Enhancing Anesthetic Techniques for Improving Rat Whisker Stimulation Responses in the Barrel Cortex

Enhancing Anesthetic Techniques for Improving Rat Whisker Stimulation Responses in the Barrel Cortex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhancing Anesthetic Techniques for Improving Rat Whisker Stimulation Responses in the Barrel Cortex Ye Yuan, Sinan Li, Jie Zhao, Tian Liu, Jue Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4778385/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This paper presents an efficient anesthetic methodology designed to enhance the quality of brain barrel cortex signals obtained during rat whisker stimulation experiments. The proposed approach effectively eliminates muscle noise interference, thereby enabling the acquisition of clear and robust brain barrel cortex signals. Initially, alpha-chloralose (ac) combined with Isoflurane is used to induce anesthesia in rats. Subsequently, Dexdomitor is administered to suppress muscular movements, further refining the signal quality. Experimental outcomes demonstrate that our anesthetic method yields significantly stronger and cleaner brain barrel cortex signals, exhibiting enhanced robustness compared to existing methods that rely solely on Isoflurane or the Ketamine-Xylazine combination. Specifically, brain barrel cortex signals obtained under alpha-chloralose anesthesia are double those obtained with Isoflurane and quadruple those with Ketamine-Xylazine. The peak amplitude latency is shorter under alpha-chloralose anesthesia than under Isoflurane and Ketamine-Xylazine anesthesia. Notably, these improvements are achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates. Finally, alpha-chloralose effectively maintains anesthesia for up to 7 hours, surpassing the shorter durations achievable with continuous Isoflurane administration or the 30-minute window offered by Ketamine-Xylazine. This highlights the practical advantages of our proposed method. Biological sciences/Drug discovery Biological sciences/Neuroscience barrel cortex signal anesthetics experiment alpha-chloralose muscle noise robustness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 I. INTRODUCTION Recently, the use of anesthesia in the process of collecting brain signals from rodents has introduced numerous potential confounding factors, making it crucial for researchers to understand the impacts of various anesthesia regimens on their experimental animals [1]. This understanding can significantly aid investigators in selecting suitable anesthesia/sedation rotocols tailored for the collection of brain signals [2]. Isoflurane, a widely utilized anesthetic agent across both rodent and non-rodent species over the past decades [3], effectively assists in maintaining a stable anesthetic state during experiments [4]. Furthermore, the combined use of ketamine and xylazine has emerged as a popular choice for inducing anesthesia in diverse animal species [5]. This combination is highly esteemed in both veterinary medicine and research circles due to its ability to consistently provide a controllable state of sedation, analgesia, and muscle relaxation, thereby enhancing the overall quality and reliability of experimental outcomes. Despite their prevalent usage in anesthetizing animals for neurophysiological experiments, Isoflurane and the Ketamine-xylazine combination both exhibit inherent limitations [6]-[7]. Isoflurane necessitates continuous administration to sustain its anesthetic effect, potentially introducing variability and impacting the animal’s physiological state over time. Conversely, Ketamine and xylazine offers a relatively brief duration of action, typically spanning approximately 30 minutes, necessitating frequent redosing and potentially contributing to fluctuations in key physiological parameters. In neurophysiological research, securing high-fidelity brain signals from animal models is paramount to ensuring the accuracy and reproducibility of experimental findings. For instance, the presence of muscle noise can significantly hinder the isolation of pristine neural signals. Thus, the adoption of an anesthetic regimen that proficiently minimizes such noise without significantly perturbing the animal’s physiological equilibrium is imperative [8]. This paper introduces a groundbreaking anesthetic approach tailored for rats, aimed at achieving unparalleled raw brain signal acquisition by effectively eliminating muscle noise interference during experiments. Our method innovatively integrates the use of alpha-chloralose (a-c) and Dexdomitor, subsequent to an initial induction phase with Isoflurane. Specifically, alpha-chloralose is administered to maintain a stable anesthetic state throughout the experiment [9]- [15], while Dexdomitor is employed to prevent muscular contractions during the crucial signal acquisition phase. This strategic combination addresses the challenges associated with conventional anesthetics by harnessing the enduring stability of alpha-chloralose and the potent muscle-relaxing effects of Dexdomitor [16]. Our method ensures a stable anesthetic condition spanning up to 7 hours, significantly mitigating the need for continuous dosing and the corresponding perturbations to the animal’s physiology. Experimental outcomes underscore the superiority of this novel anesthetic protocol. Compared to the traditional Isoflurane and Ketamine-xylazine methods, our approach yields cleaner barrel cortex signals and enhances the robustness of data acquisition. Furthermore, it minimizes alterations to critical physiological indicators, including body temperature, respiratory rate, and heart rhythm, as evidenced in Fig. 1. By refining the anesthetic strategy, we elevate the quality of neurophysiological data, thereby fostering more dependable and precise research findings in animal model-based studies. Our proposed model revolves around utilizing alpha-chloralose (a-c) as the primary anesthetic agent, coupled with a low-dose Isoflurane (0:5%) administration. This approach is inspired by the advantage of a-c in allowing animals to maintain spontaneous respiration, a crucial aspect in experiments where the preservation of natural physiological functions is imperative. In this paper, we demonstrate how the combined use of a-c and Isoflurane can yield superior raw barrel cortex signals devoid of muscle noise under whisker stimulation. Specifically, we introduce a novel combination of anesthetics, leveraging the strengths of alpha-chloralose and Isoflurane, to achieve a stable anesthetic state while minimizing muscle noise interference. By optimizing the anesthetic approach, we significantly improve the quality of neurophysiological data, enabling more reliable and accurate research outcomes. We provide experimental evidence demonstrating the effectiveness of our proposed anesthetic protocol in enhancing barrel cortex signal acquisition and minimizing alterations to critical physiological parameters. The primary contributions of this article are as follows: 1) A novel anesthetic approach utilizing alpha-chloralose (a-c) is introduced to effectively manage the anesthetic state of animals during experimental signal acquisition. This method departs from the current state-of-the-art anesthetics, such as Isoflurane and Ketamine-xylazine combinations, by integrating a-c with a low concentration of Isoflurane (0:5%). Consequently, the initial step in our protocol involves administering a combination of a-c and 0:5% Isoflurane to anesthetize the animal prior to signal acquisition, ensuring optimal conditions for accurate and reliable data collection. This improved performance is achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates. 2) The methodology employed in this paper aims to maintain a stable anesthetic state in animals during barrel cortex signal measurements, thereby ensuring the accuracy and reliability of the data collected. To achieve this, we administer Dexdomitor to mitigate muscular movements during signal acquisition, effectively reducing the potential interference of muscle noise on the raw brain signals. This approach, coupled with the use of a combination of alpha-chloralose (a-c) and 0:5% Isoflurane anesthesia, further minimizes the impact of extraneous factors, allowing for the capture of high-quality, uncontaminated brain signals. The efficacy of a-c in maintaining anesthesia for up to 7 hours stands in contrast to the shorter durations achievable with continuous Isoflurane administration or the 30-minute window offered by Ketamine-Xylazine, highlighting the practical advantages of our proposed method. 3) Experimental investigations have been conducted to validate that the methodology presented in this paper significantly outperforms existing anesthetic protocols, including Isoflurane and Ketamine-xylazine combinations, in terms of acquiring cleaner and more robust animal brain barrel cortex signals with minimal muscle noise interference. The brain barrel cortex signals under the alpha-chloralose anesthetic is double that of Isoflurane and quadruple that of Ketamine-xylazine at the whisker stimulation experiment system. Moreover, our approach demonstrates a marked improvement in signal quality and robustness, underscoring its effectiveness in minimizing extraneous factors that can compromise the integrity of brain signal measurements. The rest of this article is organized as follows: Section II states experiment setups and data acquisition. Section III introduces the different methods of anesthetics for experiment data acquisition. The experiment results are used to validate our method’s effectiveness in Section IV. Section V presents the conclusions of the paper and discusses future works. II. EXPERIMENTS SETUPS AND DATA ACQUISITION This section begins by presenting the sources of the signal data utilized in our experiments. Subsequently, we detail the preparatory steps and materials required for the experimental setup, emphasizing the necessary preparations to ensure the successful execution of our research. A. Signal Data Sources The Sprague Dawley male rats used in this study, weighing between 250-350g, were housed in an environment adhering to a strict 12-hour light-dark cycle within facilities accredited by Xi’an Jiaotong University’s Animal Protection and Use Committee. Throughout their stay, the animals had unrestricted access to food and water, and all experiments were conducted in accordance with protocols approved by the Committee. We made every effort to minimize animal suffering and ensure the ethical treatment of all subjects involved in this research. These guidelines are same with described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). All of these SD rats (31-12-002-D-000010 )were purchased from the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences. In this study, we conducted comparative experiments on three distinct groups of Sprague Dawley male rats, each comprising 12 animals (N1=12, N2=12, N3=12). These experiments aimed to compare the effects of three anesthesia methods: alphachloralose, Isoflurane, and Ketamine-xylazine. Animals were randomly assigned to receive one of these anesthesia protocols on experimental days, with the order of administration counterbalanced across groups to mitigate potential biases. Notably, rats that underwent Isoflurane anesthesia were excluded from subsequent experiments involving alpha-chloralose and Ketaminexylazine to avoid confounding factors. Upon completion of the experiments utilizing alpha-chloralose, Isoflurane, and Ketamine-xylazine, euthanasia was performed in accordance with ethical guidelines. During each experiment, an extracellular electrode was placed within the animal’s dura mater to capture raw electroencephalogram (EEG) brain signals under anesthesia. This approach enabled us to obtain a comprehensive understanding of the EEG signals under varying anesthetic conditions. B. Preparing things of experiment To accurately measure the raw EEG brain signals of Sprague Dawley male rats, we followed a series of preparatory steps for our experiments, as outlined in reference [17]. These steps were crucial to ensure the reliability and validity of our data collection process. 