Int. J. Devl Neuroscience 48 (2016) 38–49

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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Learning, memory and synaptic plasticity in hippocampus in rats exposed to sevoflurane Hongyan Xiao 1 , Bing Liu 1 , Yali Chen, Jun Zhang ∗ Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai, China

a r t i c l e

i n f o

Article history: Received 7 October 2015 Received in revised form 1 November 2015 Accepted 10 November 2015 Available online 2 December 2015 Keywords: Sevoflurane anesthesia Developing brain Neurotoxicity Synaptic plasticity Learning Memory

a b s t r a c t Purpose: Developmental exposure to volatile anesthetics has been associated with cognitive deficits at adulthood. Rodent studies have revealed impairments in performance in learning tasks involving the hippocampus. However, how the duration of anesthesia exposure impact on hippocampal synaptic plasticity, learning, and memory is as yet not fully elucidated. Methods: On postnatal day 7(P7), rat pups were divided into 3 groups: control group (n = 30), 3% sevoflurane treatment for 1 h (Sev 1 h group, n = 30) and 3% sevoflurane treatment for 6 h (Sev 6 h group, n = 28). Following anesthesia, synaptic vesicle-associated proteins and dendrite spine density and synapse ultrastructure were measured using western blotting, Golgi staining, and transmission electron microscopy (TEM) on P21. In addition, the effects of sevoflurane treatment on long-term potentiation (LTP) and long-term depression (LTD), two molecular correlates of memory, were studied in CA1 subfields of the hippocampus, using electrophysiological recordings of field potentials in hippocampal slices on P35-42. Rats’ neurocognitive performance was assessed at 2 months of age, using the Morris water maze and novel-object recognition tasks. Results: Our results showed that neonatal exposure to 3% sevoflurane for 6 h results in reduced spine density of apical dendrites along with elevated expression of synaptic vesicle-associated proteins (SNAP-25 and syntaxin), and synaptic ultrastructure damage in the hippocampus. The electrophysiological evidence indicated that hippocampal LTP, but not LTD, was inhibited and that learning and memory performance were impaired in two behavioral tasks in the Sev 6 h group. In contrast, lesser structural and functional damage in the hippocampus was observed in the Sev 1 h group. Conclusion: Our data showed that 6-h exposure of the developing brain to 3% sevoflurane could result in synaptic plasticity impairment in the hippocampus and spatial and nonspatial hippocampal-dependent learning and memory deficits. In contrast, shorter-duration exposure (1 h) results in less damage. These results provide further evidences that duration of anesthesia exposure could have differential effects on neuronal plasticity and neurocognitive performance. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Sevoflurane is a commonly used volatile anesthetic, particularly to induce anesthesia in the clinical pediatric context. The molecular mechanisms underlying its anesthetic effects remain unclear. However, it is known to activate the ␥-aminobutyric acid type A (GABAA ) receptors and/or inhibit N-Methyl-d-aspartate (NMDA) receptors – thereby depressing synaptic transmission – and may

∗ Corresponding author at: Huashan Hospital, Fudan University, No.12 Urumiqi middle Road, Jin’an District, Shanghai, China. Fax: +86 21 52887690. E-mail addresses: [email protected] (H. Xiao), [email protected] (B. Liu), [email protected] (Y. Chen), [email protected] (J. Zhang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijdevneu.2015.11.001 0736-5748/© 2015 Elsevier Ltd. All rights reserved.

lead to an anesthetic state associated with, for example, amnesia, unconsciousness, and analgesia (Mashour et al., 2005). In addition, previous studies suggested that potentiation of the GABAA receptor could also suppress neuronal plasticity and cause cognitive impairment (Möhler, 2007). Much experimental evidence has demonstrated that when the developing brain is exposed to sevoflurane, it could result in widespread neurodegeneration and neurocognitive dysfunction in rodents and non-human primates (Sinner et al., 2014; Jevtovic-Todorovic, 2012). This raises concerns about the effects of general anesthesia in children undergoing surgery, although a causal relationship between anesthetic exposure and developmental outcome in humans remains speculative (Olsen and Brambrink, 2013; Mellon et al., 2007; Loepke and Soriano, 2008; Nasr and Davis, 2015).

