statistical testing was performed with 2 statistical software packages: Statistical Package for the Social Sciences (SPSS version 17.0, IBM Corporation, Armonk, NY) and GraphPad Prism software, version 4.0 (GraphPad Software, San Diego, CA). A P value <0.05 was considered statistically significant.

RESULTS

Working Memory and Short-Term Memory Performance 24 Hours After Isoflurane

Mice were treated with isoflurane or vehicle gas for 1 hour, and memory performance was studied the following day. Working memory was assessed 1 minute after behavioral training, whereas short-term recognition was assessed 1 hour after training. To assess memory performance within a treatment group, we compared the time the mouse spent with the novel object with the time spent with the familiar object. To compare memory performance between treatment groups, we compared the discrimination ratios. Control wild-type mice that were exposed to vehicle gas and a vehicle injection demonstrated normal working memory as they spent more time exploring the novel object than the familiar object (novel versus familiar object, t = 3.30, df = 9, P = 0.005; Fig. 2A). Isoflurane-treated mice also demonstrated normal working memory (novel versus familiar object, t = 2.64, df = 9, P = 0.013; Fig. 2A). The discrimination ratios for working memory for control and isoflurane-treated groups were similar (0.68 ± 0.05 vs 0.67 ± 0.04, t = 0.026, df = 18, P = 0.979, 95% confidence interval [CI] −0.12 to 0.12; Fig. 2B). Also, time spent exploring both sample objects during the training phase did not differ between the groups (45.21 ± 8.05 seconds vs 49.28 ± 9.60 seconds, t = 0.32, df = 18, P = 0.750, 95% CI −30.39 to 22.26 seconds; Fig. 2C). However, since the 95% confidence interval for the difference between the means is quite wide, this experiment may have been underpowered to detect differences in exploratory behavior during training.

F2-26
Figure 2:
Normal working memory performance 24 hours after isoflurane anesthesia. A, One day after isoflurane exposure, mice were trained on the object recognition paradigm and tested 1 minute after training. Time spent with novel and familiar objects during testing. Both control (exposed to vehicle gas; n = 10) and isoflurane (exposed for 1 hour, 1.3% isoflurane; n = 10) mice demonstrated normal memory performance as they spent more time exploring the novel than the familiar object. B, The discrimination ratios (time spent with novel object/time spent with both objects) of control and isoflurane-treated mice. The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

Control wild-type mice demonstrated normal short-term memory as they spent more time exploring the novel object than the familiar object (novel versus familiar object, t = 4.00, df = 30, P < 0.001; Fig. 3A). In contrast, isoflurane-treated mice exhibited short-term memory deficits as they exhibited no preference for the novel object (novel versus familiar object, t = 0.40, df = 30, P = 0.345, 95% CI −2.24 to 1.51 seconds; Fig. 3A). The discrimination ratio was lower for the isoflurane-treated group than for the control group (0.51 ± 0.03 vs 0.66 ± 0.03, t = 3.66, df = 60, P < 0.001; Fig. 3B) and was similar to that predicted by chance (0.5). The impairment of short-term memory performance in the isoflurane-treated mice could not be attributed to differences in exploratory behavior during the training phase (control 74.02 ± 7.54 seconds versus isoflurane 62.3 ± 9.44 seconds, t = 0.97, df = 58, P = 0.336, 95% CI −12.47 to 35.91 seconds; Fig. 3C).

F3-26
Figure 3:
Impaired short-term recognition memory 24 hours after isoflurane anesthesia. One day after isoflurane exposure, mice were trained on the object recognition paradigm and tested 1 hour later. A, Time spent with novel and familiar objects during testing. Control (exposed to vehicle gas; n = 31) mice demonstrated normal memory performance as they spent more time exploring the novel than the familiar object, whereas isoflurane (exposed to 1 hour, 1.3% isoflurane; n = 31) mice spent an equal amount of time with each object and hence showed impaired memory performance. B, The discrimination ratios (time spent with novel object/time spent with both objects) of control and isoflurane-treated mice. The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