1)Preparation: Before commencing the experiments, it is imperative to ensure that the work area is safe and adequately ventilated. Next, prepare all necessary anesthetic instruments and medications, including alpha-chloralose, Isoflurane gas, and Ketamine-xylazine, along with the inhalation anesthesia system, appropriately sized anesthesia masks, a surgical platform, injection needles, a warming pad, and other essential equipment. Additionally, Buprenorphine and Dexmedetomidine (Dexdomitor) will be utilized in this study due to their crucial role in animal anesthesia. Set up the inhalation anesthesia system by connecting it to the gas supply and ensuring that it is calibrated to accurately control the concentration and flow rate of Isoflurane, thereby ensuring a smooth and effective anesthesia process. 2)Prepare the Rats: Prior to anesthesia, the rats are gently placed in appropriately sized anesthesia chambers designed to acclimate them to the environment. This step helps to minimize stress and ensure a smoother anesthesia process. With the gas source activated, Isoflurane gas is gradually introduced into the anesthesia chamber. The concentration of Isoflurane is incrementally increased until the rats reach a state of full anesthesia, marked by a lack of response to external stimuli. Throughout this process, it is crucial to closely monitor the rats’ breathing patterns and the depth of anesthesia, adjusting the gas concentration as needed. Toe pinch tests are periodically performed to assess the level of anesthesia, ensuring that the rats are sufficiently sedated without being overdosed. 3)Surgical Procedure: Once the animals are in a deep state of anesthesia, we start the surgical process. A portion of the skull at the Bregma location is carefully removed to expose the brain. Then, one extracellular electrode is gently placed onto the dura mater, ensuring minimal disruption to the surrounding tissue. The electrode is used to collect the barrel cortex signals, providing valuable insights into the neural activity of the whisker stimulation. 4)Maintain Anesthesia and Record EEG Signals: It is crucial to maintain an appropriate depth of anesthesia throughout the duration of the experiment by adjusting the flow rate and concentration of Isoflurane as needed. This ensures that the rats remain sufficiently sedated without experiencing undue stress or discomfort. Throughout the anesthesia and recording process, continuous monitoring of the rats’ vital signs, such as respiratory rate and heart rate, is essential to ensure their safety and well-being. The raw EEG brain signals are collected under the influence of the chosen anesthetics, including alpha-chloralose, Isoflurane, and Ketamine-xylazine, allowing for a comprehensive assessment of neural activity under various anesthetic conditions. Upon completion of the preparatory steps, we utilize the Cerebus system to gather the raw EEG brain signals from the animals. The Cerebus system is a comprehensive data acquisition hardware and software platform designed specifically for recording and analyzing signals emanating from the brain and peripheral nervous system. This system incorporates microelectrode arrays, connectors, a neural signal acquisition system, and application software, offering a seamless solution for neurophysiology experiments. As a multi-channel data acquisition system, the Cerebus is renowned for its power, ease of operation, and versatility. It is capable of recording and analyzing the electrical activity of the animal brain and peripheral nerves with unparalleled precision. The system is configured to cater to a wide range of animal models, including birds, mice, rats, cats, and primates, both under anesthesia and in their natural waking state. In real-time, the Cerebus system captures, processes, and analyzes action potentials (spikes), field potentials (LFPs), and other physiological signals pertinent to the experimental conditions. This enables researchers to gain a deeper understanding of neural function and behavior, facilitating the advancement of neuroscience research. 5)Termination of Anesthesia: When concluding the anesthesia procedure, it is crucial to gradually taper off the concentration of Isoflurane. This gradual reduction ensures a smooth transition for the rats as they gradually regain consciousness. Once the rats are fully awake and have resumed their normal activities, they can be safely removed from the anesthesia chamber. Upon removal, the rats should be provided with appropriate care and monitoring to ensure their continued safety and health. This includes monitoring their vital signs and observing for any signs of distress or discomfort. By adhering to these protocols, researchers can ensure that the animals are treated humanely and with the utmost respect throughout the experimental process. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.). C. Experimental System Rats’ whiskers are sensitive detectors for picking up tactile information, enabling them to navigate, recognize, and sense their surroundings [18]. Each whisker is highly specialized and organized, represented in a barrel-like column in the somatosensory cortex of the rat brain. The barrel cortex comprises approximately 13% of the cortical volume and 69% of the somatosensory cortex. The movement of these whiskers generates evoked potentials in the somatosensory cortex in a synchronized manner. A growing body of research explores and validates this link, demonstrating that the arrangement of the barrels in the barrel cortex corresponds clearly to the arrangement of the whiskers on the rat’s face. When electrical stimulation is applied to one barrel-like area, multiple barrel-like columns may be activated [19]. To collect specific neural electrical signals induced by the barrel cortex, this study employed mechanical stimulation of a single whisker. By stimulating the movement of a single whisker (C2), projected onto the corresponding barrel column, we could more precisely locate the evoked response. As illustrated in Fig. 2, an L-shaped tube was attached to the shaft of a stepper motor with a 1.8 stepping angle. The target whisker (C2 of the larger barrel-like column) was inserted into the tube. Using an Arduino UNO R3 controller and the stepper motor driver A4988, the motor shaft was rotated for 1 ms, triggering the acquisition of synchronous local field potentials (LFPs) from the barrel cortex, as shown in Fig. 3. III. ANESTHETICS METHODS In this section, we delve into the three anesthetic methods commonly employed in animal experiments: (a) Alpha-chloralose, (b) Isoflurane, and (c) Ketamine-xylazine. We provide a brief overview of each method, highlighting their respective applications and considerations. Understanding the nuances of each anesthetic method is crucial for ensuring the safety, comfort, and ethical treatment of the animals involved in research. By exploring the unique properties and usage protocols of these anesthetics, researchers can make informed decisions regarding the most appropriate choice for their specific experimental needs. A. Alpha-chloralose Alpha-chloralose (a-c) is a crystalline compound with limited solubility in water, categorized as a sedative-hypnotic agent. It is frequently employed for anesthesia in laboratory rodents, particularly rats and mice, due to its specific effect on the central nervous system, primarily through modulating GABA receptors. GABA, an inhibitory neurotransmitter, regulates neuronal excitability, and a-c enhances GABAergic activity in rodents, inducing sedation. The depth of anesthesia achieved with a-c varies with dosage and the route of administration, typically administered via intravenous or intraperitoneal injection in small rodents. This ensures the animals are in a sedated or unconscious state during surgical or experimental procedures, minimizing discomfort and stress. In our experiment, we prepare a-c one day prior to the procedure. During the experiment, the animals are placed in an anesthesia box for 5 minutes to ensure deep anesthesia. Subsequently, we perform an intraperitoneal injection of Buprenorphine to further alleviate pain during surgery. After a 5-minute wait for the analgesic to take effect, we proceed with the surgical procedure, carefully removing a portion of the cranium to place an extracellular electrode on the dura mater for EEG signal acquisition. Once the surgery is completed, we administer a-c to the animals, allowing a 30-minute period for it to take full effect.During this time, we adjust the concentration of Isoflurane (Iso), starting at 1 and titrating downwards to find the optimal balance for maintaining anesthesia while minimizing adverse effects. This approach ensures that the animals are adequately anesthetized throughout the experiment while we collect their raw EEG brain signals. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.). B. Isoflurane Isoflurane belongs to the halogenated ether class of compounds, characterized by its unique molecular structure incorporating halogen atoms. At room temperature, it exists as a clear, colorless liquid with a pleasant aroma, exhibiting non-flammable and stable properties under standard conditions. Widely recognized as a volatile anesthetic agent in medical and veterinary settings, Isoflurane is renowned for its efficacy in inducing and maintaining general anesthesia during surgical procedures. For mice and other small animals, Isoflurane is often administered via inhalation, leveraging its rapid absorption into the bloodstream. This method of delivery is particularly favored in veterinary medicine due to Isoflurane’s swift onset of action, allowing for precise control over the depth of anesthesia. Its primary mechanism of action involves enhancing the activity of gamma-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors in the central nervous system, leading to the suppression of neuronal activity and the induction of anesthesia. The rapid onset and offset of Isoflurane’s effects, coupled with its efficient elimination from the body, contribute to its popularity as an anesthetic agent. This facilitates a swift and smooth recovery process for the animals, minimizing potential adverse effects and enhancing overall animal welfare. In this experiment, to prevent SD rats from waking up suddenly during stimulation, Isoflurane should be maintained at an anesthesia level of 1:5%-2% for the collection of LFP signals from the barrel cortex. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.). C. Ketamine-xylazine Ketamine, a dissociative anesthetic agent, exerts its primary effects by antagonizing N-methyl-D-aspartate (NMDA) receptors within the central nervous system. This action elicits a dissociative state characterized by profound analgesia, amnesia, and catalepsy, all while preserving cardiovascular stability. Consequently, Ketamine is capable of inducing dissociative anesthesia. Meanwhile, Xylazine, an alpha-2 adrenergic agonist, possesses sedative, analgesic, and muscle relaxant properties. By stimulating alpha-2 adrenergic receptors in the central nervous system, Xylazine promotes sedation, muscle relaxation, and analgesia. Its sedative effects are particularly noteworthy, as it effectively calms animals and eases muscular tension. When combined, Ketamine and Xylazine exhibit synergistic effects, amplifying the anesthetic and sedative qualities of both drugs. Ketamine contributes to the dissociative anesthesia, while Xylazine enhances sedation and muscle relaxation. This combination is renowned for its swift onset of action and relatively brief duration of anesthesia, making it an ideal choice for procedures requiring rapid recovery. Importantly, the Ketamine-Xylazine combination exerts minimal impact on respiratory and cardiovascular functions in healthy animals, ensuring patient safety during procedures. As a result, it is widely utilized in minor surgeries and diagnostic imaging, where rapid anesthesia and recovery are crucial. During the experiment, local field potential (LFP) changes are observed as the concentration of general anesthetics (KetamineXylazine) varies. Generally, Ketamine-Xylazine is effective for only about 30 minutes of sustained anesthesia. To address this limitation, the anesthesia regimen needs to be improved by continuously administering 0:5% to 1:5% Isoflurane. This ensures that SD rats maintain a deeper anesthetic state during surgery and experiments, resulting in more stable and reliable LFP data collection. