This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

NOTE: The following protocol is in accordance with the National Institutes of Health guidelines as approved by the University of Oregon Animal Care and Use Committee.
1. Acute Surgery
<ol>
	<li>Anesthetize the animal with a ketamine-medetomidine cocktail, via intraperitoneal (i.p.) injection (Table 1). 
	NOTE: The mice used in these experiments are generated by crossing a cre-dependent ChR2-eYFP transgenic line10 to interneuron driver lines (Pvalb-iCre11, PV+; Sst-iCre12, SOM+; Cr-iCre12, CR+). Viral delivery of ChR2 or related opsins should work equally well, assuming similar expression levels are obtained.</li>
	<li>Before beginning surgery, ensure that the animal exhibits no response to a gentle toe pinch. Re-administer anesthetics throughout the experiment as necessary to maintain this depth of anesthesia. If using injectable anesthetics, optionally implant an i.p. catheter for maintenance injections.</li>
	<li>Keep the animal hydrated with saline or lactated Ringer&#8217;s solution throughout the experiment (approximately 3 ml/kg/hr), for example by using an appropriately diluted anesthetic cocktail for maintenance injections (Table 1).</li>
	<li>Place the animal in a stereotaxic or other head-holding apparatus. Ensure that the skull is well-secured. This is essential for maintaining stable single cell recordings.</li>
	<li>Apply opthalamic ointment to the eyes to prevent dryness. Maintain body temperature at 36.5-37 &#176;C.</li>
	<li>Perform a cisternal drain for additional recording stability. Using a scalpel blade, remove tissue from the posterior face of the skull to expose the cisterna magna. Make a small nick in the dura to drain the cerebrospinal fluid. Use only the very tip of the blade, and avoid contacting the cerebellum or brain stem.</li>
	<li>Using the scalpel, perform a small craniotomy (~2 mm2) over the cortical area of interest (for auditory cortex, roughly -2.3 mm posterior of bregma, 4.5 mm lateral of the midline).</li>
	<li>Remove the dura and cover the exposed cortex with a layer of warm agarose 0.5 - 1 mm thick (1.5% agarose in saline, 0.9% NaCl; apply at ~37 &#176;C). Keep the agarose moist throughout the experiment by periodically applying several drops of saline.</li>
</ol>
2. Recording Set-up
<ol>
	<li>Prepare a tungsten electrode (7-14 M&#937;, 127 &#956;m diameter, 12&#176; tapered tip, epoxy-coated). Superglue the electrode to a glass capillary tube and add heat shrink tubing for grip (Figure 1).</li>
	<li>Mount the electrode on a motorized (or hydraulic) micromanipulator, set to travel orthogonal to the cortical surface. Slide a wire ground under the skin, against the skull. Avoid contact with the muscles on the side of the head and the back of the neck as they can generate electromyographic artifacts.</li>
	<li>Amplify the electrical signal using an extracellular ampli&#64257;er suitable for single-unit recording, preferably equipped with an impedance check mode. Monitor ongoing spiking activity (band pass 300 - 5,000 Hz) with two oscilloscopes and a set of powered speakers. Here, the recorded data is digitized continuously at a sampling rate of 10 kHz; spikes are extracted offline.</li>
	<li>Advance the electrode through the agarose until the tip reaches the surface of the cortex. Observe this step through a microscope. &#8220;Zero&#8221; this position on the micromanipulator.</li>
	<li>To best approximate the depth of recording, visually confirm that the electrode tip exits the cortical surface at a depth of zero when withdrawing the electrode at the end of a penetration. 
	NOTE: A discrepancy between the manipulator reading and the observed electrode position indicates that it has drifted relative to the tissue. This can be caused by instability of the brain or animal, or manipulator drift.
	<ol>
		<li>As an additional check to ensure that this zero set-point corresponds to the surface of the cortex, monitor stimulus-evoked field potentials while advancing through the first several hundred micrometers of tissue. When monitoring field potentials, lower the lower band-pass filter cutoff of the extracellular amplifier to 10 Hz. Confirm that the polarity of the local field potential reverses around a depth of ~100 &#181;m, near the layer I/II border13. This is a reliable reference point for auditory cortex, but it may differ for other cortical areas.</li>
	</ol>
	</li>
	<li>Mount an optical fiber coupled to a blue light source (Figure 2) onto a manual micromanipulator. Using the microscope, position the tip of the fiber as close to the surface of the agarose as possible. Center the beam where the electrode will enter the tissue.</li>
	<li>Regulate the output of the light source with a control unit capable of delivering a TTL (transistor-transistor logic) pulse train of specified width and duration&#8211; e.g., an Arduino (Figure 3), a computer I/O card (as shown), or a commercially available pulse generator.