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Significant effort has been directed at identifying the mechanisms of sevoflurane-induced neurotoxicity at the cellular level, and particularly the role of neuroapoptosis and damaged neurogenesis in mediating learning and memory impairment (Stratmann et al., 2010). Some researchers have gone further recently by suggesting that persistent alternation of synaptic development and neuronal plasticity in cognition-related brain regions underlie the deficits in neurobehavioral performance, especially in hippocampus-dependent tasks (Vutskits, 2012). The hippocampus, part of a brain system responsible for learning and memory, has been shown to be an important target of general anesthetics. It has been suggested that neuronal plasticity impairment could underlie the cognitive abnormalities. However, the effects of sevoflurane exposure on synaptic plasticity in the developing brain are not completely understood. Therefore, in the present study, we investigated the role of synaptic plasticity in anesthetic-induced hippocampal-dependent impairments in learning and memory. We used neonatal rats as a model of the developing brain, and exposed them to 3% sevoflurane for 6 h. We thus explored the anesthetic’s effects on molecular, structural, and functional aspects of synaptic plasticity in the hippocampal area and on behavioral development, as manifested in performance later in adulthood. In particular, we examined the effects of a shorter sevoflurane exposure (1 h) on hippocampal synaptic plasticity, in order to compare exposure duration effects. 2. Materials and methods 2.1. Animals The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Fudan University. At P6, litters of 8–10 rat pups were obtained from each of 10 Sprague-Dawley rat dams obtained from the Animal Care Center of Fudan University. On P7, all animals from each litter were randomly assigned to either a control (Group Control) or to 2 treatment groups receiving exposure to sevoflurane: (1) for 1 h (Group Sev 1 h) or (2) for 6 h (Group Sev 6 h). For each group, subjects at P21 were either killed for molecular (western blot: n = 6) and histological (Golgi staining: n = 5 and transmission electron microscope: n = 3) assessments or were weaned and further bred until electrophysiological (n = 5) and neurobehavioral (n = 9) tests (i.e., Morris water maze, novel-object recognition) were conducted. 2.2. Anesthesia procedure The neonatal rats were separately anesthetized as we have previously described (Lei et al., 2013). Briefly, P7 rats were placed in a sealed box ventilated with 3.0% sevoflurane in 100% oxygen and treated for 6 h or 1 h at a flow rate of approximately 1 L/min. The box was adjusted to maintain a specific concentration of sevoflurane and oxygen at constant levels. Gases within the anesthetic box were monitored continuously through a gas sample line by using a monitor (Datex Ohmeda S/5, Helsinki, Finland). The temperature in the sealed box was maintained at 30 ± 1 ◦ C with a heating pad. The total survival percentage of P7 rats for 6 h anesthesia was 80% (7 deaths among the 35 pups) and at 1 h was 100% (n = 30). The control group (n = 30) received 100% oxygen but without anesthetic exposure under identical conditions as experienced by the anesthetized animals. Following anesthesia, the pups were returned to their dams for lactation. They were housed under the same standard lab housing with a 12-h light/dark cycle and a regulated temperature (20–25 ◦ C) and humidity (45–65%). After weaning, the rats were housed 3 per cage and had free access to food and water. At P21 or P35, the brain samples were prepared according to experimen-

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tal requirements, which are described in experimental methods, respectively. All behavioral experiments were performed during the light phase between 7:00 AM and 7:00 PM. 2.3. Protein extraction from hippocampal tissue and western blot analysis The rats on P21 were then sacrificed by rapid decapitation, and bilateral hippocampus areas were harvested and stored at −80 ◦ C until used in western blot analysis. Western blot was performed as described previously (Lei et al., 2013; Liu et al., 2015). Hippocampal tissues were homogenized by brief sonication in RIPA buffer (Millipore, Temecula, CA, USA) containing a complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride (PMSF) solution. The homogenate was centrifuged at 12,000 rpm for 15 min at 4 ◦ C. After the protein samples were quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), 60 ␮g of each sample was electrophoresed through a 10% sodium dodecyl sulfate-polyacrylamide gel and wet electrotransferred to nitrocellulose membranes (Millipore). The blots were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl, 0.1% Tween 20, 20 mM Tris, pH 7.4) for 1 h, then incubated overnight at 4 ◦ C with anti-syntaxin (1:200, sc-13994, Santa Cruz Biotechnology, CA, USA), anti-SNAP25 (1:4000 dilution, BD Biosciences, CA, USA), anti-synaptophysin (1:1000, Cell Signaling Technology, CA, USA), anti-␣-synuclein (1:1000, Cell Signaling Technology, CA, USA), primary antibodies and then incubated with the corresponding secondary antibody (1:5,000; Epitomics, Hangzhou, Zhejiang Province, China) at room temperature for 1 h. Protein signals were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology). A ␤-actin antibody (1:1,000; Santa Cruz Biotechnology, CA, USA) was used to normalize sample loading and transfer. Band intensities were densitometrically quantified using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA). Data were expressed as mean ± SEM. The changes were presented as a percentage of band intensity of the control group sample. For purposes of comparing protein expression to that in Group Sev 1 h and Group Sev 6 h, protein expression in the control group was set at 100%. 2.4. Golgi staining The hippocampus was collected bilaterally after execution at P21. Golgi-Cox staining to obtain hippocampal dendritic spine density was conducted with the FD Rapid GolgiStainTM Kit (FD Neuro Technologies Inc., Ellicott City, MD) following the manufacturer’s instructions. Coronal tissue sections of 100-␮m thicknesses were cut at room temperature using a vibratome (Leica VT1200S, Germany). After slides were dehydrated with a gradient of 50%, 75%, 95%, to 100% ethanol and cleared in xylene, we prepared the specimens with slide coverslips and sealed them with Permount. The slides were then viewed in detail with a light microscope (Leica DFC 420, Germany). We analyzed the stained spine using techniques similar to those described in other studies (Zhao et al., 2013; Han et al., 2013). Six pyramidal neurons that were well-impregnated and clearly distinguishable from others in each hippocampus were analyzed (20× objective lens). Five segments of 10 ␮m (or longer) of apical dendrites were randomly selected from each pyramidal neuron for inspection (via 100× oil immersion lens) to quantify the density of spines. Spine density of secondary apical dendrites was analyzed at proximal segments emerging at more than 50 ␮m away from the soma of the hippocampal CA1 neurons. All of these spines were required to have a clearly distinguishable base or origin and were isolated from neighboring dendrites. Spine density was calculated per 10 ␮m of dendritic length. The open-source ImageJ 1.48 r Java image-viewing and image-processing program (Wayne