L-655,708 Reverses Memory Deficits After Isoflurane

We next sought to determine whether L-655,708 reversed the short-term memory impairment detected 24 hours after isoflurane. Mice were exposed to isoflurane, followed by L-655,708 (0.35 mg/kg or 0.7 mg/kg, i.p.) or vehicle administered 23.5 hours after anesthesia and 30 minutes before behavioral training. Short-term memory was assessed 1 hour after training. Mice in the control group (vehicle gas + vehicle injection) spent more time with the novel object than with the familiar object (t = 4.00, df = 30, P < 0.001; Fig. 4A). As shown in Figure 3, mice exposed to isoflurane only (isoflurane + vehicle injection) exhibited short-term memory deficits and spent a similar amount of time with the novel and familiar objects (t = 0.400, df = 30, P = 0.346, 95% CI −2.24 to 1.51 seconds; Fig. 4A). L-655,708 restored normal memory performance in groups that were exposed to isoflurane (effect of isoflurane × L-655,708 F2,102 = 3.59, P = 0.032; Fig. 4B). A low dose of L-655,708 (0.35 mg/kg) increased the proportion of time that isoflurane-treated mice spent with the novel object (discrimination ratio, isoflurane + L-655,708, 0.68 ± 0.03 vs isoflurane + vehicle injection, 0.51 ± 0.03, P < 0.05, Tukey's HSD; Fig. 4B). Both control and isoflurane-treated mice that received L-655,708 at 0.35 mg/kg learned the task and spent more time with the novel object than with the familiar object (control, novel versus familiar object, t = 3.53, df = 9, P = 0.003; isoflurane + low-dose L-655,708, novel versus familiar object, t = 4.85, df = 10, P < 0.001; Fig. 4A).

F4-26
Figure 4:
Short-term memory deficits can be reversed by inhibition of α5 γ-aminobutyric acid subtype A (α5GABAA) receptors 24 hours after isoflurane anesthesia. Mice were trained on the object recognition paradigm 24 hours after anesthesia, and short-term memory was tested 1 hour after training. Mice received either an injection of vehicle (10% dimethylsulfoxide) or the α5GABAA receptor-selective inverse agonist L-655,708 (0.35 mg/kg, i.p.) 23.5 hours after anesthesia and 30 minutes before behavioral training. Control = vehicle gas (30% O2, 70% air, 1 hour) and vehicle injection (n = 31); isoflurane = isoflurane (1.3%, 1 hour) and vehicle injection (n = 31); L-655,708 = vehicle gas and L-655,708 (0.35 mg/kg, i.p.; n = 10); isoflurane + L-655,708 = isoflurane (1.3%, 1 hour) and L-655,708 (0.35 mg/kg, i.p.; n = 11). A, Time spent with novel and familiar objects during testing. B, Discrimination ratios (time spent with novel object/time spent with both objects) of control, L-655,708, and isoflurane + L-655,708 groups demonstrate learning. The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

We also tested whether a higher dose of L-655,708 (0.7 mg/kg) also reversed the short-term memory deficit. This higher dose of L-655,708 failed to reverse the memory deficit in isoflurane-treated mice (discrimination ratio, isoflurane + vehicle injection 0.54 ± 0.04; isoflurane + high-dose L-655,708 0.54 ± 0.05) and both vehicle and L-655,708-injected groups that were exposed to isoflurane spent similar amounts of time with the novel and familiar objects (isoflurane + vehicle injection, novel versus familiar object, t = 1.42, df = 9, P = 0.094, 95% CI −1.63 to 7.10 seconds; isoflurane + high dose L-655,708, novel versus familiar object, t = 1.29, df = 8, P = 0.117, 95% CI −1.60 to 5.67 seconds), although the sample sizes were small for this comparison.

The memory performance could not be attributed to changes in exploratory behavior during training, because treatment with isoflurane and L-655,708 did not influence the amount of time that mice spent with both objects (effect of isoflurane, F1,99 = 0.15, P = 0.73; effect of L-655,708, F2,99 = 2.40, P = 0.096; effect of isoflurane + L-655,708, F2,99 = 2.07, P = 0.132; Fig. 4C).