	<ol>
		<li>Monitor the signal on both oscilloscopes.</li>
	</ol>
	</li>
	<li>Measure total light power (mW) at the tip of the optical fiber using a power meter. Use this value to calculate irradiance (mW/mm2) by dividing by the cross-sectional area of the fiber core. Begin with an intensity value in the range of 10 - 15 mW/mm2 and adjust downward if necessary, until artifacts are eliminated.</li>
	<li>Check for light artifacts with the electrode in the agarose, poised to enter the tissue. Start the search pulse train (e.g., 30 msec light pulses, 500 msec interstimulus interval (ISI)).
	<ol>
		<li>Eliminate transient light artifacts (Figure 4) by repositioning the optical fiber relative to the electrode to change the angle of incident light. If artifacts persist, try decreasing the light power. In the rare case that artifacts cannot be completely eliminated (only minimized), extend the duration of the search pulse. This can aid in identifying true neuronal spikes.</li>
	</ol>
	</li>
</ol>
3. Straight PINP-in&#8217;
<ol>
	<li>Advance the electrode slowly through the brain at a rate of approximately 1 &#181;m/sec. Use one oscilloscope to monitor ongoing activity. On the other, trigger off the laser pulses.</li>
	<li>Listen for the faint &#8216;hash&#8217; of light-evoked spikes on the audio monitor, which indicates that the electrode is approaching a ChR2+ cell and can often be perceived well before the electrical signal is apparent on the oscilloscope. Slow the rate of advance. 
	NOTE: If the cell lies directly in the path of the electrode, the light-evoked activity will grow larger (and louder). As the electrode approaches a single interneuron, the hash will resolve into small but well-defined spikes of uniform size and shape.</li>
	<li>As soon as light-evoked spikes are large enough to trigger off of individually on the oscilloscope, begin doing so. Adjust the scaling on the horizontal axis to observe the exact shape of the spike waveform.</li>
	<li>Stop and wait for the tissue to settle. Resist the temptation to move the electrode closer to the cell. This step is critical for a stable recording. If after several minutes the signal-to-noise ratio has not improved &#8211; that is, the tissue has settled, but it hasn&#39;t brought the cell any closer to the tip of the electrode &#8211; advance 5 &#181;m further and wait again.
	<ol>
		<li>Repeat this process until either the peak or the trough of the action potential can be reliably captured with a voltage threshold set well above the noise floor (e.g., +/-300 &#181;V or greater). 
		NOTE: There&#8217;s little risk of false-positives or ambiguity when PINPing inhibitory interneurons. Most cells can easily follow a train of pulses with duration of 30 msec and inter-pulse interval 500 msec, or faster, with a reliable first spike latency of 2-5 msec (Figure 5). The majority of PV+ cells, in particular, can sustain firing for a full 1 sec (Figure 6). Because these are inhibitory neurons, disynaptic (indirect) activation isn&#8217;t a major concern.</li>
	</ol>
	</li>
	<li>While recording, monitor ongoing activity on the first oscilloscope. Keep a close eye on the size and shape of spikes using the second oscilloscope. Note the quality of their sound on the speaker.
	<ol>
		<li>Listen attentively for abrupt changes, which indicate that the cell is either drawing too close to the electrode (where it risks being impaled or damaged), or is drifting away. If the spikes become large and distorted, back out; if they grow smaller, advance the electrode. Move slowly in 2 &#181;m steps.</li>
		<li>Confirm the quality of the recording post-hoc by superimposing all spike waveforms, aligned to peak or trough. Vary the voltage threshold. Across a wide range of threshold values, there should only ever be one consistent spike shape of uniform height (Figure 5a).</li>
	</ol>
	</li>
	<li>If at any point the signal becomes contaminated with spikes from a neighboring neuron, move on. Advance slowly, on the off-chance that the electrode will pass out of range of the neighboring cell, but remain in range of the target neuron. (This is usually unsuccessful.)</li>
	<li>Move the electrode through the entire depth of cortex (900+ &#181;m) in each penetration. If no light-responsive neurons are encountered after many penetrations or, conversely, recordings are routinely contaminated with spikes from neighboring ChR2- cells, try a new electrode. 
