Name: recording brain electromagnetic activity during the administration of the gaseous anesthetic agents xenon and nitrous oxide in healthy volunteers
Uploaded: 2018-01-13T14:00:00
Description: Watch this Scientific Journal Video about Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers at JoVE.com

The authors would like to thank Mahla Cameron Bradley, Rachel Anne Batty and Johanna Stephens for valuable technical assistance with MEG data collection. Thanks are additionally extended to Dr. Steven Mcguigan for support as a second anesthesiologist. Paige Pappas provided invaluable anesthetic nurse oversight. Markus Stone graciously proffered his time and expertise in editing and filming the protocol. Dr. Suresh Muthukumaraswamy gave specific advice regarding data analysis and the interpretation of results. Finally, Jarrod Gott contributed many a stimulating discussion, helped in the execution of a number of pilot experiments and was central in the design of the foam head brace. 
This research was supported by a James S. McDonnell collaborative grant #220020419 "Reconstructing Consciousness" awarded to George Mashour, Michael Avidan, Max Kelz and David Liley.

The study entitled &#34;Effects of inhaled Xe and N2O on brain activity recorded using EEG and MEG&#34; was approved (approval number: 260/12) by the Alfred Hospital and Swinburne University of Technology Ethics Committee and met the requirements of the National Statement on Ethical Conduct in Human Research (2007).
1. Participant Selection and Pre-Study Requirements
<ol>
	<li>Conduct an interview to select healthy, right handed, adult males between the ages of 20 and 40 years old...

This paper has outlined a comprehensive protocol for the simultaneous recording of MEG and EEG during anesthetic gas delivery with N2O and Xe. Such a protocol will be valuable for studying the electromagnetic neural correlates of anesthetic-induced reductions in consciousness. The protocol is also expected to generalize to the delivery of other anesthetic gases such as sevoflurane or isoflurane. This will facilitate a greater understanding of the common, specific and distinct macroscopic mechanisms that underl...

