We would like to thank Andrew Cripps and the BioResource Unit at the University of Reading. This research was funded by the BBSRC (Grant number: BB/K010123/1). Data associated with this work is freely available from Y.Z. (ying.zheng@reading.ac.uk).

All experiments were carried out in accordance with the British Home Office regulations (Animals (Scientific Procedures) Act, 1986) and approved by the Research Ethics Committee at the University of Reading, UK.
1. Animal Preparation
NOTE: Female Lister Hooded rats were used for all experiments. This is a non-survival procedure.&#160;
<ol>
	<li>Record the rat&#39;s weight on a laboratory scale.</li>
	<li>Anesthetize the rat in a chamber with 5% isoflurane and an oxygen flow rate of 1 L/min.</li>
	<li>Place the rat onto a stereotaxic holder with a paper towel underneath its body and with its teeth resting via the bite bar.. The paper towel will make the insertion of a heat pad easier (see step 2.3) and catch any excrement from the rat during the experiment.</li>
	<li>Administer isoflurane continuously via a hard-plastic nose cone mounted onto the nose clamp for rat adaptor at a concentration of 3% with an oxygen flow rate of 0.5 L/min. Connect the cone to a small animal isoflurane anesthetic system.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Insert a thermostatic heating pad underneath the paper towel upon which the rat is resting, secure the rat&#8217;s head with two ear bars, and monitor the body temperature using a rectal thermometer.</li>
	<li>Shave the top of the rat&#39;s head.</li>
	<li>Apply ophthalmic ointment to the eyes to prevent corneal drying.</li>
	<li>Before exposing the cranium, apply lidocaine drops to the scalp and massage it gently into the skin.</li>
	<li>Make a midline incision of approximately 2-3 cm on the scalp using a scalpel to expose the surface of the skull.</li>
	<li>Carefully separate the temporalis muscle contra-lateral to the whisker pad to be stimulated from the skull using a Jacquette Scaler and a pair of serrated and curved dissecting forceps. Clean the skull with cotton swabs whenever necessary.</li>
	<li>Using a braided silk, non-absorbable suture, tie the separated muscle to the scalp with a tight knot and then tie the suture securely to the stereotaxic frame18.</li>
	<li>Use stereotaxic coordinates to locate the barrel cortex, 2.5 mm caudal to bregma and 6 mm lateral to midline19. Draw a dot at the location of the somatosensory cortex using a pencil or a marker.</li>
	<li>Drill a burr hole at the marked location using a dental drill. To prevent the skull from overheating during drilling, apply sterile saline (sodium chloride 0.9%) to the work area every 10-15 s. The drilling process involves the following 3 steps:
	<ol>
		<li>Drill a hole of diameter &#60; 2 mm into the skull using a drill bit #4 (0.055 in diameter). Take care not to drill into the dura.</li>
		<li>Thin the bottom of the hole to translucency using a drill bit #1/4 (0.019 in diameter).</li>
		<li>Use a 27 G needle to pierce the dura to allow the insertion of a microelectrode.</li>
	</ol>
	</li>
	<li>Transfer the rat, secured on a stereotaxic frame, to a Faraday cage mounted on top of a vibration isolation workstation.</li>
	<li>Attach an oximeter sensor clamp connected to an oximeter control unit to the rat&#39;s hind paw to monitor continuously the following physiological parameters: heart rate, breath rate, arterial oxygen saturation, pulse distention, and breath&#160;distention.&#160;These parameters were displayed continuously on a PC monitor, reflecting the physiological condition and anaesthetic depth of the rat.</li>
	<li>Replace the hard-plastic nose cone for isoflurane administration and the nose clamp for rat adaptor with a microflex breather fitted with a transparent soft nose cone which is modified (Figure 1A) to allow easy whisker stimulation to one side of the whisker pad without compromising the isoflurane administration.</li>
	<li>Insert two stainless steel stimulating electrodes to the whisker pad exposed by the cut-out on the nose cone.</li>
	<li>Connect the stimulating electrodes to an isolated current stimulator.</li>
	<li>Lift the skin of the midline of the neck with forceps and make a 1~2 cm incision with scissors ready for the placement of reference electrodes. Take care not to cut the muscle tissue.</li>
</ol>
3. Co-localized EEG/LFP Setup
<ol>
	<li>Clean and dry the skull surrounding the burr hole using a cotton swab.</li>
	<li>Carefully place the conductive EEG paste on one flat side of an EEG spider electrode. Leave a small hole clear of the EEG paste on the spider electrode to allow a multi-laminar microelectrode to pass through the hole without contacting the paste and the spider electrode. This prevents electrical contact between the EEG electrode and the microelectrode.</li>
	<li>Align the spider electrode to the burr hole in the skull, with the EEG paste facing the skull.</li>
	<li>Carefully press the spider electrode onto the skull, making firm contact with the skull via the EEG paste. Remove any paste obscuring the burr hole using a needle on a syringe.</li>
	<li>Remove excessive EEG paste beyond the periphery of the spider electrode so that the contact between the spider electrode and the skull is spatially constrained to the size of the electrode (Figure 1B).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56447/56447fig1.jpg" /> 
Figure 1: General setup for concurrent EEG/LFP recording. (A) The setup consists of a modified nose cone for ease of whisker pad stimulation under isoflurane anesthesia, two stimulating electrodes inserted into the whisker pad, a spider electrode positioned on the skull above the barrel cortex contra-lateral to the stimulating electrodes, a multi-channel microelectrode inserted into the barrel cortex through the spider electrode, and reference electrodes placed inside an incision at the back of the rat&#39;s neck. (B) A view through the microscope of the spider electrode securely positioned onto the skull by EEG paste. The microelectrode is inserted into a burr hole drilled into the skull under the spider electrode. The scalp is held back by surgical thread (suture) tied to the stereotaxic frame. <a href="//cloudfront.jove.com/files/ftp_upload/56447/56447fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Smear EEG paste onto the reference electrode for the EEG and place it securely inside the incision at the back of the rat&#8217;s neck.</li>
