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Post-anesthesia AMPA receptor potentiation preventsanesthesia-induced learning and synaptic deficitsLianyan Huang,1 Joseph Cichon,2 Ipe Ninan,3 Guang Yang1*

Accumulating evidence has shown that repeated exposure to general anesthesia during critical stages of braindevelopment results in long-lasting behavioral deficits later in life. To date, there has been no effective treatment tomitigate the neurotoxic effects of anesthesia on brain development. By performing calcium imaging in the mousemotor cortex, we show that ketamine anesthesia causes a marked and prolonged reduction in neuronal activity dur-ing the period of post-anesthesia recovery. Administration of the AMPAkine drug CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine] to potentiate AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptoractivity during emergence from anesthesia in mice enhances neuronal activity and prevents long-term motorlearning deficits induced by repeated neonatal anesthesia. In addition, we show that CX546 administration alsoameliorates various synaptic deficits induced by anesthesia, including reductions in synaptic expression ofNMDA (N-methyl-D-aspartate) and AMPA receptor subunits, motor training–evoked neuronal activity, and den-dritic spine remodeling associated with motor learning. Together, our results indicate that pharmacologicallyenhancing neuronal activity during the post-anesthesia recovery period could effectively reduce the adverseeffects of early-life anesthesia.

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INTRODUCTION

General anesthetics are commonly used to modulate the activity ofneuronal networks, producing a reversible loss of sensation and con-sciousness in surgeries (1–3). Although the vast majority of patientsrestore their physiological homeostasis soon after anesthesia, some maysuffer from long-term adverse effects of anesthesia. A higher incidenceof learning disabilities and attention deficit and hyperactivity disordershas been found in children repeatedly exposed to procedures requiringgeneral anesthesia (4–8). Animal studies further demonstrated thatprolonged exposure to anesthetics and sedatives during critical stagesof brain development causes neurodegeneration and behavioral deficitsin both rodents and nonhuman primates (9–13). So far, there has beenno effective strategy to alleviate the neurotoxic effects of anesthetic drugs.

Ketamine is a dissociative anesthetic, which depresses neuronal ac-tivity and reduces calcium influx into neurons, in part through blockadeof N-methyl-D-aspartate (NMDA)–type glutamate receptors (2). Recentin vivo imaging studies have shown that multiple exposures to generalanesthesia induced by ketamine-xylazine (KX) during early postnataldevelopment impair motor learning and learning-dependent remodel-ing of postsynaptic dendritic spines in adolescent mice (14). Given theimportant roles of neuronal activity in shaping the development ofneuronal circuits (15, 16), an intriguing possibility is that pharmaco-logically enhancing neuronal activity after anesthesia could be benefi-cial in alleviating the adverse effects of neonatal exposure toanesthesia.

Here, we examined the effects of an AMPAkine drug, 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546), on KX anesthesia–induced learning and synaptic deficits. AMPAkines are a class of compoundsthat increase a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

1Department of Anesthesiology, Perioperative Care, and Pain Medicine, New York Uni-versity School of Medicine, New York, NY 10016, USA. 2Medical Scientist TrainingProgram at New York University School of Medicine, New York, NY 10016, USA. 3De-partment of Psychiatry, New York University School of Medicine, New York, NY 10016,USA.*Corresponding author. Email: guang.yang@med.nyu.edu

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(AMPA) receptor transmission. These compounds bind to a modula-tory site on the AMPA receptor to reduce the kinetics of channel de-activation and desensitization, and they have been documented toenhance learning and memory in rodents and possibly humans(17–19). Using in vivo two-photon imaging, we show that KX causesa substantial and prolonged reduction in neuronal Ca2+ activity thatpersists into the period of post-anesthesia recovery. We found thatadministration of CX546 after anesthesia in mice restores neuronalCa2+ activity and prevents neonatal anesthesia–induced motor learningdeficits in adulthood. Furthermore, CX546 rescues KX anesthesia–induced synaptic deficits, including decreased expression of synapticprotein and impaired synaptic functional and structural plasticity as-sociated with learning. Together, our results indicate that pharmaco-logically enhancing neuronal activity by the AMPAkine CX546 duringthe post-anesthesia recovery period effectively reduces learning andsynaptic deficits caused by early exposure to anesthesia.

RESULTS

CX546 enhances neuronal activity during thepost-anesthesia periodTo determine the effect of neonatal KX anesthesia on neuronal activ-ity, we performed in vivo calcium imaging of layer V (L5) pyramidalneurons in the motor cortex of 2-week-old mice with or withoutanesthesia (Fig. 1A). We used transgenic mice expressing the genet-ically encoded calcium indicator GCaMP specifically in L5 pyrami-dal neurons to monitor neuronal activity (Fig. 1B) (20, 21). Wefound that the level of somatic Ca2+ transients was greatly reducedafter an intraperitoneal injection of KX [ketamine (20 mg/kg) andxylazine (3 mg/kg)] when compared to that under awake conditions(Fig. 1, B and C; total integrated DF/F0: 7.9 ± 0.7% versus 32.7 ±1.6%, P < 0.0001). MK801, an antagonist of the NMDA receptor,mimicked the effect of KX on somatic Ca2+ transients (Fig. 1C), sug-gesting that the reduction in neuronal activity during KX anesthesia

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is mainly mediated by NMDA receptor blockade. Although micestarted to wake up and exhibited voluntary movements within 1 hourafter KX injection as indicated by EMG recording (Fig. 1D), the levelof somatic Ca2+ transients remained significantly lower as comparedto the awake state (DFKX–1 h/DFawake: 0.52 ± 0.04; P < 0.0001; Fig. 1, Dand E). The reduction in somatic Ca2+ transients persisted for at least2 hours after the injection of KX (DFKX–2 h/DFawake: 0.71 ± 0.05; P =0.0072). Thus, KX anesthesia causes a reduction in neuronal activitythat persists into the post-anesthesia recovery period.