There is good consensus between pre-clinical and clinical neuroscientific evidence suggesting that the phenomenon of human consciousness depends on the integrity of explicit neural circuits. The observation that such circuits are systematically influenced by the descent into unconsciousness has substantiated the need for neuroimaging techniques to be utilized during anesthesia and enable &#39;navigating&#39; the search for the neural correlates of consciousness. With the possible exception of sleep, anesthesia represents the only method by which one can, in a controlled, reversible and reproducible fashion, perturb, and thus dissect, the mechanisms that sub-serve consciousness, especially at the macroscopic scale of global brain dynamics. Clinically, general anesthesia can be defined as a state of hypnosis/unconsciousness, immobility and analgesia and remains one of the most abundantly used and safest medical interventions. Despite the clarity and efficiency in the end result, there remains great uncertainty regarding the mechanisms of action of the various types of agents giving rise to anesthetic induced unconsciousness1.
Anesthetics can be divided into intravenous agents notably propofol and the barbiturates, or volatile/gaseous agents such as sevoflurane, isoflurane, nitrous oxide (N2O) and xenon (Xe). The pharmacology of anesthesia has been well established with multiple cellular targets identified as linked to anesthetic action. Most agents studied to date act principally via the agonism of &#947;-Amino-Butyric-Acid-(GABA) receptor mediated activity. In contrast, the dissociative agents ketamine, Xe and N2O are believed to exert their effects by primarily targeting N-Methyl-D-Aspartate-(NMDA) glutamatergic receptors2,3. Other important pharmacological targets include potassium channels, acetylcholine receptors and the remnant glutamate receptors, AMPA and kainate, however the extent of their contribution to anesthetic action remains elusive (for a comprehensive review see 4).
The extent of variability in the mechanism of action and the observed physiological and neural effects of the various types of agents renders the derivation of general conclusions on their influence on conscious processing difficult. Loss of consciousness (LOC) induced by GABAergic agents is typically characterized by a global change in brain activity. This is evident in the emergence of high-amplitude, low-frequency delta (&#948;, 0.5-4 Hz) waves and the reduction in high frequency, gamma (&#947;, 35-45Hz) activity in the electroencephalogram (EEG), similar to slow wave sleep5,6 as well as the widespread reductions in cerebral blood flow and glucose metabolism5,6,7,8,9,10,11,12. Boveroux et al.13 added to such observations by demonstrating a significant decrease in resting state functional connectivity under propofol anesthesia using functional magnetic resonance imaging (fMRI). In contrast, dissociative anesthetics yield a less clear profile of effects on brain activity. In some cases, they are associated with increases in cerebral blood flow and glucose metabolism14,15,16,17,18,19,20,21 while studies by Rex and colleagues22 and Laitio and colleagues23,24 looking at the effects of Xe provided evidence of both increased and decreased brain activity. A similar irregularity can be seen in the effects on the EEG signals25,26,27,28. Johnson et al.29 demonstrated an increase in total power of the low frequency bands delta and theta as well as in the higher frequency band gamma in a high density EEG study of Xe anesthesia while opposing observations were made for N2O in the delta, theta and alpha frequency bands30,31 and for Xe in the higher frequencies32. Such variability in the effects of Xe on the electrical scalp activity can be observed in the alpha and beta frequency ranges also with both increases33 and reductions34 being reported.
In spite of the discrepancies mentioned above, the picture starts to become more consistent across agents when one attempts to look at alterations in functional connectivity between brain areas. Such measures however, have been predominantly restricted to modalities that necessarily make concessions with respect to either spatial or temporal resolution. While studies using the EEG appear to reveal clear, and to some extent consistent, changes in the topological structure of functional networks during anesthesia/sedation with propofol35, sevoflurane36 and N2O37, the widely spaced sensor level EEG data has insufficient spatial resolution to meaningfully define and delineate the vertices of the corresponding functional networks. Conversely, studies utilizing the superior spatial resolution of fMRI and positron emission tomography (PET), find similar topological alterations in large-scale functional connectivity to that of EEG13,38,39,40,41, however possess insufficient temporal resolution to characterize phase-amplitude coupling in the alpha (8-13 Hz) EEG band and other dynamical phenomena that are emerging as important signatures of anesthetic action12,42. Moreover, these measures do not directly evaluate electromagnetic neural activity43.