	<li>Connect the EEG electrodes to the preamplifier via a passive signal splitter for low impedance signals (Figure 2). Make sure the impedance of the spider electrode is below 5 k&#8486;. If it is not, check that the EEG paste is in good contact with the skull and the electrode is firmly pressed to the EEG paste. Add more EEG paste if necessary.</li>
	<li>Mount a micromanipulator arm on the stereotaxic frame. Connect a linear 16-channel microelectrode (100 &#181;m spacing, area of each site 177 &#181;m2) to a 16-channel acute headstage clipped securely onto the micromanipulator arm.</li>
	<li>Smear EEG paste onto the reference electrodes for the EEG and microelectrode, then securely place them inside the incision (Figure 1A).</li>
	<li>Adjust the angle of the micromanipulator arm so that the microelectrode is perpendicular to the cortical surface. This angle is normally between 25-35 &#176;.</li>
	<li>Lower the microelectrode under a microscope by turning the micromanipulator knobs so that the tip of the microelectrode is aimed at the tiny opening at the bottom of the burr hole until the uppermost electrode just penetrates the cortical surface. Care must be taken to avoid forcing the microelectrode onto the surface of the dura as this would break the electrode.</li>
	<li>Couple the 16-channel microelectrode to a preamplifier connected to a data acquisition unit via a fiber optic cable (Figure 2).</li>
	<li>Turn on the preamplifier, the data acquisition unit, and the computer connected to the unit. Turn on the stimulator box.</li>
	<li>Insert the microelectrode normally to the cortical surface by slowly turning the z-axis knob of the micromanipulator to a depth of 1,500 &#181;m20.</li>
	<li>Micro-adjust the depth by applying a train of stimulus to the whisker pad and observing the 16-channel evoked LFP on a PC monitor using the software of the data acquisition unit installed on the PC. Carefully turn the z-axis knob on the micromanipulator until the highest amplitude of the evoked LFP occurs around channel 7 (as this coincides with layer IV in the cortex). 
	NOTE: Ipsi-lateral EEG electrode setup: For some experiments, a second spider electrode was placed on the ipsi-lateral side of the intact skull above the barrel cortex. This setup allowed bilateral EEG recording during the resting state to investigate the effect of the burr hole on the EEG signal. 
	NOTE: The surgical procedure to set up the EEG electrode is identical to that described above, except that during step 2.6, the temporalis muscle on each side of the head was carefully separated from the skull, sutured back and tied securely to the corresponding side of the stereotaxic frame. 
	NOTE: The concurrent EEG/LFP setup is also identical to that described above, with an additional step that a second spider electrode is loaded with the EEG paste, then pressed firmly to the skull above the ipsi-lateral barrel cortex.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56447/56447fig2.jpg" /> 
Figure 2. A signal flow diagram. The rat is placed inside a Faraday cage. The stimulating electrodes receive commands from the stimulator box controlled by the Data Acquisition Unit through its software installed on a PC. The neural signal recorded by the microelectrode is transmitted to a pre-amplifier inside the Faraday cage. The neural signal recorded by the EEG probe is transmitted to the pre-amplifier through a signal splitter. The pre-amplifier is connected to the Data Acquisition Unit outside the Faraday cage via a fiber optic cable. The neural data are then stored on a local drive on the PC, while they can also be displayed on a PC monitor. A mobile small animal isoflurane system administers isoflurane from outside the Faraday cage. <a href="//cloudfront.jove.com/files/ftp_upload/56447/56447fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Electrical Stimulation and Neural Recordings
NOTE: The sampling frequency for all neural data is 24.41 kHz with 16-bit resolution. A trial consists of a single electrical stimulation at the start of the trial. Each trial lasts 10 s, which is also the inter-stimulus interval (ISI). Each stimulus is a square current pulse of 1.2 mA lasting 0.3 ms. For bilateral experiments to study the effect of the burr hole, continuous resting state of 250 s is also recorded.
<ol>
	<li>Open the recording software on the computer in use.</li>
	<li>Load the correct circuit for the experiment by selecting &#39;Load Project&#8230;&#39; from the dropdown menu of &#39;OpenProject&#39;. A new window (&#39;WorkBench&#39;) will appear (Figure 3).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56447/56447fig3.jpg" /> 
Figure 3. A display of the software GUI for the Data Acquisition Unit. It allows the appropriate circuit to be uploaded, stimulation parameters to be set, and data to be recorded and visualized. <a href="//cloudfront.jove.com/files/ftp_upload/56447/56447fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Create a new directory (called a &#39;Tank&#39; by the software) to store neural recordings.
	<ol>
		<li>Click on &#39;File&#39; from the top of the window, and select &#39;Data Task Management&#39;. A new window (&#39;Tank Management&#39;) will appear.</li>
		<li>In the &#39;Tank Management&#39; window, press the right button of the mouse to display a menu. Select &#39;Create New Tank&#39;. Another new window (&#39;Create Data Tank&#39;) will appear.</li>
		<li>In the &#39;Create Data Tank&#39; window, select the path where you plan to create a new data directory, and enter the name of the new directory. Then press &#39;OK&#39;. This window will disappear.</li>
		<li>The new directory will appear in the &#39;Tank Management&#39; window but in grey. Register this directory by right-clicking on it, and select &#39;Register Tank&#39; from the dropdown menu. A red star and a green arrow will appear to the left of the new directory&#39;s name which is now in black (Figure 4).</li>