Previous studies have shown that AMPAkines are allosteric mod-ulators that directly potentiate AMPA receptor–mediated synaptictransmission (17). To test whether AMPAkine treatment is effectivein restoring neuronal activity in post-anesthesia mice, we treated micewith CX546 1 hour after KX injection (Fig. 1D). We found that asingle injection of CX546 caused a dose-dependent increase in neuronalactivity (Fig. 1E). Neuronal activity was significantly increased within15 min after CX546 injection (20 mg/kg, intraperitoneally; P <0.0001). Thirty minutes after CX546 injection (90 min after KXinjection), the level of somatic Ca2+ transients in cortical pyramidalneurons was comparable to that during the pre-KX awake state(normalized change in Ca2+, 1.14 ± 0.12), indicating that CX546was effective in restoring neuronal activity during post-anesthesia re-covery. In addition to CX546, we found that another AMPAkine drug,CX516, also increased neuronal activity in the period of post-anesthesiarecovery (fig. S1). At the same dosage (20 mg/kg, intraperitoneally), theeffect of CX516 on L5 somatic Ca2+ activity was ~68% of CX546.Together, these results demonstrate that potentiation of AMPA receptoractivity with AMPAkines helps to restore neuronal activity in post-anesthesia mice.

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CX546 rescues motor learning deficits induced byrepeated anesthesiaPrevious studies have shown that three exposures, but not a single ex-posure to KX anesthesia, during postnatal days 14 to 18 (P14–18) im-pair the animals’ motor learning in adolescence (14). We nextdetermined whether administration of AMPAkines in post-anesthesiamice may prevent motor learning deficits induced by multiple anestheticexposures (Fig. 2). Because CX546 is more effective than CX516 in en-hancing neuronal activity, we chose CX546 to test our hypothesis.Specifically, mice received one or three injections of KX [ketamine(20 mg/kg) and xylazine (3 mg/kg)] or saline during the second or thirdpostnatal week. In either adolescence (P30) or adulthood (P60), micewere trained to run on an accelerated rotarod (Fig. 2, A and B). In thisrotarod running task, the animals learn to change their gait pattern tomaintain their balance on an accelerated rotating rod (22, 23). Therotarod performance was measured by the average running speed thatmice mastered after training. Consistent with previous studies (14),although a single exposure to KX anesthesia has no apparent effecton the animals’ behavior at P30, mice subjected to multiple sessionsof anesthesia during P7–11 or P14–18 showed worse rotarodperformance after 2-day training at both 1 month (Fig. 2A) and2 months of age (Fig. 2B). Because the rotarod performance on the firsttraining day was not significantly different among all the groups, thisfinding indicates impaired motor skill learning after repeated anesthe-sia exposure. This deficit in the rotarod task was not observed ingroups of mice that received an intraperitoneal injection of CX546(20 mg/kg) 1 hour after each KX injection (Fig. 2, A and B). Admin-istration of CX546 alone had no significant effects on the animals’rotarod performance (P > 0.5, Tukey’s post hoc test).

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Fig. 1. CX546enhancesneuronal activityduring thepost-anesthesiape-riod. (A) Schematic illustrating the experimental approach for in vivo Ca2+ im-

(0.25mg/kg) injection (58 to 62 cells from fivemice for each group, unpairedt test). (D) Neuronal calcium fluorescence traces and electromyography

agingof cortical neurons in awakemice. (B) Two-photon imagesof L5neuronsexpressing GCaMP6s before KX injection (awake) and 15min and 1 hour afterKX injection. Scale bar, 10 mm. (C) Cells show a large decrease in somaticCa2+ 15min after KX [ketamine (20mg/kg) and xylazine (3mg/kg)] orMK801

(EMG) signals before and after KX injection, with and without CX546 treat-ment. Examples of 25-s traces are shown. i.p., intraperitoneally. (E) Normal-izedneuronal Ca2+ activity over time after KX andCX546 injection (n=5miceper group, unpaired t test). Data are presented as means ± SEM.