Therefore, in order to meaningfully advance the understanding of the macroscopic processes associated with the action of anesthetics, the limitations of the previously mentioned investigations need to be addressed; the restricted coverage of anesthetic agents and the insufficient spatio-temporal resolution of the non-invasive measurements. On this basis, the authors outline a method to simultaneously record magnetoencephalogram (MEG) and EEG activity in healthy volunteers that has been developed for the administration of the gaseous dissociative anesthetic agents, Xe and N2O.
The MEG is utilized as it is the only non-invasive neurophysiological technique other than the EEG that has a temporal resolution in the millisecond range. EEG has the problem of blurring of electrical fields by the skull, which acts as a low-pass filter on cortically generated activity, while MEG is much less sensitive to this issue and the issue of volume conduction44. It can be argued that MEG has higher spatial and source localization accuracy than EEG 45,46. EEG does not allow true reference-free recording37,47, however MEG does. MEG systems also typically record cortical activity in a much wider frequency range than EEG, including high gamma48(typically 70 - 90 Hz), which have been suggested to be involved in the hypnotic effects of anesthetic agents including Xe29 and N2O28. The MEG offers neurophysiological activity that compliments that conveyed by EEG, as EEG activity relates to extracellular electrical currents whereas MEG mainly reflects the magnetic fields generated by intracellular currents46,49. Furthermore, MEG is particularly sensitive to electrophysiological activity tangential to the cortex, while EEG mostly records extracellular activity radial to the cortex49. Thus combining MEG and EEG data has super-additive advantages50.
The gaseous dissociative agents Xe and N2O have been chosen for the following principle reasons: they are odorless (Xe) or essentially odorless (N2O) and thus can easily be utilized in the presence of control conditions when employed at sub-clinical concentrations. In addition, they are well suited for remote administration and monitoring in a laboratory environment due to their weak cardio-respiratory depressant effects61. Xenon and to a lesser extent N2O, retain a relatively low minimum-alveolar-concentration-(MAC)-awake at which 50% of patients become unresponsive to a verbal command with values of 32.6 &#177; 6.1%51 and 63.3 +- 7.1%52 respectively. Despite Xe and N2O both being NMDA receptor antagonists, they modulate the EEG differently - Xe appears to behave more like a typical GABAergic agent when monitored using the Bispectral Index33,53,54 (one of several approaches used to electroencephalographically monitor depth of anesthesia). In contrast, N2O produces a much less apparent electroencephalographic effect in that it is poorly, if at all, monitored using the Bispectral Index26. Because Xe has different reported electroencephalographic properties to the other dissociative agents, but possesses similar characteristics to the more commonly studied GABAergic agents, its electrophysiological study has the potential to reveal important features relating to the neural correlates of consciousness and the corresponding functional network changes. Agents that act at the NMDA receptor are likely to reveal more about the brain networks that subserve normal and altered consciousness, given the critical role that NMDA receptor mediated activity plays in learning and memory and its implicated role in a range of psychiatric disorders that include schizophrenia and depression80.
This paper focuses primarily on the demanding and complex data collection procedure associated with the delivery of gaseous anesthetic agents in a non-hospital environment while simultaneously recording MEG and EEG. Basic data analysis at the sensor level is outlined and example data are provided illustrating that high-fidelity recordings can be obtained with minimal head movement. The many potential methods for subsequent source imaging and/or functional connectivity analysis that would be typically performed using this kind of data are not described, as these methods are well described in the literature and demonstrate various options for analysis55,56.