		<li>Unregister any previous directories not in use by right-clicking in the &#39;Tank Management&#39; window and selecting &#39;Refresh Tank List&#39; in the dropdown menu.</li>
		<li>Click on &#39;OK&#39; to exit the &#39;Tank Management&#39; window.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56447/56447fig4.jpg" /> 
Figure 4: A display of the software GUI showing a registered data directory. <a href="//cloudfront.jove.com/files/ftp_upload/56447/56447fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Register the new directory in &#39;Scope&#39; to display neural signals during experiment.
	<ol>
		<li>Click on the &#39;Scope&#39; icon in the &#39;OpenProject&#39; window. A new window (&#39;Scope&#39;) will appear.</li>
		<li>Right click the mouse in the &#39;Scope&#39; window and select &#39;Refresh Tank List&#39; in the dropdown menu. The new directory&#39;s name will appear in grey.</li>
		<li>Click on the new directory. A red star and a green arrow will appear to the left of the new directory&#39;s name which is now in black.</li>
	</ol>
	</li>
	<li>Set up the experimental parameters for data acquisition in the &#39;WorkBench&#39; window by clicking on &#39;Setup&#39; from the top of the window. A new window will appear. Select &#39;Sweep loop&#39;, set the length of the trial and the number of trials to be recorded.</li>
	<li>Check that the Stimulator Box is turned on.</li>
	<li>Press the &#39;Record&#39; button in the &#39;WorkBench&#39; window. A new window will appear. Enter the name of the data file you want to save for the experimental run but do not hit the return button at this stage, as the EEG recording parameters need to be set up.</li>
	<li>Set up the EEG recording parameters using the Graphic User Interface (GUI) on the pre-amplifier. Touch the screen (anywhere) of the preamplifier to wake up the screen. Select &#39;Unlock&#39; to unlock the display (Figure 5).
	<ol>
		<li>Press the left icon in the &#39;2: EEG&#39; panel. A new display will appear.</li>
		<li>Press &#39;Coupling&#39; and select &#39;AC&#39;.</li>
		<li>Press &#39;Ref Mode&#39; and select &#39;Local&#39;.</li>
		<li>Press &#39;Samp Rate&#39; and select &#39;25KHz&#39;.</li>
		<li>Press &#39;OK&#39; to return to the original display.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56447/56447fig5.jpg" /> 
Figure 5: The GUI on the pre-amplifier. It allows EEG recording parameters (e.g., sampling frequency and referencing preference) to be set. <a href="//cloudfront.jove.com/files/ftp_upload/56447/56447fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="9">
	<li>Check the impedance of the EEG probe(s) by pressing the middle icon in the &#39;2: EEG&#39; panel. If too high, add more EEG paste to the probe. Press &#39;OK&#39; to return to the original display.</li>
	<li>Wait 20 s to avoid recording the initial fluctuation of the EEG recordings.</li>
	<li>Go back to the PC monitor (after the 20 s wait) and press the &#39;Return&#39; key on the keyboard. The EEG and the LFP signals will be recorded.</li>
</ol>
5. Data Analysis
<ol>
	<li>Pre-process the evoked LFP and EEG signals on a trial-by-trial basis using the following steps.
	<ol>
		<li>Shift back the neural data in time by 20 samples (equivalent to 0.82 ms). This is the delay produced by the circuit used to collect neural data in TDT itself. By shifting the data, the time zero point is aligned to the onset of the stimulus.</li>
		<li>Remove the stimulus artifact by replacing the neural data from 0 to 1 ms with a straight line connecting the data point at 0 ms with the data point at 1 ms.</li>
		<li>Zero-mean each trial by subtracting the mean value of the neural signal 200 ms prior to stimulus onset.</li>
		<li>Low-pass filter the data below 800 Hz using a 4th order Butterworth IIR type filter in both directions to avoid introducing any temporal shift in the data.</li>
		<li>Align the multi-laminar data across animals. For each animal&#39;s LFP data, apply the inverse Current Source Density (spline iCSD, source radius R = 0.5 mm) analysis21 with a Gaussian filter (&#955; = 50 &#181;m) to locate the layer IV sink1, which is given by the largest negative peak occurring at a cortical depth below the pial surface within the first 15 ms of stimulus onset. The CSD, and the corresponding LFP, data are then aligned according to their sink locations across animals. The common sink is located in layer IV, ~ 600 &#181;m below the pial surface.</li>
		<li>After alignment, use channels 2, 7, and 12 of the realigned LFP as representatives of neural responses of the supragranular, granular, and infragranular layers, respectively in the barrel cortex.</li>
	</ol>
	</li>
	<li>Calculate the mean evoked LFP and EEG by averaging the pre-processed data over 100 trials.</li>
	<li>To investigate the effect of the burr hole on the EEG, down-sample the EEG signals to 1,000 Hz, and compute the power spectral density (PSD) for the contra-lateral (with a hole in the skull) and ipsi-lateral (intact skull) spider electrode recordings over a 250 s period of resting state. PSD is computed from 0.1-100 Hz in Matlab using the function &#39;pmtm&#39; which is based on the multitaper method22.</li>
	<li>Divide the frequency range into the following well-known frequency bands: Delta (&#948;): 0.1-4 Hz, Theta (): 4-8 Hz, Alpha (&#945;): 8-13 Hz, Beta (&#946;): 13-31 Hz, Gamma (&#947;): 31-100 Hz. Calculate the average PSD within each band.</li>
	<li>Within each frequency band, calculate the normalized difference in PSD, Perr, between the contra- and the ipsi-lateral EEG using the equation: 
	<img alt="Equation" src="/files/ftp_upload/56447/56447eq1.jpg" /> 
	where Pc and Pi are the average PSD of the contra- and ipsi-lateral EEG, respectively in the frequency band of interest.</li>
	<li>Within each frequency band, perform a one-sample t-test to test the hypothesis that there is no significant difference (at 0.05 significance level) between the PSD of the EEG signal recorded from the two hemispheres.</li>
</ol>

