In vivo feld recordings efectively monitor the mouse cortex and hippocampus under isofurane anesthesia

Yi-qing Yin, Li-fang Wang, Chao Chen, Teng Gao, Zi-fang Zhao, Cheng-hui Li

Department of Anesthesiology, China-Japan Friendship Hospital, Beijing, China

In vivo feld recordings efectively monitor the mouse cortex and hippocampus under isofurane anesthesia

Yi-qing Yin#, Li-fang Wang#, Chao Chen, Teng Gao, Zi-fang Zhao, Cheng-hui Li*

Department of Anesthesiology, China-Japan Friendship Hospital, Beijing, China

How to cite this article:Yin YQ, Wang LF, Chen C, Gao T, Zhao ZF, Li CH (2016) In vivo feld recordings efectively monitor the mouse cortex and hippocampus under isofurane anesthesia. Neural Regen Res 11(12):1951-1955.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Graphical Abstract

A successful method to obtain in vivo feld recordings of the mouse cortex and hippocampus under isofurane (ISO) anesthesia

Isofurane is a widely used inhaled anesthetic in the clinical setting. However, the mechanism underlying its efect on consciousness is under discussion. Therefore, we investigated the efect of isofurane on the hippocampus and cortex using an in vivo feld recording approach. Our results showed that 1.3%, 0.8%, and 0.4% isofurane exerted an inhibitory infuence on the mouse hippocampus and cortex. Further, high frequency bands in the cortex and hippocampus showed greater suppression with increasing isofurane concentration. Our fndings suggest that in vivo feld recordings can monitor the efect of isofurane anesthesia on the mouse cortex and hippocampus.

nerve regeneration; neurons; isofurane; patch clamp; cell membrane; synaptic response; inhalational anesthesia; electrophysiology

Introduction

Isoflurane is a widely used inhaled anesthetic, although its mechanisms for inducing unconsciousness in the central nervous system (CNS) are poorly understood. It has been shown that presynaptic selectivity in conjunction with postsynaptic and extrasynaptic gamma-aminobutyric acid (GABA)Areceptor potentiation are potential CNS synaptic pathways (Kotani and Akaike, 2013; Westphalen et al., 2013). However, in vivo feld recordings refecting neural processing of diferent concentrations of anesthetics have not been reported.

Therefore, we used an in vivo feld recording approach to determine whether the same mechanism occurs in living anesthetized animals. Field recording methods enable CNS recordings in intact animals (Sakmann, 2006; Furue et al., 2007) in which accumulating extracellular signals due to synaptic excitation can easily be recorded with a feld electrode. Furthermore, this method refects in vivo neuronal activity, and provides sufcient signal quality to defne synaptic events for understanding neural processing during normal and drug-mediated behavior (Kolb et al., 2013). In this study, we introduce our in vivo feld recording method, and use it to examine cortical and hippocampal synaptic response aTher inhalation of diferent isofurane concentrations.

Materials and Methods

Animal preparation

All animal experiments were approved by the Animal Care Committee of the China-Japan Friendship Hospital and performed in accordance with the United States National Institutes of Health Guideline for the Care and Use of Laboratory Animals. All attempts were made to minimize the number of animals used.

A recording chamber was prepared prior to surgery. Aconical stone drill bit (CA1063, Minimo Precision Instruments and Tools, Daido Mecha Tronics Co., Ltd., Shanghai, China) and high speed dental drill (OS-40, Osada Electric. Co., Ltd., Tokyo, Japan) were used to drill a hole of approximately 2 cm in diameter through a 35 mm plastic dish. Before craniotomy, the skin above the recording area was cut using scissors, and the recording chamber placed directly onto the skull and glued using cyanoacrylic glue (UHU).

Craniotomies were performed on 10 female and male C57/BL6 mice, aged 3 to 4 months old (Chinese Academy of Medical Science Institute of Experimental Animal, Beijing, China). Mice were placed in a stereotaxic frame (New Standard 51700, Friends Honestly Life Science Company, Ltd., Beijing, China), and deeply anesthetized throughout surgery using 1.7% isofurane (YBH40052005; Abbott Laboratories, Shanghai, China) (Silva et al., 2010) delivered in 2 L/min oxygen through a securely fitted mask. Erythromycin eye ointment (150502; Beijing Shuangji Pharmaceuticals, Beijing, China) was applied to protect the eyes from injury. Anesthesia was obtained when there was no response to pinching. Mice were allowed to spontaneously breathe, and their body temperature was monitored with a rectal probe and maintained at 37°C with a thermostatically-controlled electric heating pad (CWE, Inc., Ardmore, PA, USA). Hair was removed using a trimmer and human hair removal cream (L’Oreal, Paris, France), and an incision made in the skin using a scalpel. ATher subcutaneous injection of 1% xylocaine (6H98J2, China Otsuka Pharmaceutical Co. Ltd., Tianjin, China), muscle was exposed using cotton-swab applicators. The skull was washed using the same xylocaine solution and dried with compressed air. Fascia was removed using a scalpel blade. Next, the area around the intended craniotomy (center 2.5 mm anterior of bregma, 2.5 mm lateral, and 3-4 mm diameter) was thinned using a high-speed drill (ZH-GSZ; Anhui Zhenghua Biological Equipment Co. Ltd., Anhui, China) with a small tip steel burr (0.5 mm in diameter). The area was then gently perforated with forceps to disclose the dura mater, which was removed with scissors. The cortex was cleaned with artificial cerebrospinal fluid (119 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, and 10 mM glucose). Subsequently, 5% CO2/95% O2was administered for 10-15 minutes, and then 2.5 mM CaCl2added. The fltrate was obtained using a 0.22-μm flter apparatus (597316; Friends Honestly Life Science Company, Ltd., Beijing, China) sterilized and stored at 4°C (HW Press, 2011). The cortex was kept humid. The leThand right side of the holders were gently adjusted until they were parallel to the recording chamber. Artifcial cerebrospinal fuid flled the recording chamber and ensured there was no leakage along the edge between the recording chamber and cortex.