<table border="0" cellpadding="0" cellspacing="0" width="681">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Neuromag TRIUX 306-channel MEG system</td>
			<td style="width:115px;">Elekta Oy, Stockholm, SWEDEN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polhemus Fastrak 3D system</td>
			<td style="width:115px;">Polhemus, VT, USA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEG compatible ER-1 insert headphones</td>
			<td style="width:115px;">Etymotic Research Inc., IL, USA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Low Density foam head cap, MEG compatible</td>
			<td style="width:115px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Custom made by research team</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Harness, MEG compatible</td>
			<td style="width:115px;">N/A</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">~3 m long, ~ 5 cm wide, cloth/jute strip to secure participant position on MEG chair</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambu Neuroline 720 Single Patient Surface Electrodes</td>
			<td style="width:115px;">Ambu, Copenhagen, Denmark</td>
			<td style="width:119px;">72015-K10</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">3.0T TIM Trio MRI system</td>
			<td style="width:115px;">Siemens AB, Erlangen, GERMANY</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Asalab amplifier system</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">this system is no longer manufactured and has been deprecated to 64 channel eego EEG amplifier</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">64-channel Waveguard EEG cap, MEG compatible</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">CA-138</td>
			<td style="width:231px;">size Medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magnetically shielded cordless battery box</td>
			<td style="width:115px;">ANT Neuro, Enschede, NETHERLANDS</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Magnetic shielding not provided by manufacturer &#8211; Modified by research team</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">OneStep ClearGel Electrode gel</td>
			<td style="width:115px;">H+H Medizinprodukte GbR, Munster, GERMANY</td>
			<td style="width:119px;">154547</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Akzent Xe Color Anesthesia Machine</td>
			<td style="width:115px;">Stephan GmbH, Gackenbach, GERMANY</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Omron M6-Comfort Blood Pressure Monitor</td>
			<td style="width:115px;">Omron Healthcare, Kyoto, JAPAN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Xenon gas (99.999% purity)</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">we estimate that we use approx 40 L (SATP) per participant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Nitrous Oxide</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Oxygen</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Medical Air</td>
			<td style="width:115px;">Coregas, Thomastown, VIC, AUSTRALIA</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">x2 G size cylinders</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter Respiratory &#38; HMES with Capno Port Hypnobag</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">352/5805</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yankauer High Adult</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">8888-502005</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Quadralite EcoMask anaesthetic masks</td>
			<td style="width:115px;">Intersurgical Australia Pty Ltd</td>
			<td style="width:119px;">7093000/7094000</td>
			<td style="width:231px;">size 3 and size 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Suction Canister Disp 1200 mL Medival Guardian</td>
			<td style="width:115px;">Cardinal Health, OH, USA</td>
			<td style="width:119px;">65651-212</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Catheter Mount Ext 4-13 cm with&#160; 90A elbow</td>
			<td style="width:115px;">Medtronic, MN, USA</td>
			<td style="width:119px;">330/5667</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Catheter IV Optiva 24g x 19 mm Yellow St Su</td>
			<td style="width:115px;">Smiths Medical, MN, USA</td>
			<td style="width:119px;">5063-INT</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dexamethasone Mylan Injection Vials (4 mg/1 mL)</td>
			<td style="width:115px;">Alphapharm Pty Ltd, Sydney, AUSTRALIA</td>
			<td style="width:119px;">400528517</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ondasetron (4 mg/2 mL)</td>
			<td style="width:115px;">Alphapharm Pty Ltd, Sydney, AUSTRALIA</td>
			<td style="width:119px;">400008857</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="546">
			<td height="546" style="height:546px;width:216px;">Medical resuscitation cart</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;">The medical resuscitation cart is configured according to the suggested minimal requirements for Adult resuscitation recommended in the document &#34;Standards for Resuscitation: Clinical Practice and Education; June 2014) by the Australian and New Zealand Resuscitation councils and specifically endorsed by multiple professional health care organizations including the Australian and New Zealand College of Anaesthetists.&#160; It includes all the necessary airway and circulatory equipment, as well as the associated pharmacuetical agents to enable full cardio-respiratory resuscitation and support in a non-clinical environment.&#160; Full details can be found at https://resus.org.au/standards-for-resuscitation-clinical-practice-and-education/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Maxfilter Version 2.2</td>
			<td style="width:115px;">Elekta Oy, Stockholm, SWEDEN</td>
			<td style="width:119px;">N/A</td>
			<td style="width:231px;">Data analysis software provided with Elekta&#39;s Neuromag TRIUX MEG system</td>
		</tr>
	</tbody>
</table>