Short-Term Memory Performance 72 Hours After Isoflurane

To determine whether short-term memory deficits persisted beyond the first 24 hours, mice were studied 72 hours after isoflurane treatment. At that time point, control and isoflurane-treated mice showed normal recognition memory, as evidenced by a preference for the novel object in both groups (control, novel versus familiar object, t = 2.86, df = 9, P = 0.009; isoflurane, novel versus familiar object; t = 2.45, df = 9, P = 0.018; Fig. 5A). The discrimination ratios were similar between the 2 groups (control, 0.65 ± 0.05 vs isoflurane, 0.60 ± 0.04; t = 0.787, df = 18, P = 0.441, 95% CI −0.08 to 0.17; Fig. 5B), which indicates that learning and recognition memory recovered by 72 hours after isoflurane treatment. The normal memory performance at 72 hours after anesthesia could not be attributed to differences in exploratory activity between groups, as both control and isoflurane-treated mice spent a similar amount of time exploring both objects during the training phase (control, 45.92 ± 9.44 seconds versus isoflurane, 46.65 ± 6.57 seconds; t = 0.06, df = 18, P = 0.95, 95% CI −24.89 to 23.43 seconds; Fig. 5C). However, since the 95% confidence interval for the difference between the means is quite wide, this experiment may have been underpowered to detect differences in exploratory behavior during training.

F5-26
Figure 5:
Normal recognition memory 72 hours after isoflurane anesthesia. Mice were trained on the object recognition paradigm 72 hours after anesthesia, and short-term memory was tested 1 hour after training. A, Time spent with novel and familiar objects during testing. Control (exposed to vehicle gas; n = 10) and isoflurane (exposed to 1 hour, 1.3% isoflurane; n = 10) mice demonstrated normal memory performance as they spent more time exploring the novel than the familiar object. B, The discrimination ratios (time spent with novel object/time spent with both objects) of control and isoflurane-treated mice. The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

Short-Term Memory Performance of Gabra5–/– Mice 24 Hours After Isoflurane

On the basis of our previous study, which showed that L-655,708 administered before anesthesia can prevent memory deficits, and the results presented above, we predicted that mice lacking α5GABAA receptors would not exhibit postanesthesia memory deficits. To test this postulate, we trained Gabra5–/– mice and tested them on the object recognition paradigm 24 hours after anesthesia. The performance of control Gabra5–/– mice and control wild-type mice did not differ significantly (discrimination ratio 0.66 ± 0.05 vs 0.74 ± 0.05; t = 1.24, df = 19, P = 0.230, 95% CI −0.23 to 0.06). Control Gabra5–/– mice spent more time with the novel object than with the familiar object (novel versus familiar object, t = 2.56, df = 11, P = 0.013; Fig. 6A). As predicted, Gabra5–/– mice exposed to isoflurane also showed a preference for the novel object (novel versus familiar object, t = 2.51, df = 11, P = 0.015; Fig. 6A). Isoflurane did not cause significant impairment of memory performance in Gabra5–/– mice at 24 hours after anesthesia (discrimination ratio, control, 0.66 ± 0.05; isoflurane, 0.62 ± 0.05; effect of isoflurane, F1,47 = 0.38, P = 0.544; Fig. 6B). Gabra5–/– mice treated with L-655,708 (0.7 mg/kg) 10 minutes before exposure to isoflurane or vehicle gas also learned the task and preferred the novel object over the familiar object (L-655,708, novel versus familiar object, t = 2.10, df = 11, P = 0.030; isoflurane + L-655,708, novel versus familiar object, t = 3.83, df = 10, P = 0.002). L-655,708 did not affect the memory performance of Gabra5–/– mice exposed to vehicle gas or isoflurane (discrimination ratio, 0.64 ± 0.05 vs 0.62 ± 0.05; effect of L-655,708, F1,47 = 0.02, P = 0.90; Fig. 6B). No significant interactions were observed (effect of isoflurane × L-655,708, F1,46 = 0.02, P = 0.41; Fig. 6B). Nonparametric analysis also revealed no differences in discrimination ratios between treatment groups (χ2 = 1.80, df = 3, P = 0.616). Additionally, neither isoflurane nor L-655,708 influenced exploratory behavior during training (effect of isoflurane, F1,43 = 0.04, P = 0.851; effect of L-655,708, F1,43 = 1.17, P = 0.285; effect of isoflurane × L-655,708, F1,43 = 0.66, P = 0.423; Fig. 6C).