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Fig. 2. CX546 rescues motor learning deficits induced by repeated KXanesthesia. (A andB) Animals receivedoneor three injections of KX at either

table S2). At both P30 (C) and P60 (D), mice with repeated KX exposures atP7–11 displayed higher proportions of untrained gait features such as drag,

P7–11 or P14–18 andwere tested on the rotarod running task at 1 (A) or 2 (B)months of age. Rotarod performance is expressed as the average speedreached during the first (pre-training) and last (post-training) training session(n= 7 to 10mice per group). Two-way analysis of variance (ANOVA) followedby Tukey’s post hoc test. See table S1 for statistics and details. n.s., not sig-nificant. (C and D) Analysis of the animals’ gait patterns when running on atreadmill (n = 8 to 12mice per group, individual animal values were listed in

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wobble, and sweep but less trained gait feature (steady run) after 1-hourtraining. Treadmill performance is expressed as percent difference in eachsubject’s trained gait feature between post-training and pre-training. Withineach treatment group, post-training performance was compared to pre-training baseline using paired t test. Comparisons between groupswere per-formed with one-way ANOVA followed by Tukey’s post hoc test. Data arepresented as means ± SEM.

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In addition to the rotarod task, we observed motor learning deficitsin another motor learning paradigm, treadmill running, where micelearned to change their gait patterns progressively (Fig. 2, C and D).In this treadmill running task, the animals’ gait patterns were classifiedas drag, wobble, sweep, and steady run (24). Mice displayed largeproportions of untrained gait features (drag, wobble, and sweep) whenthey first ran on the treadmill, whereas the percentage of trained gaitfeature (steady run) increased after training. In both adolescent andadult mice, when treadmill performance was assessed 5 hours afteran initial 1-hour training, we found significant increases in trained gaitfeature in saline-treated controls (post-training versus pre-training:P = 0.0005 and P < 0.0001, paired t test), whereas mice with repeatedKX exposure failed to show training-related performance improvement(Fig. 2, C and D). Mice that were given CX546 after KX anesthesiashowed similar increases in treadmill performance as the control group(Fig. 2, C and D). Together, these observations indicate that long-lastingmotor learning deficits caused by repeated KX anesthesia during earlydevelopment could be rescued by administration of CX546 during thepost-anesthesia recovery period.

CX546 treatment prevents the reduction of synaptic proteinexpression after repeated anesthesiaThe results above indicate that pharmacologically enhancing neuronalactivity with CX546 during post-anesthesia recovery effectively preventsmotor learning deficits induced by early exposure to anesthesia. To ex-plore the mechanisms underlying anesthesia-induced learning deficitsand CX546-mediated protection, we examined the amounts of variousproteins in the cortex after repeated KX exposure, focusing on thoseinvolved in synaptic activity and function. Synaptosome and whole-cell preparations were generated from the cortex of P30 and P60mice that had received three KX injections during P7–11, andprotein concentrations were determined by Western blot analysis(Fig. 3A). We found that in both adolescent and adult mice, post-synaptic glutamate NMDA receptor subunits (GluN1 and GluN2A)and the glutamate AMPA receptor subunits (GluA1 and GluA2)were significantly decreased in synaptosomes from KX-treated miceas compared to saline-treated controls (P30: P = 0.0068, 0.0344,0.0317, and 0.0400; P60: P = 0.0083, 0.05, 0.0104, and 0.0306, Tukey’spost hoc test; Fig. 3, B to D). The amounts of GluN1, GluN2A, GluA1,and GluA2 in the whole-brain fraction, however, remained unaltered(Fig. 3, E to G). These results demonstrate that repeated anesthesia atthe neonatal stage causes long-lasting reduction of glutamate receptorsat synapses. Mice that received both KX and CX546 exhibited normalsynaptic expression of NMDA and AMPA receptor subunits duringadolescence and adulthood (P > 0.2 versus saline; Fig. 3, B to D), sug-gesting that potentiating neuronal activity with CX546 during thepost-anesthesia period prevents synaptic deficits later in life.

CX546 treatment rescues training-evoked neuronal activityafter repeated anesthesiaIonotropic glutamate receptors are responsible for the glutamate-mediated postsynaptic excitation of neurons (Fig. 1C) (25, 26). Therescue of anesthesia-induced reduction of NMDA and AMPA recep-tors at synapses by CX546 suggests that deficits of neuronal functionmay also be rescued. To test this, we examined the neuronal activity inthe primary motor cortex by performing in vivo Ca2+ imaging of L5pyramidal neurons while mice performed a motor skill task. In thisexperiment, mice received three KX or saline injections at P7–11.

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At either P30 or P60, mice were head-restrained and trained to runforward on a custom-built, free-floating treadmill under a two-photonmicroscope (Fig. 4A) (24). L5 pyramidal neurons of the motor cortexwere imaged across three two-photon imaging planes, which span thecortical column (Fig. 4B). Here, we were able to record Ca2+ activity inapical tuft dendrites (10 to 50 mm from pial surface), in apical trunkdendrites (200 to 250 mm from pial surface), and at the level of the L5soma (500 to 600 mm from pial surface) during the quiet resting stateand forward running (Fig. 4C; total recording time, 2.5 min for restingand 2.5 min for treadmill running). We observed a significant reduc-tion in the number of Ca2+ transients generated in the apical tuft/trunk dendrites and soma of L5 neurons when 1-month-old mice withneonatal KX exposure were running on the treadmill as comparedwith saline-injected controls (Fig. 4, D and E; tuft, P = 0.0016; trunk,P = 0.0453; L5 soma, P = 0.0472, Tukey’s post hoc test). The peakamplitudes (DF/F0) of Ca