<ol>
	<li>Mitzdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+source-density+method+and+application+in+cat+cerebral+cortex:+investigation+of+evoked+potentials+and+EEG+phenomena.">Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena.</a> Physiol Rev. 65 (1), 37-100 (1985).</li><li>Logothetis, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Underpinnings+of+the+BOLD+Functional+Magnetic+Resonance+Imaging+Signal.">The Underpinnings of the BOLD Functional Magnetic Resonance Imaging Signal.</a> J Neurosci. 23 (10), 3963-3971 (2003).</li><li>Buzs&#225;ki, G., Anastassiou, C. A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+extracellular+fields+and+currents+—+EEG,+ECoG,+LFP+and+spikes.">The origin of extracellular fields and currents — EEG, ECoG, LFP and spikes.</a> Nat Rev Neurosci. 13 (6), 407-420 (2012).</li><li>Einevoll, G. T., Kayser, C., Logothetis, N. K., Panzeri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+and+analysis+of+local+field+potentials+for+studying+the+function+of+cortical+circuits.">Modelling and analysis of local field potentials for studying the function of cortical circuits.</a> Nat Rev Neurosci. 14 (11), 770-785 (2013).</li><li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balanced+excitation+and+inhibition:+Model+based+analysis+of+local+field+potentials.">Balanced excitation and inhibition: Model based analysis of local field potentials.</a> Neuroimage. 63 (1), 81-94 (2012).</li><li>Bruyns-Haylett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurogenesis+of+P1+and+N1:+A+concurrent+EEG/LFP+study.">The neurogenesis of P1 and N1: A concurrent EEG/LFP study.</a> Neuroimage. 146, 575-588 (2017).</li><li>Nunez, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electric Fields of the Brain: The Neurophysics of EEG. , (1981).</li><li>Jackson, A. F., Bolger, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurophysiological+bases+of+EEG+and+EEG+measurement:+A+review+for+the+rest+of+us.">The neurophysiological bases of EEG and EEG measurement: A review for the rest of us.</a> Psychophysiology. 51 (11), 1061-1071 (2014).</li><li>Cohen, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+Does+EEG+Come+From+and+What+Does+It+Mean?.">Where Does EEG Come From and What Does It Mean?.</a> Trends Neurosci. 40 (4), 208-218 (2017).</li><li>Bojak, I., Oostendorp, T., Reid, A., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+Mean+Field+Models+of+Neural+Activity+to+EEG+and+fMRI+Data.">Connecting Mean Field Models of Neural Activity to EEG and fMRI Data.</a> Brain Topogr. 23 (2), 139-149 (2010).</li><li>Coombes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+neural+dynamics:+Simple+and+complex.">Large-scale neural dynamics: Simple and complex.</a> Neuroimage. 52 (3), 731-739 (2010).</li><li>Deco, G., Jirsa, V. K., Robinson, P. A., Breakspear, M., Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+brain:+from+spiking+neurons+to+neural-masses+and+cortical+fields.">The dynamic brain: from spiking neurons to neural-masses and cortical fields.</a> PLoS Comput. Biol. 4 (8), e1000092 (2008).</li><li>Pinotsis, D. A., Friston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+fields,+spectral+responses+and+lateral+connections.">Neural fields, spectral responses and lateral connections.</a> Neuroimage. 55 (1), 39-48 (2011).</li><li>Riera, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+in+the+dipolar+model+for+the+neocortical+EEG+sources.">Pitfalls in the dipolar model for the neocortical EEG sources.</a> J Neurophysiol. 108 (4), 956-975 (2012).</li><li>Valdes, P. A., Jimenez, J. C., Riera, J., Biscay, R., Ozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+EEG+analysis+based+on+a+neural+mass+model.">Nonlinear EEG analysis based on a neural mass model.</a> Biol Cybern. 81 (5), 415-424 (1999).</li><li>Musall, S., von Pf&#246;stl, V., Rauch, A., Logothetis, N. K., Whittingstall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Neural+Synchrony+on+Surface+EEG.">Effects of Neural Synchrony on Surface EEG.</a> Cereb Cortex. 24 (4), 1045-1053 (2014).</li><li>Snyder, A. C., Morais, M. J., Willis, C. M., Smith, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+network+influences+on+local+functional+connectivity.">Global network influences on local functional connectivity.</a> Nat Neurosci. 18 (5), 736-743 (2015).</li><li>Mayhew, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+analysis+of+neural+activity+in+brain:+Increased+oxygen+consumption+following+activation+of+barrel+cortex.">Spectroscopic analysis of neural activity in brain: Increased oxygen consumption following activation of barrel cortex.</a> Neuroimage. 12 (6), 664-675 (2000).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2005).</li><li>Martindale, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hemodynamic+impulse+response+to+a+single+neural+event.">The hemodynamic impulse response to a single neural event.</a> J Cereb Blood Flow Metab. 23 (5), 546-555 (2003).</li><li>Pettersen, K. H., Devor, A., Ulbert, I., Dale, A. M., Einevoll, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current-source+density+estimation+based+on+inversion+of+electrostatic+forward+solution:+Effects+of+finite+extent+of+neuronal+activity+and+conductivity+discontinuities.">Current-source density estimation based on inversion of electrostatic forward solution: Effects of finite extent of neuronal activity and conductivity discontinuities.</a> J Neurosci Methods. 154 (1-2), 116-133 (2006).</li><li>Thomson, D. J., et al., Fitzgerald, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multitaper+analysis+of+nonstationary+and+nonlinear+time+series+data.">Multitaper analysis of nonstationary and nonlinear time series data.</a> Nonlinear and Nonstationary Signal Processing. , 317-394 (2000).</li><li>Bae, J., Deshmukh, A., Song, Y., Riera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Source+Imaging+in+Preclinical+Rat+Models+of+Focal+Epilepsy+using+High-Resolution+EEG+Recordings.">Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings.</a> Journal of Visualized Experiments : JoVE. (100), e52700 (2015).</li><li>Baumann, S. B., Wozny, D. R., Kelly, S. K., Meno, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+conductivity+of+human+cerebrospinal+fluid+at+body+temperature.">The electrical conductivity of human cerebrospinal fluid at body temperature.</a> IEEE Trans Biomed Eng. 44 (3), 220-223 (1997).</li><li>Wendel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Age+and+Skull+Conductivity+on+Surface+and+Subdermal+Bipolar+EEG+Leads.">The Influence of Age and Skull Conductivity on Surface and Subdermal Bipolar EEG Leads.</a> Computational Intelligence and Neuroscience. 2010, (2010).</li><li>Flemming, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+distortion+of+EEG+signals+caused+by+a+hole+in+the+skull+mimicking+the+fontanel+in+the+skull+of+human+neonates.">Evaluation of the distortion of EEG signals caused by a hole in the skull mimicking the fontanel in the skull of human neonates.</a> Clin Neurophysiol. 116 (5), 1141-1152 (2005).</li></ol>

