Figure 1, Figure 3, Figure 4, Figure 8 and Figure 9 were created with BioRender.com. We would like to thank Scott Kilianski for the help with MATLAB code and sharing his scripts. We thank Anna Grace Carns for the help with probe trajectory reconstruction.

All studies were approved by the Institutional Animal Care and Use Committee at the University of Virginia (Charlottesville, Virginia). Five male C57BL/6J&#160;mice, age 3-7 months, weighing 25-30 g,&#160;were used. The details of the reagents and the equipment used here are listed in the Table of Materials.
1. Headplate and headset implantation
<ol>
	<li>Make an ECoG headset by soldering perfluoroalkoxy (PFA) coated stainless steel wire to a 3-pin connector...

Angled Silicon Multielectrode Recordings for Deep Brainstem Structures in Mice

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Brainstem nuclei mediate fundamental functions such as breathing, consciousness, and sleep26,27,28. The brainstem&#39;s location (deep and posterior) presents a challenge in studying its neuronal activity in vivo using standard techniques. Here an angled anterior approach is presented to allow reproducible single unit recording in head-fixed mice.
Careful insertion of the multi-electrode sili...

Silicon multielectrode-based recordings are becoming increasingly popular to measure neuronal activity across many brain regions with single action potential resolution1,2,3,4. Over the last decade, high-density recording technology has grown considerably. Current silicon-based recording electrodes can accommodate high channel counts, optical fibers, and electrocorticography (ECoG) recording devices5,6. Moreover, chronic implantation of these electrodes allows for long-term recordings7,8.
Despite recent technological advancements, targeting deep caudal structures like the brainstem with multi-channel probes remains challenging. When targeting brainstem structures such as the ventrolateral periacqueductal gray (vlPAG), one significant obstacle is identifying a probe trajectory that avoids major blood vessels, e.g., the superior sagittal venous sinus and the transverse venous sinus. Injury to these large veins can cause extensive bleeding, damage to the underlying brain tissue, and even death9,10. We propose targeting brainstem structures from anterior coordinates at an angle, allowing the recording probe to penetrate the brain below such high-risk vascular structures (see Figure 1). This angled approach, compared to a vertical one, maximizes the number of brain regions accessible for recording. Additionally, in experimental circumstances wherein ECoG recordings are desired, the angled anterior approach affords more skull surface available for ECoG headset implantation, as the craniotomy window for probe insertion is positioned more anteriorly10,11.
Identifying the specific cell groups and circuits responsible for anesthesia-induced REM sleep changes remains a major goal of anesthesia research. Thus, the objective here was to reproducibly and reliably access the vlPAG - a brainstem region associated with REM sleep - to obtain single unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia12,13. Previous studies have used electrophysiological local field potential (LFP) measurements of the vlPAG in awake mice to identify neural state changes associated with anesthesia14,15. However, LFP measurements are primarily sensitive to synaptic activity, not action potentials, within the recorded area16. Consequently, there remains a limited understanding of how anesthetics directly affect the neural activity patterns produced by vlPAG neurons. Here, a method is described to obtain high-density single-neuron recordings from the brainstem of head-fixed mice. This method can also be adapted to record single-neuron activity from various other deep and posterior brainstem structures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1024 channel RHD Recording Controller</td>
			<td>Intan Technologies, Los Angeles, California, USA</td>
			<td>C3008</td>
			<td>Silicon probe recording; recording hardware and software</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm x 50 mm No. 1.5 VWR coverslip</td>
			<td>VWR, Radnor, Pennsylvania, USA</td>
			<td>48393-081</td>
			<td>Histology</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA in PBS</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>J61899.AK</td>
			<td>Histology; perfusion solution</td>
			<td></td>
		</tr>
		<tr>
			<td>C&#38;B metabond</td>
			<td>Patterson Dental, Richmond, Virginia, USA</td>