2+ transients in the apical tuft, apical trunk,and L5 soma were not significantly different in KX-treated mice ascompared to saline-injected controls (Fig. 4F). CX546 treatment afterrepeated KX at P7–11 rescued training-evoked Ca2+ transients generatedin the apical tuft, apical trunk, and L5 soma to the same level as the salinecontrols (Fig. 4, D and E). CX546 treatment did not alter the peak am-plitude of Ca2+ transients generated during running as compared to KX-treated mice (Fig. 4F). Similar to what was observed in 1-month-oldanimals, we found that at P60, the level of motor training–evoked Ca2+

activity in L5 neurons remained significantly lower in KX-treated micerelative to saline-treated controls (tuft, P < 0.0001; trunk, P = 0.0008;L5 soma, P < 0.0001) but not in mice with CX546 treatment (Fig. 4G).Together, these results show that mice with repeated KX anesthesia dur-ing early development have decreased motor training–evoked neuronalactivity and that CX546 reverses these changes in neuronal activity.

CX546 treatment partially rescues motor learning–induceddendritic spine formation after repeated anesthesiaThe formation and elimination of synaptic connections are thought toplay an important role in learning and memory formation (23, 27–32).Our previous studies have shown that repeated exposure to KX inmice at P14–18 results in the reduction of motor learning–dependentdendritic spine formation (14). To examine whether CX546 rescues def-icits in motor learning–induced spine plasticity, we first examined thedynamics of postsynaptic dendritic spines of L5 pyramidal neuronsin the motor cortex of Thy1-YFP (yellow fluorescent protein) micethat received a single or repeated KX exposure at P7–11 (Fig. 5A). Un-der baseline conditions (no motor training), we found that spine for-mation and elimination over 2 days in 1-month-old mice that receiveda single or repeated KX exposure at P7–11 were comparable to thosein saline-treated controls (Fig. 5, B and C). These results indicate thatearly exposure to KX does not alter the baseline dynamics of dendriticspines in the motor cortex in adolescence.

Next, we examined whether motor learning–induced spine remod-eling is altered after repeated KX exposure (Fig. 5D). We found sig-nificant decreases in motor learning–induced spine formation (7.8 ±0.4%, 742 spines, n = 5 mice; P < 0.0001, Bonferroni’s post hoc test)and spine elimination (5.1 ± 0.2%; P = 0.0125) in 1-month-old micethat have received three KX injections at P7–11 as compared withsaline-treated controls (formation, 13.4 ± 0.8%; elimination, 7.5 ± 0.5%;687 spines, n = 5 mice) (Fig. 5, D to F). A single injection of KX at P7had no effects on motor learning–induced spine remodeling. Mice withconsecutive administrations of KX and CX546 exhibited significant

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Fig. 3. CX546 treatment prevents the reduction of synaptic protein ex-pression after repeated KX anesthesia. (A) Experimental design. (B) Syn-

ANOVA followed by Tukey’s post hoc test). (E) Whole-cell lysates weregenerated from the mouse cortex and probed with indicated antibodies

aptosome fractions were generated from the cortex of P30 or P60 miceafter various treatments and probed with indicated antibodies by West-ern blot. (C andD) Densitometric quantification of Western blots from thesynaptic fractions at P30 (C) and P60 (D) (n = 4 mice per group, one-way

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by Western blot. (F and G) Western blot quantification from whole-celllysates at P30 (F) and P60 (G) (n= 4mice per group). Each circle representsan individual animal. Data are presented as means ± SEM. See table S3 forstatistics and details.

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increases in motor learning–induced spine formation (10.6 ± 0.5%,676 spines, n = 5 mice; P = 0.0198) and spine elimination (7.7 ± 0.6%;P = 0.0071) as compared to mice with KX injections only. Similar resultswere observed in adult mice at 2 months of age: Motor learning–inducedspine formation was significantly lower in mice with three KX injectionsat P7–11 (6.1 ± 0.6%; P < 0.0001) but not in mice with CX546 treatmentafter anesthesia (8.8 ± 0.3%), as compared with saline-treated controls(10.4 ± 0.3%) (Fig. 5G). We did not find significant differences in motorlearning–induced spine elimination among the three groups of mice atP60 (Fig. 5H). These results show that CX546 treatment rescues motorlearning–induced dendritic spine plasticity in the motor cortex of micesubjected to repeated neonatal anesthesia.

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DISCUSSION

With an increasing number of infants and young children exposedto anesthetic agents each year, there is a growing concern about the

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safety of general anesthesia for the developing brains. Growing pre-clinical evidence indicates that early-life anesthesia could impair thedevelopment of neuronal circuits and cause long-lasting learningdeficits (9–13). Here, we show that KX anesthesia causes a prolongedreduction of neuronal activity in the motor cortex. The reduction inneuronal activity extends well into the post-anesthesia recovery periodand can be rescued rapidly after administration of an AMPAkinedrug, CX546. Adult mice with repeated KX anesthesia during the sec-ond postnatal week display deficits in motor skill learning andlearning-induced synaptic plasticity. Administration of CX546 duringpost-anesthesia recovery prevents anesthesia-induced motor learningand synaptic impairments. Together, our studies indicate that phar-macologically enhancing neuronal activity during the post-anesthesiarecovery period could be an important strategy for reducing adverseeffects caused by early exposure to anesthesia.