recording brain electromagnetic activity during the administration of the gaseous anesthetic agents xenon and nitrous oxide in healthy volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

This section utilizes data obtained from one subject in order to demonstrate the typical features of the simultaneous recordings and the potential of such information to contribute a better understanding of anesthetic induced altered states of consciousness. To simplify the exposition, results are shown for i) recordings of the post-anti-emetic administration baseline (baseline 3), ii) 0.75 equi-MAC-awake peak gas concentrations (level 3) of N2O (47%) and Xe (24%), and iii) Xe ...

Watch this Scientific Journal Video about Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers at JoVE.com

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

The purpose of this experiment is to characterize changes in brain electromagnetic activity in response to the administration of the two gaseous dissociative agents xenon and nitrous oxide to healthy participants in a two-way repeated measures crossover design. Due to the complexity of the experiment, it is important that the majority of equipment setup occurs before participant arrival. The most important of these is the configuration of the EEG system in the magnetically shielded room or MSR.The EEG amplifier is placed in the MSR as far as practical away from the participant, and needs to be configured to run on DC power using either a built-in battery or an external shielded battery box. Fiber optic coupling from the amplifier to the data-logging computer should be checked to be operational. Finally, a video camera in the MSR should be configured such that its field of view clearly captures the participant's face and upper thorax such that the physician anesthesiologist can clearly monitor the respiratory and airway status of the participant.In order that consistent and comparable data is acquired, participants are first interviewed to confirm their eligibility for the study. Participants should be healthy males of 20 to 40 years of age with no psychiatric or neurological conditions, no recent history of drug or alcohol use, and have no respiratory or cardiac conditions. Participants should arrive clean-shaven on the day of the experiment, and having fasted for at least six hours.To ensure high quality EEG recordings, it is recommended that the subject's hair has been recently washed without conditioner, and that their scalp has been firmly brushed to remove any dried or dead skin. An MEG-compatible high-density EEG cap is applied with the appropriate use of conductive gel. Head position indicator coils are then firmly attached at predefined locations on the EEG cap.All auxiliary biopotential electrodes are then applied at the designated locations on the subject's face and body. For subsequent analysis, it is important that all electrodes and HPI coil positions are accurately registered along with digitization of the outlines of the orbits, nasal ridge and cheeks for subsequent coregistration with structural MRI. Two electrodes are placed submentally to record resting muscle tone.Electrooculogram or EOG recordings are made by placing two further electrodes above and below the eye. And finally, a three-lead ECG derivation is configured by electrodes on the left and right wrist flexor surfaces. EEG electrode impedances should then be verified as being below five kiloohms in order to yield high-fidelity recordings.The duration of the experiment as well as the sedative effects of the administered gases makes minimization of head movement vital. A memory foam head brace is applied in order to reduce lateral head movement, and a jute harness is tied around the body to minimize any vertical movement. Eyes-closed resting state and baseline auditory continuous performance task recordings are now performed.It is of vital importance that the administration of anesthetic gases is supervised by a physician anesthesiologist at all times. As well as patient monitoring, the role of the physician anesthesiologist includes ensuring the adequate configuration of the anesthetic gas delivery unit, specialized in this case to deliver and monitor xenon. The physician anesthesiologist will test the anesthesia machine, check that the mask fits comfortably and does not leak, adjust gas flows, insert a cannula, administer antiemetics, as well as commencing denitrogenation with 100%oxygen with the participant seated in the MSR before the final post-antiemetic baseline data collection and the start of the gas administration protocol.At this stage the participant will be comfortably seated in the MSR having been denitrogenated and having been administered dexamethasone and ondansetron to minimize the risk of any adverse nausea or vomiting due to gas administration. Before the MSR door is closed, the anesthetic nurse or other suitably trained medical professional is seated next to the participant. A final check is made that the blood pressure cuff is connected, the button response boxes are comfortably positioned, and the pulse oximeter on the toe or finger is securely fastened.The protocol then proceeds to deliver four Equi-MAC awake progressively increasing stepwise levels of xenon gas, or three stepwise nitrous oxide levels over a period of 15 minutes each in order to assure blood-brain-gas equilibration. Each level should contain a five-minute steady state when the desired gas concentration is reached and should be followed by a 10-minute washout period. During all levels, oxygen, carbon dioxide, and xenon or nitrous oxide levels are continuously monitored and electronically logged.At the end of each level, participants are briefly assessed before progressing to higher levels. Because at the highest xenon level, in which loss of responsiveness is achieved, airway obstruction can readily and unexpectedly occur, and subjects need to be closely monitored and