The authors have nothing to disclose.

We have described an experimental procedure for concurrent recording of co-localized EEG and LFP signals of an isoflurane anesthetized rat in response to whisker pad stimulation. A microelectrode was inserted into the neocortex through an opening in the EEG spider electrode which was aligned with a burr hole drilled into the skull. The electrode was secured to the skull by a conductive and adhesive EEG paste23. The nose cone used for the administration of isoflurane was modified so that stimulating electrodes could be inserted into the whisker pad with ease.
The EEG paste was effective at mounting the spider electrode securely to the skull, while providing excellent electrical conductivity throughout the experimental day without the need for additional application of paste. It replaced the undesirable use of glue to fix the periphery of the spider electrode to the skull, as glue is non-conductive and can increase the impedance of the electrode if it runs between the skull and the electrode. EEG paste has a number of advantages over EEG gel, which is difficult to shape around the burr hole and can dry out during experiment, resulting in poor EEG signals.
As the rat was placed inside a Faraday cage, electrical noise due to the environment was greatly attenuated. However, sometimes the neural signal was still quite noisy. In most cases, this was caused by the reference electrode not securely positioned and therefore needed to be re-adjusted or more EEG paste used. Another common problem was that the evoked LFP was small in amplitude. This could be due to the microelectrode not positioned at the center of the cortical region activated by the stimulating electrodes. Instead of re-inserting the microelectrode, which could cause more damage to the local neurons, we usually adjusted the position of the stimulating electrodes in the whisker pad until a reasonable amplitude of the LFP (&#62;3 mV) could be observed.
One of the limitations of the technique is the poor spatial resolution of the spider electrode, which has a diameter of 6 mm. This is large compared with the size of the rat&#39;s skull. Unfortunately, the spider electrode used here is the smallest available to purchase. It will be desirable to reduce the diameter of the spider electrode to 2-4 mm, thus increasing the spatial specificity of EEG recordings, making the comparison between the EEG signal and the supragranular LFP signal less ambiguous.
Several critical steps in the protocol need special attention. The first is the insertion of the microelectrode through the burr hole. As the dura is otherwise intact, the precision of the insertion is crucial. A slight resistance at the tip of the electrode usually means the electrode is not positioned correctly. It must be raised, position adjusted, and re-inserted. The second is the position of the nose cone on the rat. It must not be too loose, as isoflurane will escape from the cone. It also must not be too tight, as this can obstruct the nostrils of the rat and cause difficulty breathing. Special attention is also required to ensure that the amplitude of the EEG recording is much smaller (usually 5 to 10 times smaller) than the LFP top channel recording. If they are similar, it is an indication that the EEG probe has come into direct or indirect contact with the microelectrode. An indirect contact is usually through the cerebral spinal fluid (CSF) that sometimes fills the hole drilled in the skull. The conductivity of CSF is typically 100 times that of the skull24,25. Thus, if the level of CSF inside the burr hole is sufficiently high, it can make contact with the spider electrode. To avoid this, the hole should be frequently cleaned with super absorbent cotton sponges such as the absorption spears.
The effect of a burr hole (diameter &#60;2 mm) in the skull on the EEG recording surrounding the hole was studied by placing another spider electrode on the intact skull atop the ipsi-lateral barrel cortex so that bilateral EEG recordings could be compared. The results shown in Figure 9 and Figure 10, suggest the effect to be insignificant at the 0.05 level of significance. Other factors affecting the amplitude of the EEG include how well the EEG paste was in contact with the skull, how firm the electrode was pressed to the paste, and the spatial extent of the EEG paste on the skull.
It is also worthwhile to note that the protocol described here recorded skull EEG, which is different from scalp EEG used in human EEG studies. The scalp acts like a resistor or a low-pass filter, which will reduce the signal-to-noise ratio of the EEG recording further.
Finally, comparison of the temporal dynamics of the ERP and that of the evoked LFP across cortical layers suggest that somatosensory evoked potential reflects better the LFP in the supragranular layer of the cortex than that in the granular and infragranular layers. This is in agreement with our earlier work6, demonstrating that the initial segment (P1) of the ERP is related to the return current arising from the inflow of the excitatory synaptic current occurring in the granular layer, while the subsequent decrease (N1) in ERP may be related to the delayed arrival of thalamic afferent to cortical layers II/III and/or feedforward signals from deeper cortical layers. In conclusion, concurrent recordings of EEG/LFP can enhance understanding of the neural genesis of EEG, and facilitate the mathematical modeling of EEG in terms of neural signals across cortical layers.