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg,i.p.). IV. EXPERIMENTAL RESULTS This section begins by presenting the outcomes of barrel cortex signal collection experiments conducted on animals using various anesthetics (alpha-chloralose, Isoflurane, and Ketamine-xylazine) under whisker stimulation. Following this, we conduct a comparative analysis, emphasizing the robustness of the signals obtained through our proposed method. Finally, we demonstrate that our approach results in minimal to no significant alterations in physiological parameters, highlighting its efficacy and safety. A. The EEG signals and brain barrel cortex signals under whisker stimulation with a-c, Isoflurane and Ketaminexylazine anesthesia The barrel cortex signals under whisker stimulation and EEG brain signals of animals were recorded using three distinct anesthetic methods: alpha-chloralose (a-c), Isoflurane, and the Ketamine-xylazine combination. These signals were precisely captured by a single extracellular electrode positioned at +2.5mm and -5.5mm relative to the animal’s Bregma dura mater. A total of 36 animals were randomly assigned to three groups, with each group receiving one of the anesthetic methods. Consequently, each anesthetic method was administered to 12 animals, ensuring equal representation in the sample. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.). 1) Result 1: The brain EEG signals and barrel cortex signals under whisker stimulation with a-c anesthesia. The Cerbus system was employed to record the EEG brain signals of animals using an extracellular electrode. When combining alpha-chloralose (a-c) with Isoflurane at varying concentrations, we observed certain differences in the recorded signals. Notably, the raw EEG signals remained stable when Isoflurane was set to 0, albeit with some muscle noise present. To mitigate this, we incrementally increased the Isoflurane concentration from 0 to 0:25% and then to 0:5%, while recording the signals. Figure 4 illustrates the outcomes of these adjustments. Specifically, Figure 4 demonstrates that at 0:5% Isoflurane, there is minimal muscle noise evident in the animal’s EEG signals. This finding suggests that combining a-c with 0:5% Isoflurane represents the optimal approach for acquiring high-quality EEG signals from animal brains. Subsequently, we stimulated the C2 whisker under the aforementioned anesthetic method. Fig. 5 shows the barrel cortex signals under whisker stimulation within 1 ms. The results clearly indicate that the peak amplitude can almost reach 800 uV at 20 ms, demonstrating the efficacy of the anesthetic combination in facilitating clear and precise signal acquisition. 2) Result 2: The brain EEG signals and barrel cortex signals under whisker stimulation with Isoflurane anesthesia. Generally speaking, Isoflurane provides a stable anesthetic state during the recording of animals’ brain EEG signals. However, qualitative observations indicate that a concentration of 1:5% Isoflurane is necessary to eliminate muscle noise from the EEG signals entirely. At lower concentrations, such as 0:5% Isoflurane or when using alpha-chloralose (a-c) anesthesia, animals tend to awaken within half an hour, as evidenced by movements like toe pinching or ear twitching, which were not observed with 1:5% Isoflurane. Fig. 6 showcases the brain EEG signals acquired under 1:5% Isoflurane anesthesia, highlighting its effectiveness in ensuring a stable and artifact-free recording environment. Fig. 7 illustrates the barrel cortex signals under whisker stimulation within 1ms using Isoflurane anesthesia. The peak amplitude can reach 400 uV, which is half of what is observed with alpha-chloralose (a-c). The peak amplitude latency is 22ms, demonstrating the temporal characteristics of the signal under Isoflurane anesthesia. 3) Result 3: The brain EEG signals and barrel cortex signals under whisker stimulation with Ketamine-xylazine anesthesia. Ketamine-xylazine is a commonly preferred anesthetic for animals in various regions due to its effective sedative properties. However, its relatively short duration of anesthesia limits its applicability for prolonged brain EEG signal recording experiments. Despite offering a superior anesthetic effect compared to 1:5% Isoflurane, Ketamine-xylazine anesthesia still introduces some muscle noise into the recorded brain EEG signals. Fig. 8 depicts the brain EEG signals obtained under Ketamine-xylazine anesthesia, highlighting the presence of this noise. Figure 9 presents the barrel cortex signals under whisker stimulation within 1ms using Ketamine-xylazine anesthesia. It is evident that the peak amplitude reaches only 200 uV, which is half of the amplitude observed with Isoflurane. The peak amplitude latency is 24 ms, demonstrating the temporal response characteristics under Ketamine-xylazine anesthesia. B. The EEG brain signals robustness We conducted an experiment to validate the enhanced robustness of our proposed anesthetic method for prolonged anesthesia compared to Isoflurane and Ketamine-xylazine. This study involved monitoring the animals’ raw EEG brain signals over time while under anesthesia. Given that Ketamine-xylazine anesthesia is effective for only approximately half an hour and cannot be repeatedly administered to the same animal without causing harm or death, we focused our analysis on the other two methods. Fig. 10 showcases the results of raw EEG brain signals recorded under alpha-chloralose (a-c) anesthesia at 1 hour and 7 hours. The findings demonstrate that a-c anesthesia has a less pronounced impact on the raw EEG signals compared to Isoflurane and Ketamine-xylazine. This underscores the potential of our proposed method for facilitating extended EEG recording sessions with minimal interference. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.). C. P hysiological parameters of animals We demonstrate that our method results in minimal changes to key physiological parameters, including breathing rate, heart rate, and body temperature. 1) Result 1: Breathing rate under different anesthesia. In our comparative experiments using alpha-chloralose, Isoflurane, and Ketamine-xylazine, we observed a decrease in breathing rates among all animals as anesthesia progressed. However, Ketamine-xylazine’s effectiveness was limited to approximately 0.5 hours, precluding its repeated use on the same animal due to potential harm or fatality. Fig. 11 illustrates the breathing rates recorded over 7 hours for alpha-chloralose and Isoflurane, and over 0.5 hours for Ketamine-xylazine. Initially, all animals exhibited a breathing rate of 120 breaths per minute, which decreased to 110, 108, and 104 breaths per minute after 0.5 hours, respectively. By the 7-hour mark, the alpha-chloralose and Isoflurane groups had further decreased to 75 breaths per minute, with no significant differences observed between the alpha-chloralose and Isoflurane groups at any time point. Fig.12 quantifies the breathing rates, presenting the mean and standard error of the mean (SEM) for changes in breathing rates among the 12 animals anesthetized with each method, providing a clear comparison of their effects on respiratory function. 2) Result 2: Heart rate under different anesthesia. Throughout the anesthesia period, animals were continuously monitored for heart rate using electrocardiography (ECG). Heart rate measurements were taken at baseline (pre-anesthesia), immediately post-induction, and at regular intervals thereafter. As depicted in Fig. 13, all contrast experiments began with a baseline heart rate of 320 beats per minute (bpm). Post-induction, the heart rates for alpha-chloralose, Isoflurane, and Ketamine-xylazine anesthesia were 300 bpm, 280 bpm, and 260 bpm, respectively. During mid-anesthesia, the rates were 310 bpm, 275 bpm, and 210 bpm, while at the end of anesthesia, they were 315 bpm, 290 bpm, and 215 bpm, respectively. Notably, alpha-chloralose caused a moderate and relatively stable decrease in heart rate compared to baseline, maintaining levels close to baseline throughout the anesthesia period. Isoflurane induced a significant reduction from baseline, which remained stable but lower throughout anesthesia. Conversely, the Ketamine-xylazine combination resulted in the most pronounced decrease, with an immediate and sustained drop in heart rate post-induction. Fig.14 quantifies the heart rate, presenting the mean and standard error of the mean (SEM) for heart rate changes among the 12 animals under each anesthesia method. This offers a clear visualization of the respective effects of each anesthetic method on cardiac function. 3) Result 3: Temperature change under different anesthesia. Anesthesia can significantly impact animals’ body temperature, making the monitoring of these changes imperative for safeguarding their welfare and ensuring the accuracy of experimental outcomes. Fig. 15 illustrates the typical temperature fluctuations encountered during anesthesia using alpha-chloralose, Isoflurane, and the Ketamine-xylazine combination. Across our contrast experiments, the baseline temperature prior to anesthesia was consistently 33.0◦C. At the end of anesthesia, the temperatures dropped to 30.5◦C, 29.5◦C, and 30.0◦C, respectively, for the three anesthesia methods. Furthermore, Fig.16 quantifies the temperature changes, providing a detailed representation of the mean and standard error of the mean (SEM) for temperature variations among the group of 12 animals undergoing anesthesia with each of these agents. This offers valuable insights into their specific effects on body temperature, highlighting the importance of temperature monitoring in maintaining animal welfare and ensuring reliable experimental data. D. Discussion Alpha-chloralose demonstrated a moderate and steady decline in heart rate compared to baseline levels, preserving nearbaseline rates throughout anesthesia. In contrast, Isoflurane significantly reduced heart rate from baseline, maintaining a relatively stable but lowered rate throughout the procedure. Notably, the combination of Ketamine-Xylazine produced the most pronounced decrease in heart rate, with an immediate and sustained drop after induction. These observations underscore the necessity of selecting anesthetic protocols tailored to the specific cardiovascular requirements of the experimental or clinical setting. Specifically, Alpha-Chloralose exhibited a minimal impact on heart rate, rendering it suitable for procedures necessitating stable cardiovascular function. Isoflurane, with its noticeable yet stable depressant effect on the cardiovascular system, may necessitate vigilant monitoring and potential interventions to manage bradycardia. Ketamine-Xylazine, exhibiting strong depressant effects, efficiently induces a profound anesthetic state but requires close cardiovascular monitoring. Similarly, monitoring body temperature during anesthesia is paramount, as hypothermia can significantly impact physiological processes and experimental outcomes. Alpha-chloralose elicits the least temperature reduction, making it advantageous in studies prioritizing temperature maintenance. Isoflurane causes a moderate decrease in temperature, necessitating careful thermal environment management. Ketamine-Xylazine, however, results in the most significant drop in body temperature, emphasizing the importance of understanding these changes to safeguard animal health and ensure data reliability. A critical conclusion is that the peak amplitude of the barrel cortex signals under whisker stimulation within 1ms shows a prominent