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Rasband, NIH, USA) was used to calibrate the scale and enlarge the segment of the spines. All analyses were completed by an investigator blinded to the experimental condition. 2.5. Transmission electron microscopy (TEM) Three rats from each group at P21 were perfused transcardially with 60 mL of normal saline after isoflurane anesthesia, followed by 50 mL of 4% paraformaldehyde (PFA). Then the hippocampal tissues were separated immediately and immersion fixation was completed at around 1 mm3 size. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 ◦ C for 2 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing a graded ethanol dehydration series and was infiltrated using a mixture of half acetone and half resin overnight at 4 ◦ C. The tissue was embedded 24 h later in resin and cured fully, in turn, as follows: 37 ◦ C overnight, 45 ◦ C for 12 h, and 60 ◦ C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapses in the hippocampus were observed under TEM (FEI TECNAI-G2, Germany). We selected 20 synapses per group for ultrastructure analysis, which included synaptic cleft width, postsynaptic density, and curvature of synaptic interface. To do this, we used the image analysis software Image Pro Plus 6, in accordance with the Guldner and Jones’s methods (Guldner and Ingham, 1980; Jones and Devon, 1978).

2.7. Morris water maze task 2.7.1. Place navigation trials To test hippocampal-dependent spatial cognition, the rats were trained in the standard Morris water maze (MWM) with a hidden platform (Morris et al., 1982). A white escape platform (12-cm diameter) was submerged in the circular pool (160-cm diameter, at a 50-cm depth) that was filled with warm (23–25 ◦ C) opaque water. A at P60-64, each rat pup was given a daily four-trial session (30–35 min inter-trial interval) for 5 consecutive days. Each trial consisted of releasing the rat into the water facing the outer edge of the pool at one of the quadrants (in a random sequence) and letting the animal escape to the platform. They received 4 trials per day of training in searching for the submerged and unmarked platform, with trial durations of 60 s, respectively, on the platform at the end of trials. All trials were videotaped, and rat swimming paths were recorded with the ANY-maze video tracking system (Stoelting Co., IL, USA), which allowed us to measure the time taken (i.e., latency) to find the platform(s), as well as other behavioral information obtained during the spatial reference memory test. The animals were dried and placed beneath a heating lamp after completing each test. 2.7.2. Spatial probe test A probe trial was performed 1 day after the last trial at P65 with the platform removed from the pool to assess memory retention for the platform location. During the 60 s probe trial, we recorded and analyzed the swimming speed (cm/s), the swimming path tracks, and the number of entries into the platform quadrant zone.

2.6. Electrophysiological study 2.8. Novel-object recognition task Transverse hippocampal slices were prepared from rats at P3542, as reported previously (Gao et al., 2014). The number of slices corresponds to numbers of animals studied. Briefly, rats were sacrificed by cervical dislocation under deep isoflurane anesthesia. Brains were rapidly removed and briefly submerged in ice-cold artificial cerebrospinal fluid solution (ACSF, 25 mM glucose, 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2 PO4 , 25 mM NaHCO3 ,1 mM MgCl2 , and 2 mM CaCl2 ). All solutions were saturated with 95% O2 + 5% CO2 . For hippocampal slices, 400 ␮m-thick slices were prepared using a Leica VT 1200S sectioning system. Slices were transferred to an interface chamber (Harvard Apparatus, Holliston, MS) at 32 ◦ C, perfused with oxygenated ACSF (perfusion rate: 1–2 mL/min) and allowed to recover for a minimum of 2 h prior to recording. Extracellular recordings were obtained from the CA1 area. A bipolar enamel-coated platinum stimulating electrode was placed in Schaffer collateral/commissural fibers, and a glass recording electrode (resistance 1–4 M) filled with ACSF was placed in the pyramidal cells in CA1 under a microscope. Synaptic responses to electrical stimulation were collected every 1 min and averaged over 5 min using a stimulus intensity that produces 30–50% of the maximum initial slope of the extracellular field excitatory postsynaptic potential (fEPSP). The strength of synaptic transmission was determined by measuring the onset (a 30–70% rising phase) slope of fEPSP. The stimulus frequency at the baseline was 0.017 Hz. For LTP induction in area CA1, 3 high-frequency stimulation (HFS) trains (1 s at 100 Hz) were delivered with an inter-train interval of 5 min. LTD was induced with low-frequency stimulation (LFS, 900 pulses at 1 Hz for 15 min). After the HFS or LFS, changes in fEPSP amplitudes were recorded for a further 60 min. The data were recorded and analyzed with Clampex 9.0 and Clampfit 9.0 (AXON, USA). The average of fEPSP slopes during the 20 min prior to the induction of LTP or LTD was taken as the baseline, and all values were normalized to this baseline and were expressed as a mean percentage ± SEM%. Comparisons of groups were done by using Mann–Whitney U-test. P values of ≤0.05 were considered as statistically significant.