continuously assessed by qualified personnel, both inside and outside the MSR for all necessary action to be taken immediately. At the conclusion of the experiment, the physician anesthesiologist will enter the MSR and debrief with the patient.The participant is then brought outside the MSR where the apparatus is removed and they are administered standard postoperative anesthetic care. Before the participant leaves, ensure they are safe to travel home with a relative or friend. In the next 24 hours, the participant will be required to fill in the questionnaires regarding the procedure and any after effects.This video has been primarily prepared to illustrate the methods involved in delivering gaseous anesthesia while recording simultaneous EEG and MEG data, therefore, only very basic data analysis and representative results are presented to give the viewer a clear idea of what the recorded data should look like. The basic results illustrate examples of head movement, gas concentration timecourse and auditory task accuracy;filtered artifact-free EEG and MEG data;EEG and MEG spectra and power topography;and auditory evoked responses for both baseline and during gas administration. To create the basic results, the following pipeline is employed using a combination of data analysis software platforms and toolboxes.The raw MEG and EEG data is stored as fif and cnt file formats respectively. Head movements are computed using the MaxFilter software and a script written by the authors, both used to convert head movements from the cortonian space into Cartesian coordinates. Auditory task accuracy and reaction time are then computed and visualized, time-aligned with the head movement and gas concentration time series, in order to assess data quality before further processing the EEG and MEG data.Time alignment of the data is based on recorded trigger times. Typically, head movement will be minimal during baseline conditions, while more movement tends to occur at the higher gas levels associated with more dissociated or sedated states. This can be seen here for example in the 42%xenon recording where the top figure shows head movement, and the bottom, gas concentration and auditory task accuracy.Note that loss of responsiveness occurs between 7.5 and 12.5 minutes. Absolute head movement of less than five millimeters is considered acceptable for source imaging purposes. Data segments with movement larger than five millimeters are ignored.Preprocessing of the MEG and EEG data initially involves selection of bad channels. This is followed by the use of the tSSS algorithm to remove artifacts from the MEG recording due to external sources. The rest of the processing pipeline involves the use of data analysis software platforms and associated toolboxes for analysis.Band-pass and notch filtering is applied to both the MEG and EEG data to focus analysis on the frequency band of interest. Time periods in the data containing artifacts are rejected based on visual inspection, in addition to removing periods involving excessive head movement changes. Filtered artifact-free MEG and EEG data can then be visualized and related to anesthetic-induced changes.Amplitude spectra for each sensor and spectral band power topographies across all sensors are then computed for both MEG and EEG data. Visualization of the amplitude spectra at different sensor locations facilitates observation of the effects of different anesthetic levels on signal spectra. Visualization of sensor level power topography in different frequency bands, in this case the alpha band, enables the observation of the effects of different anesthetic levels on the spacial pattern of activity.The filtered artifact-free MEG and EEG data are then epoched using triggers linked to the time of auditory tone delivery. Average auditory evoked responses are then constructed through time on sample averaging. Visualization of sensor level auditory evoked responses allows for the characterization of the effects of anesthetics on the brain's responsiveness.In particular, awakening of the initial evoked response and the disappearance of the later evoked response peaks results from high gas concentrations. There are many other potential interesting analysis steps that can be performed subsequent to this basic analysis. Given that there are many options presented in the literature, we do not cover them in detail here.Such options include MEG and EEG source imaging using beamforming or minimum-norm estimation to gain a better understanding of the relationship between brain activity and brain anatomy. Sensor or source-level functional connectivity analysis can unravel changes in brain networks linked to anesthetic action. There is also the option to either focus analysis on the sensor or source-level auditory evoked responses, or to look at continuous data independent of the auditory stimulus in order to identify any anesthetic-induced changes in global brain dynamics.The macroscale mechanisms of anesthetic-induced reductions in consciousness are not well understood. Evidence from literature has strongly implicated a breakdown in functional connectivity underlying anesthetic-induced reductions in consciousness. Although anesthetics have a diverse range of molecular targets of action and different macroscopic effects on the EEG and MEG, it is still not resolved if there exists a universal or common macroscale mechanism across all anesthetics.Here we have outlined a protocol for performing gaseous anesthesia using the putative NMDA receptor antagonists xenon and nitrous oxide while simultaneously recording MEG and EEG data. This method will facilitate the study of different gaseous and volatile agents on simultaneously recorded MEG and EEG data, and thus the search for universal mechanisms of anesthetic-induced reductions in consciousness.

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Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...