difference among anesthetics. The brain barrel cortex signals under alpha-chloralose anesthesia (800uV) are double those under Isoflurane (400uV) and quadruple those under Ketamine-Xylazine (200uV). Additionally, the peak amplitude latency is shorter under alpha-chloralose anesthesia compared to Isoflurane and Ketamine-Xylazine anesthesia. These findings will help improve neural signal acquisition during brain stimulation techniques in other fields in vivo. V. CONCLUSIONS In this paper, we present an innovative and efficient approach for conducting rat anesthesia experiments, aimed at acquiring clearer brain signals and barrel cortex signals under whisker stimulation, devoid of muscle noise interference. Our method begins with the administration of Isoflurane as the primary anesthetic agent to induce anesthesia in the rats. Subsequently, Dexdomitor is employed to effectively suppress muscular movements during the collection of brain signals, thereby minimizing artifactual noise. Experimental outcomes have convincingly demonstrated that our proposed anesthesia protocol yields brain EEG signals and barrel cortex signals with greater clarity and improved robustness compared to conventional methods utilizing Isoflurane or the Ketamine-Xylazine combination. The brain barrel cortex signals under alpha-chloralose anesthesia are double those observed with Isoflurane and quadruple those with Ketamine-Xylazine during whisker stimulation. Additionally, the peak amplitude latency is shorter under alpha-chloralose anesthesia compared to Isoflurane and Ketamine-Xylazine anesthesia. Alpha-chloralose also maintains minimal alterations to key physiological parameters such as body temperature, respiration, and heart rates. Looking ahead, our focus will be on leveraging this refined anesthesia methodology to further enhance the purity and robustness of neural signals acquired during brain stimulation experiments. Such endeavors will facilitate deeper insights into brain functioning, particularly at the neuronal level, where data acquisition is often hindered by subtle yet significant noise sources. By minimizing these disturbances, we aim to unravel the intricacies of brain activity with unprecedented clarity. Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE The animal study (XJTUANIMAL2020) was reviewed and approved by Xi’an Jiaotong University’s Animal Protection and Use Committee. These guidelines of using animals are same with described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). CONSENT FOR PUBLICATION All authors agree for publication of this paper. AVAILABILITY OF DATA AND MATERIALS All of the data can be available when you contact the Ye Yuan at School of Life Science and Technology,Xi’an Jiaotong University: [email protected] . COMPETING INTERESTS There are no actual or apparent conflict of interest in this study for any of the co-authors. FUNDING This paper was supported by the Funding of Xi’an Jiaotong University Innovative Leading Talents Scholarship (XJTU2021). AUTHORS’ CONTRIBUTIONS YY writed and analyzed all the papers with JW’s help. JW and TL provided scientific and administrative support for YY. SL helped with paper’s pictures works. JZ helped with articles analysis. ACKNOWLEDGEMENTS This paper was supported by the Funding of Xi’an Jiaotong University Innovative Leading Talents Scholarship (XJTU2021). CONSENT TO PUBLISH DECLARATION All the authors agree that the details and images using in this paper can be published with no conflict. All the images used in this paper have no conflict for other purpose. References Sharp PS, Shaw K, Boorman L, Harris S, Kennerley AJ, Azzouz M, et al, “Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime,” Scientific Reports, vol. 5, 12621, 2015. Madularu D, Yee JR, Kenkel WM, Moore KA, Kulkarni P, Shams WM, “Integration of neural networks activated by amphetamine in females with different estrogen levels: A functional imaging study in awake rats,” Psychoneuroendocrinology, vol. 56, pp. 200-212, 2015. Antunes LM, Golledge HD, Roughan JV, Flecknell PA, “Comparison of electroencephalogram activity and auditory evoked responses during isoflurane and halothane anaesthesia in the rat,” Vet Anaesth Analg, vol. 30, pp. 15-23, 2003. P. S. Sharp, K. Shaw, L. Boorman, S. Harris, A. J. Kennerley, M. Azzouz, and J. Berwick , ”Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime,” Scientific Report, 12621, 2015. Shih Y-YI, Chang C, Chen J-C, Jaw F-S, “BOLD fMRI mapping of brain responses to nociceptive stimuli in rats under ketamine anesthesia,” Medical Engineering and Physics, vol. 30, no.8, pp. 953-958, 2008. Mizushima, N., and Ishikawa, Y, “The Effects of Isoflurane and Ketamine-Xylazine on Physiological Parameters in Rats,” Comparative Medicine, vol. 62, no. 3, pp. 209-214, 2012. Wyss, M. T., Jolivet, R., Buck, A., Magistretti, P. J., Weber, B, “ In Vivo Evidence for Lactate as a Neuronal Energy Source,” Journal of Neuroscience, vol. 31, no. 20, pp. 7477-7485, 2011. Lucie A. Low, Lucy C. Bauer, Brenda A. Klaunberg, “Comparing the Effects of Isoflurane and Alpha Chloralose upon Mouse Physiology,” Plos One, pp. 1-14, 2016. Constantin, M., ”Alpha-Chloralose: Its Use in Animal Experimentation,” Journal of the American Association for Laboratory Animal Science, vol. 50, no.6, pp. 872-875, 2011. Flecknell, P. A., Roughan, J. V., ”Use of Alpha-Chloralose and Urethane in Physiological Research: Is it Justified” Laboratory Animals, vol. 38, no.4, pp. 401-410, 2004. Silverman J, Muir WW, ”A review of laboratory animal anesthesia with chloral hydrate and chloralose” Laboratory animal science, vol. 43, no.3, pp. 210-216, 1993. Holzgrefe HH, Everitt JM, Wright EM, ” Alpha-chloralose as a canine anesthetic,” Laboratory animal science, vol. 37, no.5, pp. 587-595, 1987. Sommers MG, van Egmond J, Booij LHDJ, ” Heerschap A. Isoflurane anesthesia is a valuable alternative for a-chloralose anesthesia in the forepaw stimulation model in rats,” NMR in biomedicine, vol. 22, no.4, pp. 414-418, 2009. Shin-Lei Peng, Han Chiu, Chun-Yi Wu, Chiun-Wei Huang, Yi-Hsiu Chung, Cheng-Ting Shih, Wu-Chung Shen, ” The effect of caffeine on cerebral metabolism during alpha-chloralose anesthesia differs from isoflurane anesthesia in the rat brain,” Psychopharmacology, vol. 236, pp. 1749-1757, 2019. Scheff, J. D., Goldstein, M. A, ” Neurophysiological and Neurological Effects of Dexdomitor in Animal Models,” Veterinary Anesthesia and Analgesia, vol. 39, no.4, pp. 421-428, 2012. Dahan A, Teppema LJ, ” Influence of anaesthesia and analgesia on the control of breathing,” British journal of anaesthesia, vol. 91, no.1, pp. 40-49, 2003. White WJ, Field KJ, ” Anaesthesia and surgery of laboratory animals,” Veterinary Clinics of North America: Small Animal Practice, vol. 17, pp. 989-1017, 1987. Carl C.H. Petersen, ”The Functional Organization of the Barrel Cortex,” Neuron, 2006. Kim, C. K., A. Adhikari, K. Deisseroth, ”Integration of optogenetics with complementary methodologies in systems neuroscience,” Nature Reviews Neuroscience, vol. 18, pp. 222-235, 2017. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4778385","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":343572023,"identity":"c7d6b660-2ff5-41d3-ae0b-e2ab23912e1f","order_by":0,"name":"Ye Yuan","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Yuan","suffix":""},{"id":343572024,"identity":"b312792c-4f1d-47b1-a7fe-406f382d7ded","order_by":1,"name":"Sinan Li","email":"","orcid":"","institution":"Xi'an Jiaotong 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04:02:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52930,"visible":true,"origin":"","legend":"\u003cp\u003eAnesthetics Methods.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/d86422d76a61de2172d51e53.png"},{"id":63090222,"identity":"e0c3c66f-673d-4f67-8f2d-3c111c18b5dd","added_by":"auto","created_at":"2024-08-23 04:10:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104338,"visible":true,"origin":"","legend":"\u003cp\u003eWhisker Stimulation System.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/6a20573f560fdfd83bdc510b.png"},{"id":63090859,"identity":"a2ff3669-3b21-48cc-84eb-37f6b3b1f407","added_by":"auto","created_at":"2024-08-23 04:18:32","extension":"png","order_by":3,"title":"Figure 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7","display":"","copyAsset":false,"role":"figure","size":46383,"visible":true,"origin":"","legend":"\u003cp\u003ebarrel cortex signals under whisker stimulation with Isoflurane.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/b1d607a4e0ca2b6d87fc864e.png"},{"id":63089769,"identity":"77162fcd-e436-4411-b5f9-cb11f98a491a","added_by":"auto","created_at":"2024-08-23 04:02:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":27172,"visible":true,"origin":"","legend":"\u003cp\u003eKetamine-xylazin anesthetics EEG signal.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/62544a8bfe6c459ebc2a9cdb.png"},{"id":63091691,"identity":"f523351c-0d31-4e4f-8167-80bb44ea0ceb","added_by":"auto","created_at":"2024-08-23 04:34:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":40949,"visible":true,"origin":"","legend":"\u003cp\u003ebarrel cortex signals under whisker stimulation with Ketamine-xylazin.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/986c0b3ea40291cc8d4c609f.png"},{"id":63089765,"identity":"29c66d8f-a137-4955-a5b8-6d113a516cf8","added_by":"auto","created_at":"2024-08-23 04:02:32","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":52374,"visible":true,"origin":"","legend":"\u003cp\u003e1 hour and 7 hours anesthesia results.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/a1f8a3c109aeccaa2a02f0c7.png"},{"id":63090864,"identity":"3e953178-8e79-4f63-916f-7d9ed866a1bc","added_by":"auto","created_at":"2024-08-23 04:18:32","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":45360,"visible":true,"origin":"","legend":"\u003cp\u003e7 hours Breathing rate.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/0b0104355f2e867bd0c51b8e.png"},{"id":63089770,"identity":"f93a44ed-c4c8-49ed-a763-fb7558874307","added_by":"auto","created_at":"2024-08-23 04:02:32","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":20877,"visible":true,"origin":"","legend":"\u003cp\u003eBreathing rate change under different anesthesia.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/913fa494a4408aa92bcdabbb.png"},{"id":63090225,"identity":"f2a4e94f-b9e8-4a37-a6a6-7137c8fdf16f","added_by":"auto","created_at":"2024-08-23 04:10:32","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":48393,"visible":true,"origin":"","legend":"\u003cp\u003e7 hours Heart rate.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/a504902871d6e589ee30072b.png"},{"id":63090863,"identity":"89418f5b-1898-4230-8fba-340e2f98e104","added_by":"auto","created_at":"2024-08-23 04:18:32","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":20640,"visible":true,"origin":"","legend":"\u003cp\u003eHeart rate change under different anesthesia.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/867f62ad0254ca036b97ece7.png"},{"id":63090860,"identity":"68f5af18-7bae-4b85-9d3d-f6d02377cfa6","added_by":"auto","created_at":"2024-08-23 04:18:32","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":45943,"visible":true,"origin":"","legend":"\u003cp\u003e7 hours temperature.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/2ffaf7b45b33353e875186fb.png"},{"id":63089761,"identity":"ccf21f4d-e4d0-479c-92ae-8cb7cc9b7403","added_by":"auto","created_at":"2024-08-23 04:02:32","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":21053,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature change under different anesthesia.