The novel-object recognition task is based on the innate tendency of rodents to differentially explore novel objects over familiar ones. It has been shown to be sensitive to hippocampal damage (Bett et al., 2013; Langston and Wood, 2010). After completing MWM tests, at P68-75, the rats were assessed in a novel-object recognition test, as described previously (Lee et al., 2014). Objectrecognition testing took place in 2 separate testing arenas, hereafter referred to as “contexts,” of identical size (61-cm square base, walls 50 cm high). The two were distinct in their appearance and texture to allow testing of context-specific memory. Context 1 had yellow walls and a base covered in wood-effect vinyl lining, while context 2 had black walls and a black plastic base. Visual cues were placed on three different walls within each context. The subjects were habituated to the contexts prior to testing. Each object was validated to avoid object bias. Investigation of an object was defined as sniffing or placing the nose within 1 cm of and oriented toward the object. Subjects were video recorded and reviewed to determine their exploration time. After 2 days of acclimation to the experimental contexts, testing began at P70 with novel-object recognition. A single trial was performed, and half of the subjects were tested in context 1 and the other half in context 2. The location (left or right) of the novel object within each context was counterbalanced among subjects. In the object-context recognition task, subjects were assessed for their ability to recognize an object within a particular context. The task required 2 separate exposures, each lasting 4 min and separated by a 2-min delay. Two trials were conducted, and the test phase occurred in opposite contexts for each trial. A discrimination index (DI), representing the relative time spent exploring each object, was calculated by: (Novel object investigation time − Familiar object investigation time)/(Total investigation time). This value was compared to a theoretical value of zero, using a one-sample t-test to assess whether a preference was shown for one of the objects, and a positive DI indicates preference for the

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novel aspect of the task. For each task, the DI of the control animals was compared against the DI of animals in 2 sevoflurane exposure groups. In addition, within the 2 sevoflurane-treated groups, the DI of Sev 1 h subjects was compared to that of Sev 6 h subjects. These comparisons were made using an unpaired t-test.

aptic density (PSD) and the synaptic curvature among three groups to have no significant differences (p > 0.05, Table 1).

2.9. Statistical analysis

We next addressed the role of sevoflurane anesthesia in synaptic function by performing extracellular recording at the Schaffercollateral CA1 pathway in acute hippocampal slices from P35-42 rats in 3 groups. Our electrophysiological results revealed that we had induced LTP in all hippocampal slices in 3 groups successfully using HFS. The baseline of the EPSP slope for the control, Sev 1 h, and Sev 6 h groups before HFS was 100.58 ± 1.63%, 102.13 ± 1.01%, 100.30 ± 2.92% (mean ± SEM), respectively. There was no significant difference in the baselines (p > 0.05). Then, EPSP slopes rose to 201.41 ± 12.50%, 215.36 ± 14.99, 171.58 ± 14.55% immediately after HFS, respectively (p > 0.05). However, the EPSP slope of the Sev 6 h group (141.48 ± 7.97%) significantly decreased 20 min after HFS, compared to the control (171.20 ± 6.91%, p < 0.05) and to the Sev 1 h groups (181.24 ± 12.59%, p < 0.05). These significant inhibitions were continuously recorded until 60 min after HFS at the end of the experiment (Fig. 4). These data demonstrated that LTP in the DG area of the hippocampus was significantly impaired in the Sev 6 h slices. The EPSP slope of Sev 1 h group was comparable to that in control group (p > 0.05, Fig. 4). In contrast, the induction of NMDA receptor-dependent LTD by single-pulse LFS was intact in Sev 6 h hippocampal slices (Fig. 5). We first recorded baseline of the EPSP slope for 20 min during LTP recording. There was no significant difference in the baseline of the EPSP slope among the control (100.82 ± 1.59%), Sev 1 h (100.44 ± 2.07%), or Sev 6 h (101.81 ± 1.28%) groups before LFS was applied (p > 0.05). With 15 min of continuous LFS, LTD in all hippocampal slices in the 3 groups was successfully induced. The EPSP slopes rapidly fell to 64.36 ± 3.88%, 61.43 ± 3.75%, 62.77 ± 4.54%, respectively, and then the EPSP slope in the control, Sev 1 h, and Sev 6 h groups were 70.77 ± 4.47%, 73.74 ± 3.10%, and 71.85 ± 6.01%, respectively, of baseline EPSP after 60 min of LFS. The statistical analysis did not show any significant difference among the 3 groups (p > 0.05, Fig. 5).

All data were presented as mean ± SEM. Statistical analyses were performed using the computerized statistical package (SPSS 19.0) and Graphpad Prism software version 5.0 (Graphpad Software, Inc., San Diego, CA, USA). One-way ANOVA followed by Tukey post hoc tests was used to evaluate differences for quantity of synaptic proteins, density of dendrite spines, ultrastructural parameters of synapses, and extracellular electrophysiological parameters of the hippocampus among Control, Sev 1 h, and Sev 6 h groups. The unpaired t-test and two-way ANOVA were used to analyze the neurobehavioral data. Differences were deemed statistically significant at probability values (p) of 0.05), in the Sev 1 h group. Both the Sev 1 h and Sev 6 h groups showed no significant differences in synaptophysin and ␣-synuclein levels, compared to the control group (p > 0.05, Fig. 1). 3.2. Sevoflurane decreases the dendritic spine density Fully impregnated CA1 pyramidal cells can be detected under Golgi staining, and the spines of the apical dendrites can be analyzed under a light microscope with a 100× oil immersion objective lens. Only the density of the dendritic spines was determined in our study, because some different types of spines were not always clearly visible (e.g., thin, mushroom, or branched dendrites). In the control group, the spine density was 12.51 ± 0.38/10 ␮m. The effects of 2 sevoflurane treatments were reflected in the significant decrease in the apical dendritic spine number (9.81 ± 0.21/10 ␮m and 10.53 ± 0.29/10 ␮m, respectively, Fig. 2). 3.3. Sevoflurane induces ultrastructure changes in hippocampal synapses The synaptic ultrastructure of the hippocampus was examined using the TEM 2 weeks after sevoflurane exposure. Compared to the control group, the Sev 6 h group showed a decrease in the number of synapses (0.34 ± 0.032/␮m2 vs. 0.52 ± 0.071/␮m2 , p < 0.05), and there was an observably widened synaptic cleft (p < 0.001) (Fig. 3 and Table 1). In addition, some mitochondria within presynaptic terminals of the hippocampus in the Sev 6 h group exhibited pathological changes such as swelling, shrinkage, and vacuolization (Fig. 3). In the Sev 1 h group, the width of synaptic cleft was also markedly augmented (p < 0.01 vs. control), while the number of synapses remained unchanged (0.43 ± 0.031/␮m2 vs. 0.52 ± 0.071/␮m2 , p = 0.062). We found the thickness of postsyn-