Electrophysiological recordings

The surface of the exposed brain was irrigated with artifcial cerebrospinal fuid. In vivo feld recordings from the cortex and hippocampus were performed using a patch electrode (B100-50-7.5-HP; Friends Honestly Life Science Company, Ltd.) (thin-walled borosilicate glass capillary, tip 4-5 μm diameter, and resistance of 2-3 MΩ), flled with the following internal solution: 110 mM potassium gluconate, 20 mM KCl, 10 mM HEPES, 10 mM EGTA, 2 mM MgATP, 5 mM Na2ATP, and 0.1 mM Na-GTP; pH 7.3. The correct electrode location was determined using physiological and stereotaxic indicators. Under positive pressure (100-200 mbar), the electrode pipette was slowly advanced into the cortex and hippocampus at a speed of 0.2 mm/min to minimize trauma to the brain tissue. The hippocampal electrode was inserted into the CA1 area (2.0 mm posterior, 2.0 lateral to bregma, and 1.5 mm ventral to the dura mater) (Paxinos and Watson, 2005), with a prominent amplitude oscillation recorded at a frequency of 6-7 oscillations/second. The cortical electrode was inserted into the superfcial cortical layer (1.2 mm anterior, 2.0 lateral to bregma, and 0.5 mm ventral to the dura mater).

Cortical and hippocampal extracellular field recordings were monitored under different isoflurane concentrations, with 1.7%, 1.3%, 0.8%, and 0.4% isofurane administered via an anesthesia machine (AS-01-007; Friends Honestly Life Science Company, Ltd.). The recording time was 15 minutes except for 0.4% isofurane, which was only 5 minutes.

Current-clamp recordings were performed using an Axopatch 200B amplifier (Molecular Devices, Foster City, CA, USA). Extracellular feld potentials were recorded at a frequency band of 0.05-3,000 Hz and amplifed 200 times. No significant changes in access resistance were observed throughout the experiments. At the end of the experiment, mice were deeply anesthetized with supplemental isofurane.

Data analysis

Data were analyzed using pCLAMP software (version 10, Axon Instruments/Molecular Devices, Sunnyvale, USA). Data are expressed as the mean ± SD. Group differences were tested by one-way analysis of variance (ANOVA) and Dunnett’s test (post hoc test). P ＜ 0.05 was considered statistically signifcant.

Results

Electroencephalogram (EEG) of the cortex and hippocampus

Eight stable in vivo feld recordings of the cortex and hippocampus were obtained. Figure 1 shows in vivo cortical and hippocampal feld recordings in mice under 0.4% isofurane anesthesia.

Next, the efect of isofurane on cortical and hippocampal field recordings was investigated. As shown in Figure 2, under diferent isofurane concentrations, hippocampal and cortical feld recordings had diferent discharge levels. Further, with increasing isofurane, the discharge reduced.

Hippocampal and cortical frequency bands

The isoflurane dose-response on hippocampal and cortical frequency bands was examined. In the cortex, there were signifcant diferences in β, θ, and α signals between 1.7% and 1.3% isofurane. Further, 1.3% and 0.8% isofurane showedsignificant differences in θ signal frequency, with γ signal diferences also observed between 1.7%, 1.3%, and 0.8% isofurane. In the hippocampus, δ signal power increased as isofurane changed from 1.7% to 1.3% and 0.4%. Signifcant differences were found between 1.7% and 1.3% isofurane, and 1.3% and 0.8% isoflurane in θ and α signals. Hippocampal β and γ signal power increased as isofurane changed from 1.7% to 0.4%. Additionally, there was a signifcant diference in β signal between 1.3% and 0.8% isofurane (Table 1). Figure 3 shows the relationship between power and different frequency bands under different isoflurane concentrations delivered within the cortical and hippocampal area. There was an increase in power at about 40 Hz under all isofurane concentrations, however this did not difer signifcantly between each group.