\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/71cd7c9ffc13c7e43eb05c1c.png"},{"id":64412899,"identity":"2e51a365-b236-4756-a0d1-d01249404f14","added_by":"auto","created_at":"2024-09-12 21:53:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1298265,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4778385/v1/50cf7572-f655-4cc7-838c-dfe759f05af1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Anesthetic Techniques for Improving Rat Whisker Stimulation Responses in the Barrel Cortex","fulltext":[{"header":"I. INTRODUCTION","content":"\u003cp\u003eRecently, the use of anesthesia in the process of collecting brain signals from rodents has introduced numerous potential confounding factors, making it crucial for researchers to understand the impacts of various anesthesia regimens on their experimental animals [1]. This understanding can significantly aid investigators in selecting suitable anesthesia/sedation rotocols tailored for the collection of brain signals [2]. Isoflurane, a widely utilized anesthetic agent across both rodent and non-rodent species over the past decades [3], effectively assists in maintaining a stable anesthetic state during experiments [4]. Furthermore, the combined use of ketamine and xylazine has emerged as a popular choice for inducing anesthesia in diverse animal species [5]. This combination is highly esteemed in both veterinary medicine and research circles due to its ability to consistently provide a controllable state of sedation, analgesia, and muscle relaxation, thereby enhancing the overall quality and reliability of experimental outcomes. Despite their prevalent usage in anesthetizing animals for neurophysiological experiments, Isoflurane and the Ketamine-xylazine combination both exhibit inherent limitations [6]-[7]. Isoflurane necessitates continuous administration to sustain its anesthetic effect, potentially introducing variability and impacting the animal\u0026rsquo;s physiological state over time. Conversely, Ketamine and xylazine offers a relatively brief duration of action, typically spanning approximately 30 minutes, necessitating frequent redosing and potentially contributing to fluctuations in key physiological parameters.\u003c/p\u003e\n\u003cp\u003eIn neurophysiological research, securing high-fidelity brain signals from animal models is paramount to ensuring the accuracy and reproducibility of experimental findings. For instance, the presence of muscle noise can significantly hinder the isolation of pristine neural signals. Thus, the adoption of an anesthetic regimen that proficiently minimizes such noise without significantly perturbing the animal\u0026rsquo;s physiological equilibrium is imperative [8]. This paper introduces a groundbreaking anesthetic approach tailored for rats, aimed at achieving unparalleled raw brain signal acquisition by effectively eliminating muscle noise interference during experiments. Our method innovatively integrates the use of alpha-chloralose (a-c) and Dexdomitor, subsequent to an initial induction phase with Isoflurane. Specifically, alpha-chloralose is administered to maintain a stable anesthetic state throughout the experiment [9]- [15], while Dexdomitor is employed to prevent muscular contractions during the crucial signal acquisition phase. This strategic combination addresses the challenges associated with conventional anesthetics by harnessing the enduring stability of alpha-chloralose and the potent muscle-relaxing effects of Dexdomitor [16].\u003c/p\u003e\n\u003cp\u003eOur method ensures a stable anesthetic condition spanning up to 7 hours, significantly mitigating the need for continuous dosing and the corresponding perturbations to the animal\u0026rsquo;s physiology. Experimental outcomes underscore the superiority of this novel anesthetic protocol. Compared to the traditional Isoflurane and Ketamine-xylazine methods, our approach yields cleaner barrel cortex signals and enhances the robustness of data acquisition. Furthermore, it minimizes alterations to critical physiological indicators, including body temperature, respiratory rate, and heart rhythm, as evidenced in Fig. 1. By refining the anesthetic strategy, we elevate the quality of neurophysiological data, thereby fostering more dependable and precise research findings in animal model-based studies. Our proposed model revolves around utilizing alpha-chloralose (a-c) as the primary anesthetic agent, coupled with a low-dose Isoflurane (0:5%) administration. This approach is inspired by the advantage of a-c in allowing animals to maintain spontaneous respiration, a crucial aspect in experiments where the preservation of natural physiological functions is imperative.\u003c/p\u003e\n\u003cp\u003eIn this paper, we demonstrate how the combined use of a-c and Isoflurane can yield superior raw barrel cortex signals devoid of muscle noise under whisker stimulation. Specifically, we introduce a novel combination of anesthetics, leveraging the strengths of alpha-chloralose and Isoflurane, to achieve a stable anesthetic state while minimizing muscle noise interference. By optimizing the anesthetic approach, we significantly improve the quality of neurophysiological data, enabling more reliable and accurate research outcomes. We provide experimental evidence demonstrating the effectiveness of our proposed anesthetic protocol in enhancing barrel cortex signal acquisition and minimizing alterations to critical physiological parameters. The primary contributions of this article are as follows:\u003c/p\u003e\n\u003cp\u003e1) A novel anesthetic approach utilizing alpha-chloralose (a-c) is introduced to effectively manage the anesthetic state of animals during experimental signal acquisition. This method departs from the current state-of-the-art anesthetics, such as Isoflurane and Ketamine-xylazine combinations, by integrating a-c with a low concentration of Isoflurane (0:5%). Consequently, the initial step in our protocol involves administering a combination of a-c and 0:5% Isoflurane to anesthetize the animal prior to signal acquisition, ensuring optimal conditions for accurate and reliable data collection. This improved performance is achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates.\u003cbr\u003e2) The methodology employed in this paper aims to maintain a stable anesthetic state in animals during barrel cortex signal measurements, thereby ensuring the accuracy and reliability of the data collected. To achieve this, we administer Dexdomitor to mitigate muscular movements during signal acquisition, effectively reducing the potential interference of muscle noise on the raw brain signals. This approach, coupled with the use of a combination of alpha-chloralose (a-c) and 0:5% Isoflurane anesthesia, further minimizes the impact of extraneous factors, allowing for the capture of high-quality, uncontaminated brain signals. The efficacy of a-c in maintaining anesthesia for up to 7 hours stands in contrast to the shorter durations achievable with continuous Isoflurane administration or the 30-minute window offered by Ketamine-Xylazine, highlighting the practical advantages of our proposed method.\u003cbr\u003e3) Experimental investigations have been conducted to validate that the methodology presented in this paper significantly outperforms existing anesthetic protocols, including Isoflurane and Ketamine-xylazine combinations, in terms of acquiring cleaner and more robust animal brain barrel cortex signals with minimal muscle noise interference. The brain barrel cortex signals under the alpha-chloralose anesthetic is double that of Isoflurane and quadruple that of Ketamine-xylazine at the whisker stimulation experiment system. Moreover, our approach demonstrates a marked improvement in signal quality and robustness, underscoring its effectiveness in minimizing extraneous factors that can compromise the integrity of brain signal measurements.\u003c/p\u003e\n\u003cp\u003eThe rest of this article is organized as follows: Section II states experiment setups and data acquisition. Section III introduces the different methods of anesthetics for experiment data acquisition. The experiment results are used to validate our method\u0026rsquo;s\u003cbr\u003eeffectiveness in Section IV. Section V presents the conclusions of the paper and discusses future works.\u003c/p\u003e"},{"header":"II. EXPERIMENTS SETUPS AND DATA ACQUISITION","content":"\u003cp\u003eThis section begins by presenting the sources of the signal data utilized in our experiments. Subsequently, we detail the preparatory steps and materials required for the experimental setup, emphasizing the necessary preparations to ensure the successful execution of our research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Signal Data Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Sprague Dawley male rats used in this study, weighing between 250-350g, were housed in an environment adhering to a strict 12-hour light-dark cycle within facilities accredited by Xi\u0026rsquo;an Jiaotong University\u0026rsquo;s Animal Protection and Use Committee. Throughout their stay, the animals had unrestricted access to food and water, and all experiments were conducted in accordance with protocols approved by the Committee. We made every effort to minimize animal suffering and ensure the ethical treatment of all subjects involved in this research. These guidelines are same with described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). All of these SD rats (31-12-002-D-000010 )were purchased from the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences.\u003c/p\u003e\n\u003cp\u003eIn this study, we conducted comparative experiments on three distinct groups of Sprague Dawley male rats, each comprising 12 animals (N1=12, N2=12, N3=12). These experiments aimed to compare the effects of three anesthesia methods: alphachloralose, Isoflurane, and Ketamine-xylazine. Animals were randomly assigned to receive one of these anesthesia protocols on experimental days, with the order of administration counterbalanced across groups to mitigate potential biases. Notably, rats that underwent Isoflurane anesthesia were excluded from subsequent experiments involving alpha-chloralose and Ketaminexylazine to avoid confounding factors.\u003cbr\u003eUpon completion of the experiments utilizing alpha-chloralose, Isoflurane, and Ketamine-xylazine, euthanasia was performed in accordance with ethical guidelines. During each experiment, an extracellular electrode was placed within the animal\u0026rsquo;s\u003cbr\u003edura mater to capture raw electroencephalogram (EEG) brain signals under anesthesia. This approach enabled us to obtain a comprehensive understanding of the EEG signals under varying anesthetic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Preparing things of experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo accurately measure the raw EEG brain signals of Sprague Dawley male rats, we followed a series of preparatory steps for our experiments, as outlined in reference [17]. These steps were crucial to ensure the reliability and validity of our data collection process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1)Preparation:\u0026nbsp;\u003c/strong\u003eBefore commencing the experiments, it is imperative to ensure that the work area is safe and adequately ventilated. Next, prepare all necessary anesthetic instruments and medications, including alpha-chloralose, Isoflurane gas, and Ketamine-xylazine, along with the inhalation anesthesia system, appropriately sized anesthesia masks, a surgical platform, injection needles, a warming pad, and other essential equipment. Additionally, Buprenorphine and Dexmedetomidine (Dexdomitor) will be utilized in this study due to their crucial role in animal anesthesia. Set up the inhalation anesthesia system by connecting it to the gas supply and ensuring that it is calibrated to accurately control the concentration and flow rate of Isoflurane, thereby ensuring a smooth and effective anesthesia process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2)Prepare the Rats:\u003c/strong\u003e Prior to anesthesia, the rats are gently placed in appropriately sized anesthesia chambers designed to acclimate them to the environment. This step helps to minimize stress and ensure a smoother anesthesia process. With the gas source activated, Isoflurane gas is gradually introduced into the anesthesia chamber. The concentration of Isoflurane is incrementally increased until the rats reach a state of full anesthesia, marked by a lack of response to external stimuli. Throughout this process, it is crucial to closely monitor the rats\u0026rsquo; breathing patterns and the depth of anesthesia, adjusting the gas concentration as needed. Toe pinch tests are periodically performed to assess the level of anesthesia, ensuring that the rats are sufficiently sedated without being overdosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3)Surgical Procedure:\u003c/strong\u003e Once the animals are in a deep state of anesthesia, we start the surgical process. A portion of the skull at the Bregma location is carefully removed to expose the brain. Then, one extracellular electrode is gently placed onto the dura mater, ensuring minimal disruption to the surrounding tissue. The electrode is used to collect the barrel cortex signals, providing valuable insights into the neural activity of the whisker stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4)Maintain Anesthesia and Record EEG Signals:\u0026nbsp;\u003c/strong\u003eIt is crucial to maintain an appropriate depth of anesthesia throughout the duration of the experiment by adjusting the flow rate and concentration of Isoflurane as needed. This ensures that the rats remain sufficiently sedated without experiencing undue stress or discomfort. Throughout the anesthesia and recording process, continuous monitoring of the rats\u0026rsquo; vital signs, such as respiratory rate and heart rate, is essential to ensure their safety and well-being. The raw EEG brain signals are collected under the influence of the chosen anesthetics, including alpha-chloralose, Isoflurane, and Ketamine-xylazine, allowing for a comprehensive assessment of neural activity under various anesthetic conditions.