F6-26
Figure 6:
α5 γ-aminobutyric acid subtype A (α5GABAA) receptor null mutant mice exhibit no short-term recognition memory deficits 24 hours after isoflurane anesthesia. Mice were trained on the object recognition paradigm 24 hours after anesthesia, and short-term memory was tested 1 hour after training. Mice received either an injection of vehicle (10% dimethylsulfoxide) or the α5GABAA receptor-selective inverse agonist L-655,708 (0.7 mg/kg, i.p.) 10 minutes before gas exposure. Control = vehicle gas (30% O2, 70% air, 1 hour) and vehicle injection (n = 12); isoflurane = isoflurane (1.3%, 1 hour) and vehicle injection (n = 12); L-655,708 = vehicle gas and L-655,708 (0.7 mg/kg, i.p.; n = 12); isoflurane + L-655,708 = isoflurane (1.3%, 1 hour) and L-655,708 (0.7 mg/kg, i.p.; n = 11). A, Time spent with novel and familiar objects during testing. All groups spent more time with the novel than the familiar object. B, The discrimination ratios (time spent with novel object/time spent with both objects) of all groups demonstrate normal memory performance. The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

Prevention of Postanesthesia Memory Deficits

L-655,708, administered before isoflurane, has been shown to prevent memory deficits in the early postanesthetic period.16 We also sought to determine whether the deficit in recognition memory could be prevented by administering L-655,708 before isoflurane as a positive control. L-655,708 (0.7 mg/kg) was injected, and 10 minutes later, mice were exposed to isoflurane. Memory performance was studied 24 hours later using the object recognition paradigm. Control mice showed the predicted preference for the novel object (time spent with novel versus familiar object, t = 3.43, df = 8, P = 0.045, Fig. 7A; discrimination ratio 0.74 ± 0.04, Fig. 7B). In contrast, mice exposed to isoflurane did not prefer the novel object (time spent with novel versus familiar object, t = 0.64, df = 10, P = 0.268, 95% CI −4.90 to 8.85 seconds; Fig. 7A), and the discrimination ratio was 0.53 ± 0.05, similar to a chance level of interaction with the novel object (Fig. 7B). Again, isoflurane administered to wild-type mice 24 hours before training impaired their performance in the object recognition task (effect of isoflurane, F1,38 = 10.39, P = 0.003; Fig. 7B).

F7-26
Figure 7:
Short-term recognition memory deficits can be prevented by inhibition of α5 γ-aminobutyric acid subtype A (α5GABAA) receptors prior to anesthesia. Mice were trained on the object recognition paradigm 24 hours after anesthesia, and short-term memory was tested 1 hour after training. Mice received either an injection of vehicle (10% dimethylsulfoxide) or the α5GABAA receptor-selective inverse agonist L-655,708 (0.7 mg/kg, i.p.) 10 minutes before gas exposure. Control = vehicle gas (30% O2, 70% air, 1 hour) and vehicle injection (n = 9); isoflurane = isoflurane (1.3%, 1 hour) and vehicle injection (n = 11); L-655,708 = vehicle gas and L-655,708 (0.7 mg/kg, i.p.; n = 10); isoflurane + L-655,708 = isoflurane (1.3%, 1 hour) and L-655,708 (0.7 mg/kg, i.p.; n = 9). A, Time spent with novel and familiar objects during testing. All groups spent more time with the novel than the familiar object. B, The discrimination ratio (time spent with novel object/time spent with both objects). The dotted line represents a chance level of interaction with the novel object (discrimination ratio = 0.5). C, Time spent exploring identical sample objects during training. Data are represented as mean ± SEM. *Significance at P < 0.05.

Mice treated with L-655,708 before exposure to vehicle gas showed normal learning and preference for the novel object (time spent with novel versus familiar object, t = 4.91, df = 9, P < 0.001; Fig. 7A). Mice treated with L-655,708 before exposure to isoflurane preferred the novel object (time spent with novel versus familiar object, t = 2.48, df = 8, P = 0.019; Fig. 7A). L-655,708 alone did not significantly enhance or diminish discrimination ratios across any of the groups (effect of L-655,708, F1,39 = 1.59, P = 0.215; Fig. 7B). There was no significant interaction between L-655,708 and isoflurane (F1,39 = 0.614, P = 0.439; Fig. 7B), although the study may have been underpowered to detect a difference between groups. Neither isoflurane nor L-655,708 influenced the amount of time that mice spent with both objects during training (effect of isoflurane, F1,38 = 1.43, P = 0.240; effect of L-655,708, F1,38 = 1.54, P = 0.223; effect of isoflurane × L-655,708, F1,38 = 0.10, P = 0.752; Fig. 7C).