It is generally accepted that LFPs recorded via microelectrodes primarily reflect the weighted sum of synchronized excitatory and inhibitory synaptic activities of local pyramidal neural populations1,2,3,4. Our recent research demonstrated that the profile of the LFP signal could be separated into components of excitation and inhibition5,6. However, as LFP is normally measured via an invasive procedure, it is not suited for most studies of the human brain.
On the other hand, EEG is a non-invasive technique for measuring the electrical activity of the brain. It is widely used as a diagnostic tool for certain types of neurological diseases such as epilepsy, and as a research tool in human cognitive studies. Despite its popularity, a major limitation of EEG is the inability to interpret its temporal profiles precisely in terms of the underlying neural signals7,8,9.
Increasingly, mathematical models of EEG are developed to enhance understanding of brain function10,11,12,13,14,15. Most of the existing EEG models are developed based on fitting frequency domain characteristics of the model predicted output to the EEG data spectrum during spontaneous activity, and very few EEG models can generate realistic sensory evoked potentials. In this context, concurrent recordings of EEG and LFP will provide important insight and constraints for developing more accurate mathematical models of EEG.
To address this need for concurrent recordings to further explore the neural origin of EEG, we developed a methodology to simultaneously record EEG and multi-laminar LFP signals in the neocortex of the anesthetized rat. The setup is similar to previous concurrent EEG/LFP studies conducted in primates16,17. We further investigated the effect of a burr hole drilled into the skull on EEG recordings surrounding the hole, by comparing bilateral EEG recordings (i.e., one hemisphere with a burr hole, the other hemisphere intact) in the absence of sensory stimulation. Our results demonstrate that concurrent EEG/LFP recordings can be conducted simply and effectively, with little EEG signal distortion from the burr hole in the skull.