3.4. Prolonged sevoflurane exposure inhibits hippocampal LTP but not LTD

3.5. Hippocampal-dependent learning and memory after sevoflurane exposure 3.5.1. Morris water maze task Morris water maze test data showed that sevoflurane exposure had no significant main effects on escape latency during spatial training during testing days 1–3, (F1 = 0.315, F2 = 1.831, F3 = 2.177, p > 0.05). However, since day 4, we found that the probe time (p = 0.032) and escape latency (F4 = 3.211, F5 = 5.703, p < 0.05, see Fig. 6A and B) in the Sev 6 h group were significantly prolonged, compared to those of the control group. In the spatial probe test data, the number of entries into the platform quadrant zone in the Sev 6 h group (1.67 ± 0.37) is less than that in the Sev 1 h group (3.11 ± 0.39) or the control group (3.12 ± 0.45; F = 4.199, p = 0.027; Fig. 6C and D). These findings indicated an impaired spatial learning and memory ability at P60-65 following neonatal exposure to 3.0% sevoflurane for 6 h. In contrast, the escape latency did not show a significant difference between the control group and the Sev 1 h group during the place navigation trials (p = 0.121) and spatial probe test (p = 0.051). It is important to note that there were no significant differences in animals’ swimming speeds (control: 26.00 ± 0.96 cm/s; Sev 1 h: 26.00 ± 1.03 cm/s; Sev 6 h: 25.22 ± 1.09 cm/s, according to a one-way ANOVA: F = 0.191, p = 0.827) between the control and both sevoflurane-exposure groups.

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Fig. 1. Sevoflurane increased syntaxin and SNAP-25 levels in hippocampus. (A) Representative immunoblots for the expression levels of syntaxin 1A, SNAP-25, synaptophysin and ␣-synucleinin response to sevoflurane exposure in hippocampus 2 weeks later (P21). (B) Quantification of syntaxin, SNAP-25, synaptophysin and ␣-synuclein normalized to ␤-actin (n = 6-8/group). Data are expressed as mean percentage ± SEM of control mean values. One-way ANOVA, Tukey post hoc test, a: synaptophysin F = 0.254, p = 0.784; b: syntaxin F = 12.818, p = 0.007; c: SNAP-25 F = 7.386, p = 0.024; d: ␣-synuclein F = 1.866, p = 0.234; *p < 0.05 vs. control, **p < 0.01 vs. control.

3.5.2. Novel-object recognition task 3.5.2.1. Novel-object recognition test. Rats from the control group were able to distinguish familiar and novel objects, as revealed by their increased exploration time of the novel object (p = 0.007; paired t-test, familiar vs. novel, Fig. 7A). While rats from the Sev 1 h

and Sev 6 h groups failed to distinguish familiar and novel objects (Sev 1 h p = 0.296; Sev 6 h p = 0.810, paired t-test, familiar vs. novel, Fig. 7A), suggesting an impaired memory recall for familiar objects. The DI in the control group was greater than 0 for novel-object recognition (p = 0.004, one sample t-test). The control group’s DI

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Fig. 2. Sevoflurane decreased the spine density of pyramidal neurons from the hippocampal CA1 subfield. Fifteen apical dendritic sections were measured from one rat (5 rats/group). The analysis revealed that both sevoflurane treatments induced a significant reduction in spine density 2 weeks following anesthesia. (A) Representative spine morphology of hippocampus in three groups. (B) The histogram shows mean ± SEM of the dendritic spine numbers per 10 ␮m dendrite length. There are significant differences between the Sev groups and control at the p = 0.001 level (one-way ANOVA, Tukey post hoc test). ***p < 0.001 vs control. However, there is no difference between Sev 6 h group and Sev 1 h group (p = 0.145). Scale bar = 5 ␮m.

Fig. 3. Effects of sevoflurane on synaptic ultrastructure of hippocampus in TEM analysis. (A) Representative pictures (X10000) showed the difference on the number of synapses (4–6 synapses) in per area (10 ␮m2 ) in three groups. (Black arrows count the number of synapse) Scale bars = 500 nm; (B) control and 1 h Sev groups: synaptic structural integrity, vesicles clear and dense; 6 h Sev group: vesicles dispersed, structure blurred. Representative pictures (X15000) showed the difference on the synaptic cleft width in three groups, Sev 1 h and Sev 6 h showed widened synaptic clefts. (Black arrows show the synaptic linkages) Scale bars = 200 nm. Table 1 Structural parameters of the synaptic interface in the hippocampus (N = 20 synapses). Group

Control

Sev 1 h

Sev 6 h

PSD thickness (nm) Synaptic cleft width (nm) Synaptic curvature

45.73 ± 4.12 17.64 ± 0.97 1.0155 ± 0.0045

41.17 ± 1.71 20.33 ± 048** 1.0152 ± 0.0031

43.78 ± 2.07 21.80 ± 0.61*** 1.0113 ± 0.0028

P value p = 0.439 p < 0.001 p = 0.601

Data were presented as the means ± SEM. **p < 0.01 vs control.; ***p < 0.001 vs. control (One-way ANOVA, Tukey post hoc test). N: the number of synapses, PSD: postsynaptic density.