Ketamine is a dissociative anesthetic that primarily blocks NMDAreceptors (2, 33). In rodents, ketamine is frequently used together withthe a2-adrenoreceptor agonist xylazine to decrease the ketamine dose

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Fig. 4. CX546 treatment rescues training-evokedneuronal activity afterrepeated KX anesthesia. (A) Experimental design. (B) Two-photon Ca2+ im-

change in Ca2+ transient generation in tuft/trunk dendrites and soma ofP30 mice during running compared to rest (n = 4 to 8 mice per group;

aging in the primary motor cortex of awake, head-restrained mice runningon a treadmill. (C) Examples of calcium images acquired from tuft, trunk, andsoma of the L5 neurons (shown by arrowheads). (D) Calcium fluorescencetraces of apical tuft dendrites, apical trunk dendrites, and L5 soma under var-ious treatment conditions. Examples of 2.5-min traces are shown. (E) Fold

one-way ANOVA followed by Tukey’s post hoc test). (F) Peak amplitude ofCa2+ transients generated during running. (G) Fold change in totalintegrated Ca2+ activity in tuft, trunk, and soma of P60 mice during runningcompared to rest (n=4miceper group). Data are presented asmeans±SEM.See table S4 for statistics and details.

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for anesthesia induction. Although a single injection of ketamine(20 mg/kg) combined with xylazine (3 mg/kg) produces a light sur-gical level of anesthesia in young mice for less than 1 hour (14), wefound that neuronal activity in the cortex remains decreased for atleast 2 hours, suggesting a prolonged aftereffect of KX on brain activ-ity. Blockade of NMDA receptors with MK801 produced a similar ef-fect on neuronal Ca2+ dynamics as KX, suggesting that the effect ofKX on neuronal activity is largely caused by the blockade of NMDAreceptors. Although the KX combination is not used clinically, keta-mine is widely used in pediatric patients for either anesthetic or sed-ative purposes. It is possible that pediatric patients undergoingketamine anesthesia or sedation may suffer from reduced neuronalactivity as we observed in the mouse brain. Furthermore, other anes-thetics used in pediatric medicine could also cause prolonged reduc-tion in neuronal activity during emergence from anesthesia and mayimpair brain development.

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Given the important role of neuronal activities in neural develop-ment, we reasoned that under the circumstance when anesthesia is notoptional, swift restoration of neuronal activity and calcium influx intoneurons during the recovery phase of general anesthesia could be aneffective strategy to reduce its adverse effects. Realizing well-documentedneurotoxic effects of some NMDA receptor agonists, we focusedour attention on compounds that potentiate AMPA receptor activity.AMPAkines are a group of small compounds that slow the onset ofAMPA receptor desensitization and/or deactivation, thereby in-creasing fast excitatory transmission (17). These compounds do nothave agonistic or antagonistic properties but instead modulate the re-ceptor rate constants for transmitter binding, channel opening, and de-sensitization. They can freely cross the blood-brain barrier (34) andhave been documented to enhance learning and memory in animaland human studies (19). Our studies show that CX546 is effective inrestoring neuronal activity during the post-anesthesia recovery period

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and preventing repetitive KX-inducedmotor learning deficits.

Although it has become increasinglyclear that prolonged exposure to generalanesthesia could cause substantialchanges in the developing brain (9–12, 35),the precise neuropathology underlyingcognitive dysfunction in adulthood isnot clear. Here, we found decreases ina number of synaptic proteins (GluN1,GluN2A, GluA1, and GluA2) in the cor-tex of both adolescent and adult micewith repeated neonatal exposure to KXanesthesia. There were no changes inthe abundance of GluN1, GluN2A,GluA1, and GluA2 in the whole-cell frac-tion. The differences in glutamate re-ceptor subunit expression betweensynaptosome and whole-brain fractioncould be due to the fact that these pro-teins are not only expressed at synapticsites but also present in nonsynaptic sitesof neurons, as well as in glial cells. Giventhe pivotal roles of ionotropic glutamatereceptors in fast excitatory synaptictransmission in the brain (25), reducedexpression of NMDA and AMPA recep-tor subunits identified here can have amajor impact on the level of neuronalnetwork activity. Using in vivo Ca2+ im-aging, we found that motor training–evoked Ca2+ transients in L5 pyramidalneurons of the motor cortex were re-duced in mice with repeated ketamineexposure. Together, these findings showthat repeated exposure to anesthesiacauses long-lasting changes in synapsematuration, which in turn contributes tomotor learning deficits observed in ourstudy.

Synaptic structural plasticity in theprimary motor cortex is important for

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Fig. 5. CX546 treatment partially rescues motor learning–induced dendritic spine remodeling af-ter repeated KX anesthesia. (A) Experimental design. (B and C) Percentages of dendritic spines formed

(B) and eliminated (C) over 2 days in saline- and KX-treated mice at 1 month of age (n = 4 to 5 mice pergroup). (D) In vivo time-lapse imaging of the same dendritic segments before and 2 days after rotarodmotor training in the primary motor cortex of 1-month-old animals that received various treatments.Filled and empty arrowheads indicate dendritic spines that were formed and eliminated between thetwo views, respectively. Asterisks indicate dendritic filopodia. (E and F) Percentages of dendritic spinesformed (E) and eliminated (F) over 2-day motor training in 1-month-old adolescent mice (n = 4 to 5 miceper group, one-way ANOVA followed by Bonferroni’s post hoc test). (G and H) Percentages of dendriticspines formed (G) and eliminated (H) over 2-day motor training in 2-month-old adult mice (n = 4 micefor each group). Each circle represents an individual animal. Data are presented as means ± SEM. Seetable S5 for statistics and details.