\u003c/p\u003e\n\u003cp\u003eUpon completion of the preparatory steps, we utilize the Cerebus system to gather the raw EEG brain signals from the animals. The Cerebus system is a comprehensive data acquisition hardware and software platform designed specifically for recording and analyzing signals emanating from the brain and peripheral nervous system. This system incorporates microelectrode arrays, connectors, a neural signal acquisition system, and application software, offering a seamless solution for neurophysiology experiments. As a multi-channel data acquisition system, the Cerebus is renowned for its power, ease of operation, and versatility. It is capable of recording and analyzing the electrical activity of the animal brain and peripheral nerves with unparalleled precision. The system is configured to cater to a wide range of animal models, including birds, mice, rats, cats, and primates, both under anesthesia and in their natural waking state. In real-time, the Cerebus system captures, processes, and analyzes action potentials (spikes), field potentials (LFPs), and other physiological signals pertinent to the experimental conditions. This enables researchers to gain a deeper understanding of neural function and behavior, facilitating the advancement of neuroscience research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5)Termination of Anesthesia:\u0026nbsp;\u003c/strong\u003eWhen concluding the anesthesia procedure, it is crucial to gradually taper off the concentration of Isoflurane. This gradual reduction ensures a smooth transition for the rats as they gradually regain consciousness. Once the rats are fully awake and have resumed their normal activities, they can be safely removed from the anesthesia chamber. Upon removal, the rats should be provided with appropriate care and monitoring to ensure their continued safety and health. This includes monitoring their vital signs and observing for any signs of distress or discomfort. By adhering to these protocols, researchers can ensure that the animals are treated humanely and with the utmost respect throughout the experimental process. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Experimental System\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats\u0026rsquo; whiskers are sensitive detectors for picking up tactile information, enabling them to navigate, recognize, and sense their surroundings [18]. Each whisker is highly specialized and organized, represented in a barrel-like column in the somatosensory cortex of the rat brain. The barrel cortex comprises approximately 13% of the cortical volume and 69% of the somatosensory cortex. The movement of these whiskers generates evoked potentials in the somatosensory cortex in a synchronized manner. A growing body of research explores and validates this link, demonstrating that the arrangement of the barrels in the barrel cortex corresponds clearly to the arrangement of the whiskers on the rat\u0026rsquo;s face.\u003c/p\u003e\n\u003cp\u003eWhen electrical stimulation is applied to one barrel-like area, multiple barrel-like columns may be activated [19]. To collect specific neural electrical signals induced by the barrel cortex, this study employed mechanical stimulation of a single whisker. By stimulating the movement of a single whisker (C2), projected onto the corresponding barrel column, we could more precisely locate the evoked response. As illustrated in Fig. 2, an L-shaped tube was attached to the shaft of a stepper motor with a 1.8 stepping angle. The target whisker (C2 of the larger barrel-like column) was inserted into the tube. Using an Arduino UNO R3 controller and the stepper motor driver A4988, the motor shaft was rotated for 1 ms, triggering the acquisition of synchronous local field potentials (LFPs) from the barrel cortex, as shown in Fig. 3.\u003c/p\u003e"},{"header":"III. ANESTHETICS METHODS","content":"\u003cp\u003eIn this section, we delve into the three anesthetic methods commonly employed in animal experiments: (a) Alpha-chloralose, (b) Isoflurane, and (c) Ketamine-xylazine. We provide a brief overview of each method, highlighting their respective applications and considerations. Understanding the nuances of each anesthetic method is crucial for ensuring the safety, comfort, and ethical treatment of the animals involved in research. By exploring the unique properties and usage protocols of these anesthetics, researchers can make informed decisions regarding the most appropriate choice for their specific experimental needs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Alpha-chloralose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlpha-chloralose (a-c) is a crystalline compound with limited solubility in water, categorized as a sedative-hypnotic agent. It is frequently employed for anesthesia in laboratory rodents, particularly rats and mice, due to its specific effect on the central nervous system, primarily through modulating GABA receptors. GABA, an inhibitory neurotransmitter, regulates neuronal excitability, and a-c enhances GABAergic activity in rodents, inducing sedation.\u003c/p\u003e\n\u003cp\u003eThe depth of anesthesia achieved with a-c varies with dosage and the route of administration, typically administered via intravenous or intraperitoneal injection in small rodents. This ensures the animals are in a sedated or unconscious state during surgical or experimental procedures, minimizing discomfort and stress.\u003c/p\u003e\n\u003cp\u003eIn our experiment, we prepare a-c one day prior to the procedure. During the experiment, the animals are placed in an anesthesia box for 5 minutes to ensure deep anesthesia. Subsequently, we perform an intraperitoneal injection of Buprenorphine to further alleviate pain during surgery. After a 5-minute wait for the analgesic to take effect, we proceed with the surgical procedure, carefully removing a portion of the cranium to place an extracellular electrode on the dura mater for EEG signal acquisition.\u003c/p\u003e\n\u003cp\u003eOnce the surgery is completed, we administer a-c to the animals, allowing a 30-minute period for it to take full effect.During this time, we adjust the concentration of Isoflurane (Iso), starting at 1 and titrating downwards to find the optimal balance for maintaining anesthesia while minimizing adverse effects. This approach ensures that the animals are adequately anesthetized throughout the experiment while we collect their raw EEG brain signals. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Isoflurane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsoflurane belongs to the halogenated ether class of compounds, characterized by its unique molecular structure incorporating halogen atoms. At room temperature, it exists as a clear, colorless liquid with a pleasant aroma, exhibiting non-flammable and\u003cbr\u003estable properties under standard conditions. Widely recognized as a volatile anesthetic agent in medical and veterinary settings, Isoflurane is renowned for its efficacy in inducing and maintaining general anesthesia during surgical procedures.\u003c/p\u003e\n\u003cp\u003eFor mice and other small animals, Isoflurane is often administered via inhalation, leveraging its rapid absorption into the bloodstream. This method of delivery is particularly favored in veterinary medicine due to Isoflurane\u0026rsquo;s swift onset of action, allowing for precise control over the depth of anesthesia. Its primary mechanism of action involves enhancing the activity of gamma-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors in the central nervous system, leading to the suppression of neuronal activity and the induction of anesthesia.\u003c/p\u003e\n\u003cp\u003eThe rapid onset and offset of Isoflurane\u0026rsquo;s effects, coupled with its efficient elimination from the body, contribute to its popularity as an anesthetic agent. This facilitates a swift and smooth recovery process for the animals, minimizing potential adverse effects and enhancing overall animal welfare.\u003c/p\u003e\n\u003cp\u003eIn this experiment, to prevent SD rats from waking up suddenly during stimulation, Isoflurane should be maintained at an anesthesia level of 1:5%-2% for the collection of LFP signals from the barrel cortex. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Ketamine-xylazine\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKetamine, a dissociative anesthetic agent, exerts its primary effects by antagonizing N-methyl-D-aspartate (NMDA) receptors within the central nervous system. This action elicits a dissociative state characterized by profound analgesia, amnesia, and catalepsy, all while preserving cardiovascular stability. Consequently, Ketamine is capable of inducing dissociative anesthesia.\u003c/p\u003e\n\u003cp\u003eMeanwhile, Xylazine, an alpha-2 adrenergic agonist, possesses sedative, analgesic, and muscle relaxant properties. By stimulating alpha-2 adrenergic receptors in the central nervous system, Xylazine promotes sedation, muscle relaxation, and analgesia. Its sedative effects are particularly noteworthy, as it effectively calms animals and eases muscular tension.\u003c/p\u003e\n\u003cp\u003eWhen combined, Ketamine and Xylazine exhibit synergistic effects, amplifying the anesthetic and sedative qualities of both drugs. Ketamine contributes to the dissociative anesthesia, while Xylazine enhances sedation and muscle relaxation. This\u003cbr\u003ecombination is renowned for its swift onset of action and relatively brief duration of anesthesia, making it an ideal choice for procedures requiring rapid recovery.\u003c/p\u003e\n\u003cp\u003eImportantly, the Ketamine-Xylazine combination exerts minimal impact on respiratory and cardiovascular functions in healthy animals, ensuring patient safety during procedures. As a result, it is widely utilized in minor surgeries and diagnostic imaging, where rapid anesthesia and recovery are crucial.\u003c/p\u003e\n\u003cp\u003eDuring the experiment, local field potential (LFP) changes are observed as the concentration of general anesthetics (KetamineXylazine) varies. Generally, Ketamine-Xylazine is effective for only about 30 minutes of sustained anesthesia. To address this limitation, the anesthesia regimen needs to be improved by continuously administering 0:5% to 1:5% Isoflurane. This ensures that SD rats maintain a deeper anesthetic state during surgery and experiments, resulting in more stable and reliable LFP data collection. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg,i.p.).\u003c/p\u003e"},{"header":"IV. EXPERIMENTAL RESULTS","content":"\u003cp\u003eThis section begins by presenting the outcomes of barrel cortex signal collection experiments conducted on animals using various anesthetics (alpha-chloralose, Isoflurane, and Ketamine-xylazine) under whisker stimulation. Following this, we conduct a comparative analysis, emphasizing the robustness of the signals obtained through our proposed method. Finally, we demonstrate that our approach results in minimal to no significant alterations in physiological parameters, highlighting its efficacy and safety.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. The EEG signals and brain barrel cortex signals under whisker stimulation with a-c, Isoflurane and Ketaminexylazine anesthesia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe barrel cortex signals under whisker stimulation and EEG brain signals of animals were recorded using three distinct anesthetic methods: alpha-chloralose (a-c), Isoflurane, and the Ketamine-xylazine combination. These signals were precisely captured by a single extracellular electrode positioned at +2.5mm and -5.5mm relative to the animal\u0026rsquo;s Bregma dura mater. A total of 36 animals were randomly assigned to three groups, with each group receiving one of the anesthetic methods. Consequently, each anesthetic method was administered to 12 animals, ensuring equal representation in the sample. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e1) Result 1: The brain EEG signals and barrel cortex signals under whisker stimulation with a-c anesthesia.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Cerbus system was employed to record the EEG brain signals of animals using an extracellular electrode. When combining alpha-chloralose (a-c) with Isoflurane at varying concentrations, we observed certain differences in the recorded signals. Notably, the raw EEG signals remained stable when Isoflurane was set to 0, albeit with some muscle noise present. To mitigate this, we incrementally increased the Isoflurane concentration from 0 to 0:25% and then to 0:5%, while recording the signals. Figure 4 illustrates the outcomes of these adjustments. Specifically, Figure 4 demonstrates that at 0:5% Isoflurane, there is minimal muscle noise evident in the animal\u0026rsquo;s EEG signals. This finding suggests that combining a-c with 0:5% Isoflurane represents the optimal approach for acquiring high-quality EEG signals from animal brains.\u003c/p\u003e\n\u003cp\u003eSubsequently, we stimulated the C2 whisker under the aforementioned anesthetic method. Fig. 5 shows the barrel cortex signals under whisker stimulation within 1 ms. The results clearly indicate that the peak amplitude can almost reach 800 uV at 20 ms, demonstrating the efficacy of the anesthetic combination in facilitating clear and precise signal acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2) Result 2: The brain EEG signals and barrel cortex signals under whisker stimulation with Isoflurane anesthesia.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenerally speaking, Isoflurane provides a stable anesthetic state during the recording of animals\u0026rsquo; brain EEG signals. However, qualitative observations indicate that a concentration of 1:5% Isoflurane is necessary to eliminate muscle noise from the EEG\u003cbr\u003e\u0026nbsp;signals entirely. At lower concentrations, such as 0:5% Isoflurane or when using alpha-chloralose (a-c) anesthesia, animals tend to awaken within half an hour, as evidenced by movements like toe pinching or ear twitching, which were not observed\u003cbr\u003e\u0026nbsp;with 1:5% Isoflurane. Fig. 6 showcases the brain EEG signals acquired under 1:5% Isoflurane anesthesia, highlighting its effectiveness in ensuring a stable and artifact-free recording environment.\u003c/p\u003e\n\u003cp\u003eFig. 7 illustrates the barrel cortex signals under whisker stimulation within 1ms using Isoflurane anesthesia. The peak amplitude can reach 400 uV, which is half of what is observed with alpha-chloralose (a-c). The peak amplitude latency is 22ms, demonstrating the temporal characteristics of the signal under Isoflurane anesthesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3) Result 3: The brain EEG signals and barrel cortex signals under whisker stimulation with Ketamine-xylazine anesthesia.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eKetamine-xylazine is a commonly preferred anesthetic for animals in various regions due to its effective sedative properties. However, its relatively short duration of anesthesia limits its applicability for prolonged brain EEG signal recording experiments. Despite offering a superior anesthetic effect compared to 1:5% Isoflurane, Ketamine-xylazine anesthesia still introduces some muscle noise into the recorded brain EEG signals. Fig. 8 depicts the brain EEG signals obtained under Ketamine-xylazine anesthesia, highlighting the presence of this noise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 9 presents the barrel cortex signals under whisker stimulation within 1ms using Ketamine-xylazine anesthesia. It is evident that the peak amplitude reaches only 200 uV, which is half of the amplitude observed with Isoflurane. The peak amplitude latency is 24 ms, demonstrating the temporal response characteristics under Ketamine-xylazine anesthesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. The EEG brain signals robustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted an experiment to validate the enhanced robustness of our proposed anesthetic method for prolonged anesthesia compared to Isoflurane and Ketamine-xylazine. This study involved monitoring the animals\u0026rsquo; raw EEG brain signals over time while under anesthesia. Given that Ketamine-xylazine anesthesia is effective for only approximately half an hour and cannot be repeatedly administered to the same animal without causing harm or death, we focused our analysis on the other two methods. Fig. 10 showcases the results of raw EEG brain signals recorded under alpha-chloralose (a-c) anesthesia at 1 hour and 7 hours. The findings demonstrate that a-c anesthesia has a less pronounced impact on the raw EEG signals compared to Isoflurane and Ketamine-xylazine. This underscores the potential of our proposed method for facilitating extended EEG recording sessions with minimal interference. At the end of the experiment, the rats were euthanized by intraperitoneal injection of pentobarbital (180 mg/kg, i.p.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. P\u003c/strong\u003e\u003cstrong\u003ehysiological parameters of animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe demonstrate that our method results in minimal changes to key physiological parameters, including breathing rate, heart rate, and body temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;1) \u003cem\u003eResult 1: Breathing rate under different anesthesia.\u003c/em\u003e\u003cbr\u003e\u0026nbsp;In our comparative experiments using alpha-chloralose, Isoflurane, and Ketamine-xylazine, we observed a decrease in breathing rates among all animals as anesthesia progressed. However, Ketamine-xylazine\u0026rsquo;s effectiveness was limited to approximately 0.5 hours, precluding its repeated use on the same animal due to potential harm or fatality. Fig. 11 illustrates the breathing rates recorded over 7 hours for alpha-chloralose and Isoflurane, and over 0.5 hours for Ketamine-xylazine. Initially, all animals exhibited a breathing rate of 120 breaths per minute, which decreased to 110, 108, and 104 breaths per minute after 0.5 hours, respectively. By the 7-hour mark, the alpha-chloralose and Isoflurane groups had further decreased to 75 breaths per minute, with no significant differences observed between the alpha-chloralose and Isoflurane groups at any time point. Fig.12 quantifies the breathing rates, presenting the mean and standard error of the mean (SEM) for changes in breathing rates among the 12 animals anesthetized with each method, providing a clear comparison of their effects on respiratory function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2) Result 2: Heart rate under different anesthesia.\u003c/em\u003e\u003cbr\u003e\u0026nbsp;Throughout the anesthesia period, animals were continuously monitored for heart rate using electrocardiography (ECG). Heart rate measurements were taken at baseline (pre-anesthesia), immediately post-induction, and at regular intervals thereafter. As depicted in Fig. 13, all contrast experiments began with a baseline heart rate of 320 beats per minute (bpm). Post-induction, the heart rates for alpha-chloralose, Isoflurane, and Ketamine-xylazine anesthesia were 300 bpm, 280 bpm, and 260 bpm, respectively. During mid-anesthesia, the rates were 310 bpm, 275 bpm, and 210 bpm, while at the end of anesthesia, they were 315 bpm, 290 bpm, and 215 bpm, respectively. Notably, alpha-chloralose caused a moderate and relatively stable decrease in heart rate compared to baseline, maintaining levels close to baseline throughout the anesthesia period. Isoflurane induced a significant reduction from baseline, which remained stable but lower throughout anesthesia. Conversely, the Ketamine-xylazine combination resulted in the most pronounced decrease, with an immediate and sustained drop in heart rate post-induction. Fig.14 quantifies the heart rate, presenting the mean and standard error of the mean (SEM) for heart rate changes among the 12 animals under each anesthesia method. This offers a clear visualization of the respective effects of each anesthetic method on cardiac function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3) Result 3: Temperature change under different anesthesia.\u003c/em\u003e\u003cbr\u003e\u0026nbsp;Anesthesia can significantly impact animals\u0026rsquo; body temperature, making the monitoring of these changes imperative for safeguarding their welfare and ensuring the accuracy of experimental outcomes. Fig. 15 illustrates the typical temperature fluctuations encountered during anesthesia using alpha-chloralose, Isoflurane, and the Ketamine-xylazine combination. Across our contrast experiments, the baseline temperature prior to anesthesia was consistently 33.0◦C. At the end of anesthesia, the temperatures dropped to 30.5◦C, 29.5◦C, and 30.0◦C, respectively, for the three anesthesia methods. Furthermore, Fig.16 quantifies the temperature changes, providing a detailed representation of the mean and standard error of the mean (SEM) for temperature variations among the group of 12 animals undergoing anesthesia with each of these agents. This offers valuable insights into their specific effects on body temperature, highlighting the importance of temperature monitoring in maintaining animal welfare and ensuring reliable experimental data.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eD. Discussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlpha-chloralose demonstrated a moderate and steady decline in heart rate compared to baseline levels, preserving nearbaseline rates throughout anesthesia. In contrast, Isoflurane significantly reduced heart rate from baseline, maintaining a relatively stable but lowered rate throughout the procedure. Notably, the combination of Ketamine-Xylazine produced the most pronounced decrease in heart rate, with an immediate and sustained drop after induction. These observations underscore the necessity of selecting anesthetic protocols tailored to the specific cardiovascular requirements of the experimental or clinical setting.\u003c/p\u003e\n\u003cp\u003eSpecifically, Alpha-Chloralose exhibited a minimal impact on heart rate, rendering it suitable for procedures necessitating stable cardiovascular function. Isoflurane, with its noticeable yet stable depressant effect on the cardiovascular system, may necessitate vigilant monitoring and potential interventions to manage bradycardia. Ketamine-Xylazine, exhibiting strong depressant effects, efficiently induces a profound anesthetic state but requires close cardiovascular monitoring.