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Fig. 4. Neonatal exposure to 3.0% sevoflurane for 6 h inhibits long-term potentiation (LTP) in hippocampus during P35-42. (A). Hippocampal slices were stimulated at CA3 afferents and responses were recorded from area CA1 dendrites. Three trains of high frequency stimulation (HFS:1 s at 100 Hz; indicated by up arrows) spaced 5 min apart induced LTP. Sev 6 h attenuated the maintenance of LTP since 20 min after HFS (p < 0.05). (B). The sample traces showed representative fEPSP waves recorded during baseline (the solid line) and 1 h after third HFS (the dotted line). n = 5 slices from 5 rats/group (one-way ANOVA, Tukey post hoc test). *p < 0.05 vs control. Data were presented as the means ± SEM.

Fig. 5. Neonatal sevoflurane exposure does not affect the long-term depression (LTD) in hippocampus during P35-42. (A). LTD was induced with low-frequency stimulation (LFS, 900 pulses at 1 Hz for 15 min). Neither Sev 6 h nor Sev 1 h group had no significant effect on LTD compared with control (p > 0.05). (B). The sample traces showed representative fEPSP waves recorded during baseline (the solid line) and 1 h after LFS (the dotted line). n = 5 slices from 5 rats/group (one-way ANOVA, Tukey post hoc test). Data were presented as the means ± SEM.

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Fig. 6. Effects of neonatal sevoflurane anesthesia on escape latency in Morris water maze test (n = 9 per group). (A) Place trials were performed to examine rat’ learning ability and measurement of spatial information acquisition and as indicated by the latency to find the platform; (B) escape latency to find the hidden platform during navigation probe tests. (C) The number of entries into the platform quadrant in three groups. (D) The representative swimming paths during the probe trial within 60 s for each group. One-way ANOVA, Tukey post hoc test, *p < 0.05, **p < 0.01 vs control. Data were presented as the means ± SEM.

was higher than that in the Sev 6 h group (p < 0.05, unpaired t-test), while comparisons between 0 and the DI values in the Sev 1 h group or in the Sev 6 h group did not reveal a difference (Sev 6 h p = 0.876; Sev 1 h p = 0.121; Fig. 7B).

one sample t-test), while the DIs in both of the sevofluraneexposure groups were not greater than 0 (Sev 6 h p = 0.202; Sev 1 h p = 0.099; one sample t-test; Fig. 7D). No significant difference in DIs was identified among three groups (p > 0.05).

3.5.2.2. Object-context recognition test. The results seemed similar to those in the novel-object recognition test. Only the rats from the control group were able to distinguish familiar and novel objects, and animals spent more time with the object in a novel context (p < 0.05). There was no difference in object exploration time in either sevoflurane groups (Sev 1 h p = 0.666; Sev 6 h; p = 0.363; Fig. 7C). The DI in the control group was greater than 0 (p = 0.004,

4. Discussion In the current study, our results showed that neonatal rats exposed to 3% sevoflurane for a 6-h duration experienced hippocampal-dependent learning and memory deficits. In addition, these deficits may have been the result of changes in the density of dendritic spines, synaptic ultrastructure, expression of synaptic

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Fig. 7. Effects of sevoflurane exposure on novel object and object-context recognition. (A–B) Novel object recognition Rats in Sevoflurane-treated groups demonstrated failure in object recognition test and revealed an impaired memory recalling for familiar objects. Only control group’s DI was significantly greater than zero (unpaired t test), and DI in control group was significantly greater than that in Sev 6 h group (two-way ANOVA, Tukey post hoc test). (C–D) Object-context recognition Rats in Sevoflurane-treated groups demonstrated failure in context recognition test and revealed an impaired memory recalling for familiar context. Rats in Sevoflurane-treated groups were also unable to identify an object in a novel context. Only control group’s DI was significantly greater than zero (unpaired t test), and there was no difference in DIs among three groups (two-way ANOVA, Tukey post hoc test). *p < 0.05.

vesicle-associated proteins, and LTP, thus impairing the synaptic plasticity in the hippocampus. Furthermore, shorter-duration (1 h) of 3% sevoflurane exposure produces less severe damage on synaptic plasticity and neurocognitive performance, compared to rats exposed to prolonged sevoflurane. The information in the present study complements findings from prior studies and provides a more complete picture of the behavioral deficits associated with neonatal sevoflurane exposure-induced hippocampal synaptic plasticity impairment in adulthood.

4.1. Neonatal sevoflurane exposure changes synaptic structure During the synaptic growth-spurt period, dendritic spines of pyramidal neurons in the hippocampus have long been thought to provide a morphological basis for synaptic plasticity (Kasai et al., 2010). Alterations of spine shape and density are the most consistent anatomical correlates of altered cognitive function associated with multiple human neurologic disorders (Kaufmann and Moser, 2000). This structural plasticity can be blocked by inhalational anesthetics, which is considered to be associated with blocking actin-based motility (Kaech et al., 1999). In contrast, Briner et al. (2010) found that both inhalational and intravenous anesthetics (De Roo et al., 2009) acutely increased dendritic spine density in the rat medial prefrontal cortex at P16. However, in another study, Yang et al. (2011) revealed that isoflurane has no signifi-