Table 1 Power spectrum in the hippocampus and cortex using diferent isofurane concentrations

In summary, low frequency bands were relatively resistant to isoflurane in both hippocampal and cortical recordings, except for 1.7% isofurane. The suppression efect of isofurane on high frequency bands was dose-dependent.

Discussion

Here, we have used in vivo feld recording to monitor changes in the mouse cortex and hippocampus. Cortical and hippocampal neurons are involved in physiological CNS processes such as learning and memory. Consequently, studying the efect of isofurane on spontaneous electrical brain activity may help understanding of neural events during anesthetic-induced unconsciousness. We found that with increasing isoflurane concentration, spontaneous cortical and hippocampal activity was suppressed. Specifically, we observed a decrease in low frequency bands in the hippocampus (including δ, θ, and α signals) between 1.7% and 1.3% isofurane, and 1.3% and 0.8% isofurane. Similarly, higher frequency bands showed a declining trend in a dose-dependent manner. With regards cortical frequency bands, only higher frequency bands showed a decrease between 1.7% and 1.3% isofurane. Further, we also found an increasing trend at 40 Hz under all isoflurane concentrations in both the hippocampus and cortex, although none were signifcantly diferent. These fndings are consistent with our unpublished data on EEG recordings in the mouse hippocampus and cortex following midazolam treatment.

It is known that the δ, θ, and α powers are commonly used signal variables for assessing cognitive function. Accumulative evidence supports hippocampal θ as a synchronized rhythmic oscillation of feld potentials at 14-12 Hz in memory formation (LuTh et al., 2013). In this study, we found that high isofurane concentrations depressed δ, θ, and α power, which contributes to anesthetic-induced amnesia (Perouansky et al., 2010). In the mammalian cortex, neural communication is organized by 30-100 Hz γ oscillation, with γ frequency related to processing speed in neural networks (Insel et al., 2012). Indeed, 40-Hz oscillation is associated with various aspects of the conscious process (Desmedt and Tomberg, 1994; Joliot et al.,1994; Tallon-Baudry et al., 1996). Numerous studies have addressed the effect of anesthetic agents on oscillations in both humans and animals (Uchida et al., 2000; John et al., 2001; Sleigh et al., 2001). Hudetz et al. (2011) found that isofurane at moderate concentrations did not suppress cortical oscillations at the 40 Hz frequency. In contrast, high-frequency γ power activity at 70-140 Hz was notably suppressed by isoflurane in a concentration-dependent manner. Similar observations were previously shown in the rat hippocampus (Ma et al., 2002). Sensory stimulation may produce cortical arousal even during isofurane anesthesia, which may be responsible for enhanced γ oscillations under anesthesia (Hudetz, 2008). Consistent with these studies, we show that cortical and hippocampal γ signals are sensitive to isoflurane and decreased with increasing concentration, thereby inducing unconsciousness.

Figure 1 In vivo feld recording of the cortex and hippocampus (A) and their spontaneous discharges (B).

Figure 2 Extracellular cortical and hippocampal feld recordings under diferent isofurane concentrations.

Figure 3 Power spectrum in the hippocampus and cortex under diferent isofurane concentrations.

We also applied electrophysiological methods to examine CNS mechanisms underlying anesthetic agents. Intracranial techniques such as local feld potentials (LFPs) are not infuenced by electromyographic activity, and can analyze higher frequencies such as γ-bands. Thus, LFPs may more accurately refect the efect of anesthetics on the brain than electroencephalograms, which enables them to precisely examine anesthetic performance (Silva et al., 2010). There are three points to attain a successful stable recording: first, duringrecordings, experimental animals should be maintained in a physiological situation; second, setting of recording chambers should be accurate and careful; third, placing of recording electrodes onto designated areas must be based on good anatomical knowledge and recognition of characteristic neuronal rhythms. Although we examined features and changes in electrophysiological activity of cortical and hippocampal neurons following isofurane anesthesia, behavioral manifestations or single cell electrophysiological changes still needs to be investigated in detail.

In conclusion, our study provides a successful method to obtain stable in vivo feld recordings in the mouse cortex and hippocampus under isofurane anesthesia. Furthermore, we show that high frequency cortical and hippocampal bands are more sensitive to the suppression of diferent isofurane concentrations.

Author contributions:All authors put forward the conception, designed the study, performed the experiment, collected the data, and wrote the paper. All authors approved the fnal version of the paper.

Conficts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:This paper was double-blinded and stringently reviewed by international expert reviewers.

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Copyedited by James R, Stow A, Yin YQ, Yu J, Li CH, Li JY, Song LP, Zhao M

*Correspondence to: Cheng-hui Li, M.D., chenghui_li@sina.com.

#These authors contributed equally to this study.

orcid: 0000-0002-8531-2489 (Cheng-hui Li)

10.4103/1673-5374.197136

Accepted: 2016-11-28