\u003c/p\u003e\n\u003cp\u003eSimilarly, monitoring body temperature during anesthesia is paramount, as hypothermia can significantly impact physiological processes and experimental outcomes. Alpha-chloralose elicits the least temperature reduction, making it advantageous in studies prioritizing temperature maintenance. Isoflurane causes a moderate decrease in temperature, necessitating careful thermal environment management. Ketamine-Xylazine, however, results in the most significant drop in body temperature, emphasizing the importance of understanding these changes to safeguard animal health and ensure data reliability.\u003c/p\u003e\n\u003cp\u003eA critical conclusion is that the peak amplitude of the barrel cortex signals under whisker stimulation within 1ms shows a prominent difference among anesthetics. The brain barrel cortex signals under alpha-chloralose anesthesia (800uV) are double those under Isoflurane (400uV) and quadruple those under Ketamine-Xylazine (200uV). Additionally, the peak amplitude latency is shorter under alpha-chloralose anesthesia compared to Isoflurane and Ketamine-Xylazine anesthesia. These findings will help improve neural signal acquisition during brain stimulation techniques in other fields in vivo.\u003c/p\u003e"},{"header":"V. CONCLUSIONS","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eIn this paper, we present an innovative and efficient approach for conducting rat anesthesia experiments, aimed at acquiring clearer brain signals and barrel cortex signals under whisker stimulation, devoid of muscle noise interference. Our method begins with the administration of Isoflurane as the primary anesthetic agent to induce anesthesia in the rats. Subsequently, Dexdomitor is employed to effectively suppress muscular movements during the collection of brain signals, thereby minimizing artifactual noise. Experimental outcomes have convincingly demonstrated that our proposed anesthesia protocol yields brain EEG signals and barrel cortex signals with greater clarity and improved robustness compared to conventional methods utilizing Isoflurane or the Ketamine-Xylazine combination. The brain barrel cortex signals under alpha-chloralose anesthesia are double those observed with Isoflurane and quadruple those with Ketamine-Xylazine during whisker stimulation. Additionally, the peak amplitude latency is shorter under alpha-chloralose anesthesia compared to Isoflurane and Ketamine-Xylazine anesthesia. Alpha-chloralose also maintains minimal alterations to key physiological parameters such as body temperature, respiration, and heart rates.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eLooking ahead, our focus will be on leveraging this refined anesthesia methodology to further enhance the purity and robustness of neural signals acquired during brain stimulation experiments. Such endeavors will facilitate deeper insights into brain functioning, particularly at the neuronal level, where data acquisition is often hindered by subtle yet significant noise sources. By minimizing these disturbances, we aim to unravel the intricacies of brain activity with unprecedented clarity.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/p\u003e\n\u003cp\u003eThe animal study (XJTUANIMAL2020) was reviewed and approved by Xi\u0026rsquo;an Jiaotong University\u0026rsquo;s Animal Protection and Use Committee. These guidelines of using animals are same with described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010).\u003c/p\u003e\n\u003cp\u003eCONSENT FOR PUBLICATION\u003c/p\u003e\n\u003cp\u003eAll authors agree for publication of this paper.\u003c/p\u003e\n\u003cp\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/p\u003e\n\u003cp\u003eAll of the data can be available when you contact the Ye Yuan at School of Life Science and Technology,Xi\u0026rsquo;an Jiaotong University: [email protected].\u003c/p\u003e\n\u003cp\u003eCOMPETING INTERESTS\u003c/p\u003e\n\u003cp\u003eThere are no actual or apparent conflict of interest in this study for any of the co-authors.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThis paper was supported by the Funding of Xi\u0026rsquo;an Jiaotong University Innovative Leading Talents Scholarship (XJTU2021).\u003c/p\u003e\n\u003cp\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eYY writed and analyzed all the papers with JW\u0026rsquo;s help. JW and TL provided scientific and administrative support for YY. SL helped with paper\u0026rsquo;s pictures works. JZ helped with articles analysis.\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eThis paper was supported by the Funding of Xi\u0026rsquo;an Jiaotong University Innovative Leading Talents Scholarship (XJTU2021).\u003c/p\u003e\n\u003cp\u003eCONSENT TO PUBLISH DECLARATION\u003c/p\u003e\n\u003cp\u003eAll the authors agree that the details and images using in this paper can be published with no conflict. All the images used in this paper have no conflict for other purpose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSharp PS, Shaw K, Boorman L, Harris S, Kennerley AJ, Azzouz M, et al, \u0026ldquo;Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime,\u0026rdquo; Scientific Reports, vol. 5, 12621, 2015.\u003c/li\u003e\n\u003cli\u003eMadularu D, Yee JR, Kenkel WM, Moore KA, Kulkarni P, Shams WM, \u0026ldquo;Integration of neural networks activated by amphetamine in females with different estrogen levels: A functional imaging study in awake rats,\u0026rdquo; Psychoneuroendocrinology, vol. 56, pp. 200-212, 2015.\u003c/li\u003e\n\u003cli\u003eAntunes LM, Golledge HD, Roughan JV, Flecknell PA, \u0026ldquo;Comparison of electroencephalogram activity and auditory evoked responses during isoflurane and halothane anaesthesia in the rat,\u0026rdquo; Vet Anaesth Analg, vol. 30, pp. 15-23, 2003.\u003c/li\u003e\n\u003cli\u003eP. S. Sharp, K. Shaw, L. Boorman, S. Harris, A. J. Kennerley, M. Azzouz, and J. Berwick , \u0026rdquo;Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime,\u0026rdquo; Scientific Report, 12621, 2015.\u003c/li\u003e\n\u003cli\u003eShih Y-YI, Chang C, Chen J-C, Jaw F-S, \u0026ldquo;BOLD fMRI mapping of brain responses to nociceptive stimuli in rats under ketamine anesthesia,\u0026rdquo; Medical Engineering and Physics, vol. 30, no.8, pp. 953-958, 2008.\u003c/li\u003e\n\u003cli\u003eMizushima, N., and Ishikawa, Y, \u0026ldquo;The Effects of Isoflurane and Ketamine-Xylazine on Physiological Parameters in Rats,\u0026rdquo; Comparative Medicine, vol.\u003cbr\u003e 62, no. 3, pp. 209-214, 2012.\u003c/li\u003e\n\u003cli\u003eWyss, M. T., Jolivet, R., Buck, A., Magistretti, P. J., Weber, B, \u0026ldquo; In Vivo Evidence for Lactate as a Neuronal Energy Source,\u0026rdquo; Journal of Neuroscience, vol. 31, no. 20, pp. 7477-7485, 2011.\u003c/li\u003e\n\u003cli\u003eLucie A. Low, Lucy C. Bauer, Brenda A. Klaunberg, \u0026ldquo;Comparing the Effects of Isoflurane and Alpha Chloralose upon Mouse Physiology,\u0026rdquo; Plos One, pp. 1-14, 2016.\u003c/li\u003e\n\u003cli\u003eConstantin, M., \u0026rdquo;Alpha-Chloralose: Its Use in Animal Experimentation,\u0026rdquo; Journal of the American Association for Laboratory Animal Science, vol. 50, no.6, pp. 872-875, 2011.\u003c/li\u003e\n\u003cli\u003eFlecknell, P. A., Roughan, J. V., \u0026rdquo;Use of Alpha-Chloralose and Urethane in Physiological Research: Is it Justified\u0026rdquo; Laboratory Animals, vol. 38, no.4, pp. 401-410, 2004.\u003c/li\u003e\n\u003cli\u003eSilverman J, Muir WW, \u0026rdquo;A review of laboratory animal anesthesia with chloral hydrate and chloralose\u0026rdquo; Laboratory animal science, vol. 43, no.3, pp. 210-216, 1993.\u003c/li\u003e\n\u003cli\u003eHolzgrefe HH, Everitt JM, Wright EM, \u0026rdquo; Alpha-chloralose as a canine anesthetic,\u0026rdquo; Laboratory animal science, vol. 37, no.5, pp. 587-595, 1987.\u003c/li\u003e\n\u003cli\u003eSommers MG, van Egmond J, Booij LHDJ, \u0026rdquo; Heerschap A. Isoflurane anesthesia is a valuable alternative for a-chloralose anesthesia in the forepaw stimulation model in rats,\u0026rdquo; NMR in biomedicine, vol. 22, no.4, pp. 414-418, 2009.\u003c/li\u003e\n\u003cli\u003eShin-Lei Peng, Han Chiu, Chun-Yi Wu, Chiun-Wei Huang, Yi-Hsiu Chung, Cheng-Ting Shih, Wu-Chung Shen, \u0026rdquo; The effect of caffeine on cerebral metabolism during alpha-chloralose anesthesia differs from isoflurane anesthesia in the rat brain,\u0026rdquo; Psychopharmacology, vol. 236, pp. 1749-1757, 2019.\u003c/li\u003e\n\u003cli\u003eScheff, J. D., Goldstein, M. A, \u0026rdquo; Neurophysiological and Neurological Effects of Dexdomitor in Animal Models,\u0026rdquo; Veterinary Anesthesia and Analgesia, vol. 39, no.4, pp. 421-428, 2012.\u003c/li\u003e\n\u003cli\u003eDahan A, Teppema LJ, \u0026rdquo; Influence of anaesthesia and analgesia on the control of breathing,\u0026rdquo; British journal of anaesthesia, vol. 91, no.1, pp. 40-49, 2003.\u003c/li\u003e\n\u003cli\u003eWhite WJ, Field KJ, \u0026rdquo; Anaesthesia and surgery of laboratory animals,\u0026rdquo; Veterinary Clinics of North America: Small Animal Practice, vol. 17, pp. 989-1017, 1987.\u003c/li\u003e\n\u003cli\u003eCarl C.H. Petersen, \u0026rdquo;The Functional Organization of the Barrel Cortex,\u0026rdquo; Neuron, 2006.\u003c/li\u003e\n\u003cli\u003eKim, C. K., A. Adhikari, K. Deisseroth, \u0026rdquo;Integration of optogenetics with complementary methodologies in systems neuroscience,\u0026rdquo; Nature Reviews Neuroscience, vol. 18, pp. 222-235, 2017. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"barrel cortex signal, anesthetics experiment, alpha-chloralose, muscle noise, robustness","lastPublishedDoi":"10.21203/rs.3.rs-4778385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4778385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis paper presents an efficient anesthetic methodology designed to enhance the quality of brain barrel cortex signals obtained during rat whisker stimulation experiments. The proposed approach effectively eliminates muscle noise interference, thereby enabling the acquisition of clear and robust brain barrel cortex signals. Initially, alpha-chloralose (ac) combined with Isoflurane is used to induce anesthesia in rats. Subsequently, Dexdomitor is administered to suppress muscular movements, further refining the signal quality. Experimental outcomes demonstrate that our anesthetic method yields significantly stronger and cleaner brain barrel cortex signals, exhibiting enhanced robustness compared to existing methods that rely solely on Isoflurane or the Ketamine-Xylazine combination. Specifically, brain barrel cortex signals obtained under alpha-chloralose anesthesia are double those obtained with Isoflurane and quadruple those with Ketamine-Xylazine. The peak amplitude latency is shorter under alpha-chloralose anesthesia than under Isoflurane and Ketamine-Xylazine anesthesia. Notably, these improvements are achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates. Finally, alpha-chloralose effectively maintains anesthesia for up to 7 hours, surpassing the shorter durations achievable with continuous Isoflurane administration or the 30-minute window offered by Ketamine-Xylazine. This highlights the practical advantages of our proposed method.\u003c/p\u003e","manuscriptTitle":"Enhancing Anesthetic Techniques for Improving Rat Whisker Stimulation Responses in the Barrel Cortex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-23 04:02:27","doi":"10.21203/rs.3.rs-4778385/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b1c0410-2d83-4db1-848d-7a4d6b6de52a","owner":[],"postedDate":"August 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36404354,"name":"Biological sciences/Drug discovery"},{"id":36404355,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2024-09-12T21:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-23 04:02:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4778385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4778385","identity":"rs-4778385","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}