cant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice. The selection of animal model or exposure concentration and duration of anesthetics may contribute to the difference in our results with others. Because the apical dendrite spine is more important in modulating the processing of input signals than is the basal dendrite spine, and has a higher susceptibility to changes induced by environmental factors (De Giorgio and Granato, 2015), we only measured the apical section in this study. Recent studies have demonstrated that early exposure to commonly used general anesthetics induces marked hippocampal ultrastructure damage (Amrock et al., 2015; Lunardi et al., 2010). Here TEM photomicrographs showed the ultrastructure of synapses in sevoflurane-exposed groups was negatively affected, including reduced numbers of synapses, widened synaptic clefts, and other pathological features. Previous studies have shown that sevoflurane could reduce levels of postsynaptic density protein 95 (Zhang et al., 2015; Wang et al., 2013); however, we did not observe differences in thickness of PSD among three groups. This may be because PSD-95 expression level could not fully reflect the thickness of PSD. We also investigated the expression of synaptic vesicleassociated proteins (SVAPs) which are involved in docking, fusion, and the recycling of the synaptic vesicles, as well as neurotransmitter release (exocytosis). In this study, expressions

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of SNAP-25 and syntaxin, proteins required for membrane fusion and that are anchored in the vesicle transport membranes, are significantly elevated in the Sev 6 h group compared to the control group. These proteins are two components of SNARE (Soluble NSF [N-ethylmaleimide sensitive fusion proteins] Attachment Protein Receptor), which are mandatory for stimulating vesicular neurotransmitter release at synapses (Ahnert-Hilger et al., 2013). Although moderately elevated, their expression levels may affect regular coupling of SNARE complex formation (Kavanagh et al., 2014). Synaptophysin and ␣-synuclein, which play a role in the regulation of the exocytosis process (Bellani et al., 2010), were found unchanged after sevoflurane exposure. It should be noted that we measured only 4 of all SVAPs, so whether changes in the total number of vesicles upon sevoflurane exposure remains to be investigated. We hypothesize that an imbalance exists in the relative levels of synapse-bound proteins that may parallel morphological indicators of synaptic destruction. Therefore, alterations in the anatomical substrate for memory storage and synaptic transmission imply the possibility of long-lasting disturbed synapse development that account for long-lasting impairment in cognitive processes (Jevtovic-Todorovic et al., 2013; Zheng et al., 2013). 4.2. Impaired LTP following sevoflurane anesthesia LTP inhibition following anesthesia might account for postoperative cognitive deficit as LTP is widely assumed to be a correlate of memory consolidation and recall. The studies have reported sevoflurane could enhance or impair hippocampal LTP under concentrations ranging from subclinical to clinical ones (Ishizeki et al., 2008; Otsubo et al., 2008). Consistent with previous findings (Haseneder et al., 2009; Piao et al., 2013), our electrophysiological results revealed that 3% sevoflurane exposure for 6 h significantly inhibited LTP in hippocampal slice preparations. Synaptic plasticity is usually accompanied by the structural modification of synapses (Gomez-Palacio-Schjetnan and Escobar, 2008); for example, LTP requires the functional integrity of dendritic spines (Segal, 2005). sevoflurane-induced changes in the morphological and structural development of synapses observed on P21 may be associated with LTP inhibition on P35-42, which lead to learning and memory impairment on P60-75. As there was a reduction in the density of dendritic spines also in the Sev 1 h group in the absences of a change in LTP, it suggest that sevoflurane-induced LTP impairment results from multiple, but not single, effects on synaptic development. In contrast, formation of LTD, another facet of synaptic plasticity, is intact in sevoflurane-exposed rats, suggesting neonatal sevoflurane exposure differentially influences LTP and LTD. 4.3. Hippocampal-dependent neurobehavioral impairments Growing evidence has shown that neonatal exposure to general anesthesia in rodents and nonhuman primates produces neurobehavioral defects persisting into adulthood (Lei et al., 2013; Wang et al., 2013; Jevtovic-Todorovic et al., 2003; Satomoto et al., 2009; Paule et al., 2011; Shih et al., 2012). Recent epidemiological studies also have shown that anesthetic treatment in young children could possibly be associated with neurocognitive impairment later in life (Wang et al., 2014). Given the plethora of studies supporting the role of hippocampal synaptic plasticity in memory acquisition and encoding (Bliss and Collingridge, 1993), we tested whether neonatal sevoflurane exposure could affect two different forms of hippocampal-dependent learning and memory. The performance in the Morris water maze task, widely used in the study of spatial learning and memory, seems to correlate with LTP in the Schaffer-collateral CA1 pathway (McHugh et al., 1996), while performance in a nonspatial hippocampal-dependent new-object recognition memory task, is