			<td>powder: 5533559, quick base: 5533492, catalyst: 55335007</td>
			<td>Headplate &#38;Headset Implantation</td>
			<td></td>
		</tr>
		<tr>
			<td>C57/6J mice 4-6 weeks, males</td>
			<td>The Jackson Laboratory, Bar Harbor, Maine, USA</td>
			<td>000664</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capnomac Ultima</td>
			<td>Datex, Helsinki, Finland&#160;</td>
			<td>ULT-SVi-27-07</td>
			<td>Gas Analyzer; discontinued; alternative gas analyzer can be purchased from Bionet America&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>V22888</td>
			<td>Red fluorescent dye for coating of the silicon probe</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector Header</td>
			<td>DigiKey, Thief River Falls, Minnesota, USA</td>
			<td>1212-1788-ND</td>
			<td>ECoG Headset</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, Alabama, USA</td>
			<td>0100-20</td>
			<td>Histology</td>
			<td></td>
		</tr>
		<tr>
			<td>iBOND Universal</td>
			<td>Patterson Dental, Richmond, Virginia, USA</td>
			<td>044-1113</td>
			<td>Headplate &#38;Headset Implantation; for&#160; securing stainless steel wires to the skull</td>
			<td></td>
		</tr>
		<tr>
			<td>Low toxicity silicon adhesive</td>
			<td>World Precision Instruments, Sarasota, Florida, USA</td>
			<td>KWIK-SIL</td>
			<td>Headplate</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Manipulator System</td>
			<td>New Scale Technologies, Victor, New York, USA</td>
			<td>Multi-Probe Manipulator: XYZ Stage Assembly: 06464-0000, MPM System Kit: 06267-3-0001, MPM-Platform-360, MPM ring for MPM Manual Arms, MPM_Ring-72 DEG: 06262-3-0000</td>
			<td>Silicon probe recording; inserting the probe into the brain</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>UCLA, Los Angeles, California, USA</td>
			<td>256 ANS, 64M</td>
			<td>Discontinued; alternative silicon probes can be purchased from Neuropixels</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma Aldrich, Saint Luis, Missouri, USA</td>
			<td>M8410-100ML</td>
			<td>Silicon probe recording; preventing the tissue from drying during the recording</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>Z1376</td>
			<td>Headplate &#38;Headset Implantation; preventing the brain from drying during the surgery</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA-Coated Stainless Steel Wire-Diameter 0.008 in. coated with striped ends</td>
			<td>A-M systems, Sequim, Washington, USA</td>
			<td>791400</td>
			<td>ECoG Headset &#38; reference electrode for ECoG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire 24AWG&#160;</td>
			<td>World Precision Instruments, Sarasota, Florida, USA</td>
			<td>PTP201</td>
			<td>Reference electrode for the silicon probe recording&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Colorfrost Plus microscope slides</td>
			<td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td>
			<td>99-910-01</td>
			<td>Histology</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel Headplate</td>
			<td>Star Rapid, China</td>
			<td>custom made part</td>
			<td>Headplate &#38;Headset Implantation; design available upon request</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>KOPF, Tujunga, California, USA</td>
			<td>Model 940 Small Animal Stereotaxic Instrument with Digital Display Console</td>
			<td>Headplate &#38;Headset Implantation</td>
			<td></td>
		</tr>
	</tbody>
</table>

acute single-unit multi-electrode recordings from the brainstem of head-fixed mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

Author Spotlight: Investigating Anesthesia-Induced Sleep Pathways and Neuronal Excitability in Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Five male C57BL/6J were implanted with an ECoG headset and headplate (Figure 4A). After recovery, mice were habituated to head-fixation and the electrophysiology recording rig during two 1.5 h sessions on separate days (Figure 4B). Next, a 2 mm x2 mm craniotomy window was created (Figure 4C) and a silicon probe was inserted with the mouse awake and head-fixed (Figure 4D). Two types of silicon UCLA probe...

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

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Neuroscience

Electrocorticography Headplate Implantation and Electrode Placement for Neuronal Activity Recording

Silicon Probe Placement and Single Unit Recording from the Brainstem of Head-Fixed Mice

300 Views.  University of Virginia.  This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Angled Silicon Multielectrode Recordings for Deep Brainstem Structures in Mice

Summary