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motor skill learning (23, 28, 30). Previous studies have demonstratedthat rotarod training over 2 days results in a 5 to 7% increase in newspines in the motor cortex (23, 30). A fraction of training-associatednew spines persist over weeks, and the amount of persistent newspines strongly correlates with the animals’ performance (23, 30). Al-though baseline spine dynamics remain unaltered in mice with re-peated KX exposures, we observed a reduction in learning-induceddendritic spine remodeling after 2-day training. Together with thefinding of decreased neuronal activity during training in these mice,these results suggest that after early anesthetic exposure, corticalcircuits are less responsive to modulation by learning experienceand exhibit reduced synaptic plasticity in adulthood. The reductionsin both motor learning–induced neuronal activity and synaptic struc-tural plasticity can be rescued by CX546 treatment, further supportingthe therapeutic potential of CX546 in ketamine anesthesia–inducedcognitive impairments.

The mechanisms by which CX546 alleviates anesthesia-inducedsynaptic and learning deficits remain to be determined. The beneficialeffect of CX546 could be simply due to the shortening of the post-anesthesia depression of cortical activity as a result of potentiation ofAMPA receptor activity by CX546. In addition to restoring corticalactivity, CX546 may cause other changes in the brain to offset the del-eterious effects of anesthetic exposure on the developing brain. Forexample, it has been shown that AMPAkine treatments increase theexpression of brain-derived neurotrophic factor (BDNF) (36–39).BDNF is a potent regulator of synaptic plasticity (40) and thereforecould have an important role in the rescue of synaptic and behavioralphenotypes associated with early anesthetic exposure. Future studiesare needed to determine whether brief AMPAkine treatments wouldelicit the positive trophic effects associated with the neurotrophin inthe developing brain.

Our experiments with CX546 treatment are proof-of-principle stu-dies, which support the hypothesis that pharmacologically enhancingneuronal activity is an effective strategy for the treatment of long-lastingeffects of early exposure to anesthesia. Over the past decade, AMPA-kines have been favored for the treatment of cognitive deficits. Severalcompounds have generated very promising preclinical results in thetreatment of a number of neurological disorders such as Alzheimer’s dis-ease, Huntington’s disease, schizophrenia, and depression (19, 39, 41).They have also been used to improve cognition in healthy and elderlyhuman volunteers (42). Drugs like CX546 and CX516 appear to be in-herently safe because their ability to prolong AMPA receptor activity iskinetically limited. In the future, it would be of great interest and impor-tance to test whether these drugs are effective at minimizing the risk forthe development of learning disabilities in children who receive multipleand prolonged anesthesia.

MATERIALS AND METHODS

Study designThe objective of this study was to investigate the effects of potentiatingAMPA receptor activity on mitigating learning and synaptic deficitsinduced by neonatal anesthesia. Mice were randomly assigned to threeexperimental groups: (i) saline-treated controls, (ii) three episodes ofanesthesia, and (iii) three episodes of anesthesia plus AMPAkine treat-ment. For all three groups of mice, motor learning performance wasexamined by rotarod and treadmill running tasks, synapse protein ex-

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pression was measured byWestern blot, and training-evoked neuronalactivity and synapse structural remodeling were determined by in vivotwo-photon imaging. No statistical methods were used to pre-determine sample size. Group sample size was chosen on the basisof previous studies using the same methodologies, and variance wassimilar between groups being statistically compared. The researcherswere blind to group assignment, and no data points were excludedfrom the statistical analysis.

Experimental animalsThy1-YFPmice (H line) (43) were purchased fromThe Jackson Laboratoryand used for dendritic spine imaging experiments. Thy1-GCaMP6slowmice (line 1) and Thy1-GCaMP2.2c mice (21) were used for Ca2+ im-aging experiments. Mice were group-housed in New York UniversitySkirball animal facility. All experiments were performed in accordancewith institutional guidelines. For early anesthesia treatment, mice weregiven an intraperitoneal injection of KX mixture [ketamine (20 mg/kg)and xylazine (3 mg/kg)] every other day (P7, P9, and P11 or P14, P16,and P18) for a total of three injections. One injection of KX at this doseinduces anesthesia and provides immobilization in neonatal mice for~1 hour and does not cause cell apoptosis (14). Control animals receivedsaline injections. For CX546 treatment, mice received an intraperitonealinjection of CX546 (20 mg/kg) 1 hour after each KX injection. Duringanesthesia, a heating pad was used to maintain the animal’s body tem-perature at about 37°C.