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more likely to correlate with LTP in the dentate gyrus CA3 “Mossy Fiber” pathway (Boekhoorn et al., 2006). Our experiments only showed LTP in the Schaffer-collateral CA1 pathway, which can explain spatial learning and memory impairment in the Sev 6 h group compared to the control and Sev 1 h groups. Because of the complexity of early neurodevelopment and the pleiotropic nature of general anesthetics, it is possible that their adverse behavioral phenotype results from subtle differences in the agent, timing, and length of exposure (Zhou and Ma, 2014). 4.4. Short-duration vs. prolonged-duration sevoflurane exposure Recent studies which showed prolonged inhalational anesthetic exposure caused altered dendritic spine morphology, synaptic loss (Amrock et al., 2015) and a neurocognitive deficit in spatial reference memory and spatial working memory (Stratmann et al., 2009) compared with a shorter one. We also observed the effects of sevoflurane exposure duration on synaptic plasticity and cognitive performance, and found structural and morphological alterations of synapse showed similar damage although expression of SNAP-25 is higher in Sev 6 h group compared with Sev 1 h group. However, it is important to note that impairment of LTP, a surrogate of synaptic plasticity, is restricted to the rats in the Sev 6 h group. Accordingly, we found that acquisition of spatial memory is impaired only in the Sev 6 h group, but not in the Sev 1 h group, in the Morris water maze test. Strangely, in the novel-object recognition task, the rats from both sevoflurane-treated groups failed to distinguish the novel object from a familiar one, which suggested even a single brief anesthesia experience could impair simple nonspatial task performance. The notion of a “threshold effect” may explain the functional differences of exposure duration on synaptic plasticity (Shen et al., 2013). Platholi et al. (2014) indicated that inhalational anesthetic-induced spine shrinkage and loss were reversible upon anesthetic elimination. The similarity of structural and morphological changes at adulthood in two sevoflurane groups may attribute from partial synapse recovery with brain maturation (Qiu et al., 2015). Why does behavioral impairment occur in novel-object recognition but not in the Morris water maze in the Sev 1 h rat group? One possible reason is that different parts of the brain are responsible for the two behavioral tasks. Novel-object recognition is dependent on the hippocampus and the perirhinal cortex (Dere et al., 2007), whereas lesions in the anterior thalamic nuclei and the prefrontal cortex can mimic spatial learning and memory deficits that occur during the Morris water maze test (Aggleton and Brown, 1999). The two sevoflurane exposure durations differentially cause damages to brain regions involved in memory formation. Another explanation is this discrepancy may be due to task complexity, wherein some degree of plasticity is maintained in accordance with an exposure-duration-dependent function. 4.5. Study limitations There are some important limitations in our study. First, the experiments on synaptic protein expression, dendritic spine density, and ultrastructure of synapses, as well as the hippocampal electrophysiological measurements associated with plasticity, were not performed at a same time point with the neurobehavioral tests. This may weaken our proof regarding the causal link between impaired synaptic plasticity in the hippocampus following sevoflurane exposure and deficits in hippocampal-dependent learning and memory. However, this may also suggest synaptic plasticity in the hippocampus is persistently impaired in the Sev 6 h group. Second, sevoflurane anesthesia has important influences on physiological parameters in P7 rats (Meuwly et al., 2015), as our previous results indicated (Lei et al., 2013; Liu et al., 2015). However, Satomoto et al.

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(2009) has demonstrated that neonatal exposure to 3% sevoflurane for 6 h did not induce a significant disturbance in ventilation, oxygenation, or cerebral blood flow in mice. This led us to conclude that it was unlikely that synaptic plasticity impairment in our protocol was caused by hypoxia/hypoventilation. Finally, experimental and human evidence suggests that hyperoxia may be harmful to the developing brain (Saugstad et al., 2012; Felderhoff-Mueser et al., 2004), we subjected the neonatal rats to 100% oxygen may be questioned although control group also received 100% oxygen for the same period of time. In conclusion, our research showed that 3% sevoflurane exposure for 6 h on P7 resulted in learning and memory deficits in adult rats. These deficits may have occurred as a consequence of structural and functional impairments in hippocampal synaptic plasticity. Furthermore, the result showed even shorter sevoflurane exposure (1 h) also could influence hippocampal synaptic plasticity, and thereby hippocampal-dependent cognitive dysfunction. Although the mechanisms need to be further explored, this evidence suggested the potential role of synaptic plasticity in sevoflurane-induced hippocampal-dependent learning and memory impairment in this animal model. Author contributions Hongyan Xiao has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Bing Liu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files. Yali Chen has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files. Jun Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Conflict of interest All authors reported no conflict of interest. Acknowledgments We are very grateful to Professor Jianhai Jiang from Department of Biochemistry, School of Medicine, Fudan University for his technical assistance. This work is supported by grant (No. 81171020) from Nature Scientific Foundation of China (To Jun Zhang). References Aggleton, J.P., Brown, M.W., 1999. Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav. Brain Sci. 22, 425–444. Ahnert-Hilger, G., Münster-Wandowski, A., Höltje, M., 2013. Synaptic vesicle proteins: targets and routes for botulinum neurotoxins. Curr. Top. Microbiol. Immunol. 364, 159–177. Amrock, L.G., Starner, M.L., Murphy, K.L., Baxter, M.G., 2015. Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure. Anesthesiology 122, 87–95. Bellani, S., Sousa, V.L., Ronzitti, G., Valtorta, F., Meldolesi, J., Chieregatti, E., 2010. The regulation of synaptic function by alpha-synucein. Commun. Integr. Biol. 3, 106–109. Bett, D., Stevenson, C.H., Shires, K.L., Smith, M.T., Martin, S.J., Dudchenko, P.A., Wood, E.R., 2013. The postsubiculum and spatial learning: the role of postsubicular synaptic activity and synaptic plasticity in hippocampal place cell, object, and object-location memory. J. Neurosci. 33, 6928–6943. Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39. Boekhoorn, K., Terwel, D., Biemans, B., Borghgraef, P., Wiegert, O., Ramakers, G.J., de Vos, K., Krugers, H., Tomiyama, T., Mori, H., Joels, M., van Leuven, F., Lucassen, P.J., 2006. Improved long-term potentiation and memory in young tau-P301L transgenic mice before onset of hyperphosphorylation and tauopathy. J. Neurosci. 26, 3514–3523.

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