Short-Term Memory Performance 24 Hours After Sevoflurane

Finally, to determine whether the postanesthetic impairment in recognition memory was seen after exposure to another commonly used inhaled anesthetic, mice were treated with sevoflurane then trained on the object recognition task 24 hours later. Control, vehicle gas-treated mice demonstrated normal memory performance and preferred the novel object (novel 33.92 ± 5.03 seconds; familiar 20.03 ± 3.03 seconds, t = 5.00, df = 9, P < 0.001). In contrast, sevoflurane-treated mice spent a similar amount of time with the novel and familiar object and, hence, did not learn the task (novel 15.41 ± 2.25 seconds; familiar 13.13 ± 1.95 seconds, t = 1.07, df = 9, P = 0.157, 95% CI −2.56 to 7.12 seconds). The discrimination ratio for the sevoflurane-treated group was lower than that for the control group and was similar to that predicted by chance (sevoflurane 0.53 ± 0.03 vs control 0.63 ± 0.02, t = 2.22, df = 18, P = 0.039). A nonparametric analysis revealed that the difference between the discrimination ratios of control and sevoflurane-treated groups only approached significance (Z = −1.66, P = 0.096). Exploratory behavior during the training phase was not affected by exposure to sevoflurane (sevoflurane 23.45 ± 3.88 seconds versus control 36.46 ± 4.77 seconds, t = 2.14, df = 13, P = 0.052, 95% CI −0.14 to 26.16 seconds).

DISCUSSION

Our results show that isoflurane-treated mice exhibited short-term recognition memory deficits when compared with vehicle-treated controls as evidenced by the lower discrimination ratios. In contrast, working memory was not impaired 24 hours after isoflurane because the discrimination ratios were similar between isoflurane- and vehicle-treated mice. A low dose of the α5GABAA receptor-selective inverse agonist, L-655,708, administered 24 hours after isoflurane fully reversed the short-term memory deficits. Changes in memory performance could not be attributed to changes in exploratory activity because exposure to isoflurane or L-655,708 did not alter the time that mice spent interacting with the objects. Consequently, all mice had an equal opportunity to perceive and learn the characteristics of the objects. Short-term memory deficits resolved within 72 hours. The expression of α5GABAA receptors was necessary for the isoflurane-induced deficits in recognition memory to occur, because Gabra5–/– mice exhibited no memory impairment. Finally, recognition memory deficits also occurred 24 hours after sevoflurane.

The most novel and important finding of this study is that a low dose of L-655,708 (0.35 mg/kg) administered 24 hours after isoflurane completely reversed the deficit in recognition memory. This result was unexpected, given the widely believed mechanism by which volatile anesthetics block memory. The concentration-dependent suppression of memory during acute exposure to isoflurane has been attributed, at least in part, to increased activity of GABAA receptors.6,7,32 Isoflurane and other volatile anesthetics, including desflurane and sevoflurane, enhance GABAA receptor function, and the resulting increase in chloride conductance reduces neuronal excitability.6 In brain networks, such as the cornus ammonis 1 subfield of the hippocampus, the enhanced chloride conductance prevents the synaptic plasticity that subserves memory formation.33 Once the anesthetic has been eliminated, it is assumed that GABAA receptor activity returns to baseline and memory recovers.7,34 However, in these experiments, memory performance was impaired 24 hours after exposure to isoflurane, when the concentration of isoflurane in the brain had declined to the limits of detection (0.0095%).16 This low concentration of isoflurane is orders of magnitude less than the concentration (0.4%) required for memory blockade during anesthesia.35 Taken together, the available data suggest that a simple interaction between isoflurane and GABAA receptors would not account for the memory deficits at 24 hours.

An analogous and surprising long-term effect of the IV anesthetic ketamine on cognitive function has been reported.36,37 Ketamine is a noncompetitive antagonist of the N-methyl-D-aspartate subtype of glutamate receptors.38,39 A single dose of ketamine causes long-lasting effects that persist after the drug has been eliminated, specifically a rapid and sustained reversal of depression that lasts for weeks to months.36,40,41 The sustained effect of ketamine involves the rapamycin intracellular signaling pathway, which increases synaptic signaling proteins and the number and function of synapses in the cortex.37

Modulation of α5GABAA receptors by isoflurane plays a crucial role in initiating the memory deficits that were evident at 24 hours, because genetic and pharmacological inhibition of these receptors prevented memory impairment. L-655,708, administered after isoflurane, may counteract an unrecognized increase in the function or expression of α5GABAA receptors that persists after anesthesia in the absence of isoflurane binding to the receptor. Alternatively, L-655,708 may cause a nonspecific compensatory enhancement of memory processes. The molecular mechanisms that are triggered during periods of high GABAergic activity during anesthesia, and cause α5GABAA receptor-dependent memory deficits, remain to be determined.