In vivo Ca2+ imagingIn vivo Ca2+ imaging was performed in awake, head-restrained mice.The surgical procedure for preparing awake animal imaging has beendescribed previously (44). In brief, a head holder composed of twoparallel micro-metal bars was attached to the animal’s skull to reducemotion-induced artifact during imaging. Surgical anesthesia wasachieved with an intraperitoneal injection of KX [ketamine (100 mg/kg)and xylazine (15 mg/kg)]. A midline incision of the scalp exposedthe periosteum, and a small skull region (~0.2 mm in diameter)was located over the right motor cortex based on stereotacticcoordinates (0.5 mm posterior from the bregma and 1.5 mm lateral fromthe midline) and marked with a pencil. A thin layer of cyanoacrylate-based glue was first applied to the top of the entire skull surfaceand to the metal bars, and the head holder was then further fortifiedwith dental acrylic cement. The dental cement was applied so that awell was formed, leaving the motor cortex with the marked skullregion exposed between the two bars. All procedures were performedunder a dissection microscope. After the dental cement was com-pletely dry, a cranial window was created over the previously markedregion. The procedures for preparing a thinned-skull cranial windowfor two-photon imaging have been described in detail in previouspublications (45). The completed cranial window was covered withsilicon elastomer, and mice were given at least 1 day to recover fromthe surgery-related anesthesia. Later, mice with head mounts werehabituated to the imaging apparatus a few times (10 min each time)to minimize potential stress effects of head restraining and imaging.

To image neuronal Ca2+ activity in the cortex of awake mice, thehead holder was screwed to two metal cubes attached to a solid metalbase, and the silicon elastomer was peeled off to expose the thinnedskull region and artificial cerebrospinal fluid was added to the well.The head-restrained animal was then placed on the stage of a two-photon microscope. These in vivo Ca2+ imaging experiments were

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performed using an Olympus two-photon system equipped with aDeepSee Ti:Sapphire laser (Spectra-Physics). The average laserpower on the sample was ~20 to 30 mW. Most experiments wereacquired at frame rates of 2 Hz at a resolution of 256 × 256 pixelsusing a 25× water immersion objective. Image acquisition was per-formed using Olympus FV1000 software and analyzed post hocusing National Institutes of Health (NIH) ImageJ software. DF/F0was calculated by (F − F0)/F0, where F0 is the baseline fluorescencesignal averaged over a 2-s period. In Fig. 4E, Ca2+ transients weredefined as the events when changes of fluorescence (DF/F0) observedin dendrites or soma were >20% during the 2.5-min imagingsessions.

Motor skill trainingTwo motor tasks were used in this study. For the rotarod running task,we used an EZRod system with a test chamber (44.5 cm × 14 cm × 51 cmdimensions) to perform the test. Animals were placed on a motorizedrod (30 mm in diameter) in the chamber. The rotation speedincreased gradually from 0 to 100 rpm over the course of 3 min.The time latency and rotation speed were recorded when the animalswere unable to keep up with the increasing speed and fell. Rotarodtraining/testing was performed in one 30-min session (20 trials) perday. Rotarod performance was measured as the average speed animalsachieved during the 20-trial training session. A treadmill running taskwas introduced to provide a different motor training, as well as to per-form two-photon Ca2+ imaging and motor training at the same time.A custom-built, free-floating treadmill (96 cm × 56 cm × 61 cmdimensions) was used to allow head-fixed mice to move their fore-limbs freely to perform motor running tasks. At the onset of a trial,the motor was turned on and the belt speed gradually increased from0 to 8 cm/s within ~3 s, and the speed of 8 cm/s was maintained forthe rest of the trial.

Isolation of synaptosome fractions and Western blotMice were deeply anesthetized and perfused with 40 ml of Ca2+/Mg2+-freeDulbecco’s phosphate-buffered saline. The cortex was dissected andhomogenized with a dounce homogenizer in buffer A (320 mM su-crose, 1 mM NaHCO3, 1 mMMgCl2, and 0.5 mM CaCl2) and clearedof nuclei and insoluble material by centrifugation. The resulting super-natant was centrifuged at 30,000g to pellet membranes, and the pelletwas subsequently resuspended in buffer B (320 mM sucrose and1 mMNaHCO3). Membranes were fractionated using a discontinuoussucrose gradient consisting of 1.0 and 1.2 M sucrose at 120,000g for2 hours at 4°C. After centrifugation, the synaptosomal fraction wasisolated at the 1.0/1.2 interface, diluted in buffer B, and pelleted at120,000g for 45 min at 4°C. The resulting synaptosomes were so-lubilized in 25 mM tris (pH 7.4) and 2% SDS. Protein content wasdetermined by bicinchoninic acid assay (Thermo Scientific), and 10 mgof total protein was loaded per lane on a 10% SDS–polyacrylamide gelelectrophoresis gel. Separated proteins were transferred to a poly-vinylidene difluoride membrane (Millipore) and blocked for 30 minwith 2% bovine serum albumin in tris-buffered saline–Tween 20.Blocked membranes were probed overnight with the following anti-bodies: GluN1, GluN2A, GluA1, GluA2 (NeuroMab), and actin(Sigma). After washing, membranes were incubated with anti-rabbitor anti-mouse immunoglobulin G–horseradish peroxidase secondaryantibodies (Jackson ImmunoResearch), washed, and incubated withenhanced chemiluminescence reagent (GE Life Sciences) before ex-

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posure to film. Densitometry analysis was performed by manualscanning and digitalization of film, and quantified using the gel anal-ysis plugin for NIH ImageJ software.