<table>
	<tbody>
		<tr>
			<td>Female Lister Hood rats</td>
			<td>Charles Rivers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spider electrode</td>
			<td>Unimed Electrode Supplies Ltd</td>
			<td>SCS24-426</td>
			<td></td>
		</tr>
		<tr>
			<td>EEG paste: Ten20</td>
			<td>Unimed Electrode Supplies Ltd</td>
			<td>10-20-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic holder with dual micromanipulator arms: Dual Manipulator Stereotaxic Frame with 18&#176; Ear Bars</td>
			<td>WPI (World Precision Instruments)</td>
			<td>502603</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>National Vet Services Limited</td>
			<td>50878</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard plastic nose cone: Anasthesia Gas Mask for Rat</td>
			<td>WPI</td>
			<td>502054</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal isoflurane anaesthetic system</td>
			<td>WPI</td>
			<td>EZ-B800A</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic heating pad: Rat Blanket System 230V</td>
			<td>Harvard Apparatus UK</td>
			<td>50-7221-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment: Optixcare eye lube</td>
			<td>Viovet</td>
			<td>203865</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine Hydrochloride (Injection 2%)</td>
			<td>Larkmead Vets</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jacquette Scaler #1SSE, 18cm, Hollow</td>
			<td>WPI</td>
			<td>503421</td>
			<td></td>
		</tr>
		<tr>
			<td>Serrated and curved dissecting forceps</td>
			<td>WPI</td>
			<td>15915</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided silk, non-absorbable suture: Mersilk Suture W502H</td>
			<td>National Vet Services Limited</td>
			<td>153746</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill: BONE MICRO DRILL SYST 230 VAC</td>
			<td>Harvard Apparatus UK</td>
			<td>72-4860</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Saline: Sodium chloride 0.9%</td>
			<td>Animalcare Ltd</td>
			<td>14K26BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bit #4 : Ball Mill, Carbide, #4</td>
			<td>Harvard Apparatus UK</td>
			<td>72-4958</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bit #4 : Ball Mill, Carbide, #1/4</td>
			<td>Harvard Apparatus UK</td>
			<td>72-4962</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Newport Corporation</td>
			<td>VIS-FDC-3600</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation workstation: Vision IsoStation</td>
			<td>Newport Corporation</td>
			<td>M-VIS3660-RG4-325A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oximeter Control Unit and sensor: MouseOxPlus, Starr Life Sciences Corp.</td>
			<td>WPI</td>
			<td>O15001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent soft nose cone: Microflex Non-Rebreathing Unit with a Rat Nosecone</td>
			<td>WPI</td>
			<td>EZ-103A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel stimulating electrodes</td>
			<td>PlasticsOne</td>
			<td>E363/1/SPC</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated current stimulator</td>
			<td>Made in House</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16-channel micro-electrode, 100 &#956;m spacing, area of each site 177 &#956;m2</td>
			<td>NeuroNexus</td>
			<td>A1x16-10mm-100-177-A16</td>
			<td></td>
		</tr>
		<tr>
			<td>16-channel acute headstage</td>
			<td>Tucker David Technologies Inc., TDT</td>
			<td>RA16AC-Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Amplifier: Z-Series 64-Channel Neuro-Digitizing Preamp</td>
			<td>TDT</td>
			<td>PZ5-64</td>
			<td></td>
		</tr>
		<tr>
			<td>Passive signal splitter: 32-Channel Splitter Box for PZ5</td>
			<td>TDT</td>
			<td>S-BOX_PZ5</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition unit: RZ2 BioAmp Processor. Z-Series 4-DSP ultra high performance processor</td>
			<td>TDT</td>
			<td>RZ2-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for Neurophysiology: OpenEX</td>
			<td>TDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorption spears</td>
			<td>Fine Sicence Tools</td>
			<td>18105-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

concurrent recording of co-localized electroencephalography and local field potential in rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