On the basis of the current study, we propose that inhibition of α5GABAA receptors is a plausible strategy for reversing memory deficits after general anesthesia in patients. Inverse agonists that preferentially target α5GABAA receptors lack the adverse effects that typify nonselective GABAA receptor antagonists, such as anxiogenesis and seizures.29,42,43 Several human trials have studied this class of drugs.43,44 The inverse agonist α5IA, which is functionally selective for α5GABAA receptors, attenuated ethanol-induced impairment of word recall when administered before ethanol and was well tolerated by volunteers.44 Also, the α5GABAA receptor–selective inverse agonist RG1662 (Roche Pharmaceuticals, Basel, Switzerland) is currently undergoing phase 1 clinical trials for treatment of cognitive deficits in patients with Alzheimer disease.43 Our results suggest that the dose of inverse agonist must be selected carefully, because a high dose administered immediately before learning may actually impair memory performance.45 Higher doses of inverse agonist may exert agonist-like effects on non-α5GABAA receptors, thus increasing the activity of GABAA receptors.45

Several of our results are consistent with previous studies. The interaction times and discrimination ratios for object recognition measured under baseline, and control conditions in the current study were comparable to those reported by others for rodents and nonhuman primates.4648 Also, the memory deficits observed in isoflurane-treated mice were consistent with a deficit in retrograde memory observed in mice treated with sevoflurane (2.6% for 2 hours) and then conditioned with 2 object learning sessions.26 Notably, we observed that isoflurane impaired short-term recognition memory, whereas working memory remained intact. These results suggest that isoflurane spares the perception and encoding of information but disrupts consolidation of memory into long-term storage or memory retrieval. Our results predict that patients exposed to isoflurane could exhibit normal recall for immediate events that are accessible to working memory but might exhibit deficits in recall for events after a longer delay. Similar effects on working memory have been found after exposure to the benzodiazepines alprazolam and diazepam: object recognition was intact when rats were tested 10 minutes after training but impaired when they were tested 1 hour after training.46

The current study raises many additional important questions for future study. It remains to be determined whether isoflurane triggers downstream events that impair memory through processes initially requiring the interaction between isoflurane and α5GABAA receptors. A causal role for these receptors in memory deficits is supported by results that show that inhibition and genetic deletion of α5GABAA receptors prevent the deficits. In addition, although short-term memory deficits resolved spontaneously within 72 hours, it will be critical to determine whether higher doses of isoflurane (due to higher concentrations and/or longer durations of treatment) prolong the memory deficit. Also, it must be determined whether factors that impair recognition memory performance, such as age and inflammation, exacerbate isoflurane-induced memory loss. Previous studies have shown that age exacerbates postanesthetic memory loss; for example, aged rats had impaired anterograde memory for up to 2 weeks after anesthesia, whereas adult rats were no longer impaired at that time point.49,50 The object recognition task is a versatile experimental model to study such interactions, because it requires no appetitive or aversive reinforcement, and it has the potential for high throughput.21,51

In summary, isoflurane impairs short-term memory but not working memory after anesthesia in an ethologically relevant paradigm. Furthermore, α5GABAA receptors are necessary for the development of postanesthetic memory deficits and are a potential therapeutic target for restoring memory after general anesthesia.

DISCLOSURES

Name: Agnieszka A. Zurek, BSc.

Contribution: Study design, conduct of study, data analysis, and manuscript preparation.

Name: Erica M. Bridgwater, BSc.

Contribution: Conduct of study.

Name: Beverley A. Orser, MD, PhD, FRCPC.

Contribution: Developed study rationale and study design, and manuscript preparation.

This manuscript was handled by: Gregory J. Crosby, MD.

ACKNOWLEDGMENTS

We would like to thank Mr. Marko Katic for his assistance with the statistical methods.

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