In vivo imaging of dendritic spines and data analysisThe surgical procedure for chronic transcranial two-photon imaginghas been described previously (45). While the animal was under deepanesthesia, the skull surface was exposed with a midline scalp incision,and a small skull region (~0.2 mm in diameter) was located over theprimary motor cortex based on stereotaxic coordinates. A custom-made, stainless steel plate was glued to the skull with a central openingover the cortical region of interest. To create a cranial window for im-aging, the skull surface was immersed in artificial cerebrospinal fluid,and a high-speed drill and a microsurgical blade were used to carefullyreduce the skull thickness to about 20 mm under a dissection micro-scope. The entire surgical procedure usually took less than 30 min,and the two-photon imaging took place immediately after the skullthinning. During imaging, the animal was placed under an Olympustwo-photon microscope with the laser tuned to the optimal excitationwavelength for YFP (920 nm). Low laser power (20 to 30 mW at thesample) was used during imaging to minimize phototoxicity. Theimages were acquired with a 60× water immersion objective (numer-ical aperture, 1.1) at a zoom of 1.0 to 3.0. A stack of image planeswithin a depth of 100 mm from the pial surface was collected, yieldinga full three-dimensional data set of dendrites in the area of interest.The step size was 2 mm for the initial low-magnification image (nozoom) for relocation at later time points and 0.75 mm for all the otherexperiments (×3.0 zoom). After imaging, the plate was gently detachedfrom the skull, and the scalp was sutured with 6-0 silk. The animalswere returned to their home cages until the next view.

Data analysis was performed with NIH ImageJ software as de-scribed previously (23, 46). The same dendritic segments were identi-fied from three-dimensional image stacks taken at both time points. Thenumber and location of dendritic protrusions were identified in eachview. Filopodia were identified as long, thin structures without enlargedheads, and the rest of the protrusions were classified as spines. Spineswere considered the same between two views based on their spatial re-lationship to adjacent landmarks and spines. Spines in the second viewwere considered different if they were more than 0.7 mm away fromtheir expected positions based on the first view. The formation or elim-ination rates of spines were measured as the number of spines formedor eliminated divided by the number of spines existing in the first view.

StatisticsPrism software (GraphPad 6.0) was used to conduct the statisticalanalysis. Data were presented as means ± SEM. Tests for differencesbetween populations were performed using Student’s t test or a one-or two-way ANOVA followed by Tukey or Bonferroni post hoc test asspecified in the text. For the post hoc multiple comparisons, all thedrug treatment groups were compared with the control group. Signif-icant levels were set at P ≤ 0.05.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/344/344ra85/DC1Fig. S1. CX516 enhances neuronal activity during the post-anesthesia period.Table S1. Statistics and details for rotarod behavioral data.Table S2. Individual animals’ gait patterns when running on a treadmill.

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Table S3. Statistics and details for Western blot data.Table S4. Statistics and details for Ca2+ imaging data.Table S5. Statistics and details for dendritic spine data.

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Acknowledgments: We thank W.-B. Gan for providing Thy1-GCaMP6smice, G. Feng for providingThy1-GCaMP2.2c mice, and T. Blanck for helpful discussions. Funding: This study was supported byNIH grants GM107469, AG048410 (to G.Y.), and HD076914 (to I.N.). Author contributions: L.H. andG.Y. designed the experiments. L.H. performed andanalyzed all the experiments on animal behavior,spine imaging, andprotein level examination. L.H. and J.C. conducted andanalyzed calcium imagingexperiments. I.N. helped with the neuronal activity data analysis. G.Y. wrote the article. Competinginterests: The authors declare that they have no competing interests.

Submitted 18 March 2016Accepted 23 May 2016Published 22 June 201610.1126/scitranslmed.aaf7151

Citation: L. Huang, J. Cichon, I. Ninan, G. Yang, Post-anesthesia AMPA receptor potentiationprevents anesthesia-induced learning and synaptic deficits. Sci. Transl. Med. 8, 344ra85 (2016).

ceTranslationalMedicine.org 22 June 2016 Vol 8 Issue 344 344ra85 10

synaptic deficitsPost-anesthesia AMPA receptor potentiation prevents anesthesia-induced learning and

Lianyan Huang, Joseph Cichon, Ipe Ninan and Guang Yang

DOI: 10.1126/scitranslmed.aaf7151, 344ra85344ra85.8Sci Transl Med

patients exposed to anesthesia as well.the promising results suggest that AMPAkines may be worth evaluating as neuroprotective agents for human deficits even after repeated episodes of anesthesia exposure. Although this study was performed entirely in mice,exposure to anesthesia not only restored the animals' neuronal activity but also prevented subsequent learning of drugs that can potentiate synaptic transmission of neuronal impulses. Treatment with AMPAkines shortly afterresulting from early exposure to ketamine anesthesia. The authors then treated the mice with AMPAkines, a class

. demonstrated impaired neuronal activityet alwhere urgent surgery is needed. By studying neonatal mice, Huang subsequent brain development, but the use of neonatal anesthesia can be unavoidable, for example, in situations

Numerous studies have suggested that exposure to anesthesia in early childhood adversely affectsAMPAkines protect young brains

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