Data from 4 rats were averaged to obtain mean time series where applicable. The amplitude of the evoked EEG response, also known as the event related potential (ERP), is typically much smaller than that of the LFP. Figure 6 shows the average ERP and LFP in the supragranular, granular, and infragranular layers of the barrel cortex, respectively. The error band in each plot is the corresponding standard error. It can be seen that ERP is approximately 10 times smaller than the evoked LFP.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56447/56447fig6.jpg" /> 
Figure 6: Mean (n = 4) neural signals of ERP, supragranular, granular, and infragranular LFP. Shadow indicates standard error. <a href="https://cloudflare.jove.com/files/ftp_upload/56447/56447fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Comparisons of the temporal dynamics of ERP and LFP are shown in Figure 7. Direct superposition of ERP and the supragranular LFP in Figure 7A illustrates the order of amplitude differences between these two types of neural signals. To compare the temporal dynamics, both ERP and LFP are normalized with respect to their negative peak amplitude. Figure 7B and 7C show the normalized ERP superimposed with the normalized supragranular LFP and normalized granular LFP, respectively.
It can be seen from Figure 7B that the peaks of P1 and N1 for ERP are more delayed than the corresponding peaks of LFP in the supragranular layer. However, the temporal profiles of these two neural signals are similar, with P1 preceding N1. On the other hand, the temporal profile of ERP is markedly different from that of the granular (layer IV of the barrel cortex) LFP (Figure 7C). Importantly they are not mirror images of each other, with granular LFP dominated by a single negative peak (reflecting a major sink in cortical layer IV), whereas ERP consisted primarily of two peaks with opposite polarity.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56447/56447fig7.jpg" /> 
Figure 7: Comparison of the temporal dynamics of ERP and LFP. (A) ERP (solid line) superimposed with supragranular LFP (dashed line). Shadow indicates standard error. (B) Normalized ERP (solid line) superimposed with normalized supragranular LFP (dashed line). (C) Normalized ERP (solid line) superimposed with normalized granular LFP (dashed line). <a href="https://cloudflare.jove.com/files/ftp_upload/56447/56447fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The ERP signal was measured via a spider electrode placed on the skull with a burr hole drilled into it. To investigate the effect of the hole on EEG recordings, another spider electrode was placed on the intact skull above the ipsi-lateral barrel cortex. Care was taken to ensure that the impedances of the two spider electrodes were comparable in magnitude by adjusting the amount of EEG paste used. Data from four rats (which were not the same rats used above) are presented here.
Figure 8 shows the concurrent resting state EEG recordings from both electrodes of one rat, with 100 s data displayed in Figure 8A, and the data in the rectangular frame (20 s) are expanded in Figure 8B. The two EEG signals largely co-vary, within similar range of amplitude. Figure 9 shows the PSD of the four rats, with the top row using a linear scale on the frequency axis, and the bottom row using a logarithmic scale on the frequency axis to provide an expanded view in the lower frequency range. From Figure 9, there does not appear to be consistent bias in the PSD across subjects. This was confirmed by performing one-sample t-tests on the normalized differences in the averaged PSD in the five frequency bands, shown in Figure 10. None of the normalized PSD differences in these frequency bands were significantly different from zero (p = 0.32, 0.46, 0.85, 0.69, and 0.97, respectively).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56447/56447fig8.jpg" /> 
Figure 8: Bilateral EEG recordings. (A) Skull EEG recording during resting state with a burr hole in the skull (black) and a simultaneous EEG recording on the opposite hemisphere with the skull intact (grey). (B) Expanded view of the waveforms within the rectangular frame in (A). <a href="https://cloudflare.jove.com/files/ftp_upload/56447/56447fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56447/56447fig9.jpg" /> 
Figure 9: Power spectral density (PSD) of the contra- (blue) and the ipsi-lateral (red) EEG. Each column shows the PSD for one rat. The top panels use linear frequency scale, while the bottom panels use logarithmic frequency scale to allow the PSD in the lower frequency range to be visualized. <a href="https://cloudflare.jove.com/files/ftp_upload/56447/56447fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56447/56447fig10.jpg" /> 
Figure 10: Group analysis. Normalized difference between the contra- and the ipsi-lateral PSD with the five frequency bands: Delta, Theta, Alpha, Beta, and Gamma. Each bar shows the mean normalized differences within the frequency band, with the standard error shown as the error bar. <a href="https://cloudflare.jove.com/files/ftp_upload/56447/56447fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent at JoVE.com

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

concurrent-recording-co-localized-electroencephalography-local-field

Research

JoVE Journal

Neuroscience

12.2K Views.  University of Reading. This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Video: Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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 ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

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) ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.