	NOTE: Even within a single batch, the impedance and tip geometry of individual electrodes can vary considerably. Both factors contribute to the effective &#8220;listening radius&#8221; of the electrode, which must be large enough to detect light-evoked spikes while searching, yet restricted enough to enable recording a single interneuron in isolation.</li>
	<li>Test the impedance of electrodes as desired. To avoid damaging the tissue, do this with the tip of the electrode in the upper part of the agarose layer, well outside the brain. Within the range of 7-14 M&#937;, the exact impedance is not a sure predictor of performance, or yield, for this application. That said, it&#8217;s a reasonable proxy for listening radius: lower-impedance electrodes pick up more units; higher-impedance, fewer.</li>
	<li>At the end of the experiment, euthanize the animal by institutionally-approved means, such as anesthetic overdose, or cervical dislocation under a deep plane of anesthesia.</li>
</ol>

<ol>
	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., 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=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. V., Bruns, W., Carandini, M., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+interneurons+linearly+transform+cortical+responses+to+visual+stimuli.">Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli.</a> Neuron. 73, 159-170 (2012).</li><li>Wilson, N. R., Runyan, C. A., Wang, F. L., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+subtraction+by+distinct+cortical+inhibitory+networks+in+vivo.">Division and subtraction by distinct cortical inhibitory networks in vivo.</a> Nature. 488, 343-348 (2012).</li><li>Letzkus, 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=A+disinhibitory+microcircuit+for+associative+fear+learning+in+the+auditory+cortex.">A disinhibitory microcircuit for associative fear learning in the auditory cortex.</a> Nature. 480, 331-335 (2011).</li><li>Pi, H. 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=Cortical+interneurons+that+specialize+in+disinhibitory+control.">Cortical interneurons that specialize in disinhibitory control.</a> Nature. 503, 521-524 (2013).</li><li>Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+circuit+for+spatial+summation+in+visual+cortex.">A neural circuit for spatial summation in visual cortex.</a> Nature. 490, 226-231 (2012).</li><li>Madisen, 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=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Hippenmeyer, S., 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=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3, 159 (2005).</li><li>Taniguchi, H., 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=A+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71, 995-1013 (2011).</li><li>Christianson, G. 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The authors have no competing financial interests.

Although PINP is conceptually straightforward, it can be challenging in practice. A major determinant of success is the choice of electrode. The electrical listening radius is the critical parameter. It must be sufficiently large to detect light-evoked spikes when the tip is still some distance away from a ChR2+ cell, so that one can adjust the rate of advance accordingly. At the same time, it must be restricted enough to enable good single-unit isolation. That is, the electrode must not also pick up spikes from neighbor...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table border="0" cellpadding="0" cellspacing="0" width="1482">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:341px;">Product/Stock Number</td>
			<td style="width:708px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChR2-EYFP Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">12569</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pvalb-iCre (PV) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">8069</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sst-iCre (SOM) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">13044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cr-iCre (CR) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td>10774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Agarose</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:341px;">A9793</td>
			<td>Type III-A, High EEO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
			<td style="width:171px;">FST</td>
			<td style="width:341px;">10066-15</td>
			<td style="width:708px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical Scissors</td>
			<td style="width:171px;">FST</td>
			<td>14084-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Scalpel</td>
			<td style="width:171px;">FST</td>
			<td>10003-12 (handle), 10011-00 (blades)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
			<td style="width:171px;">Foster &#38; Smith</td>
			<td style="width:341px;">9N-76855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Homeothermic Blanket</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:341px;"><a name="RANGE!C11">507220F</a></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tungsten Microelectrodes</td>
			<td style="width:171px;">A-M Systems</td>
			<td>577200</td>
			<td>12 M&#937; AC resistance, 127 &#956;m diameter, 12&#176; tapered tip, epoxy-coated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Capillary Glass Tubing</td>
			<td style="width:171px;">Warner Instruments</td>
			<td style="width:341px;">G150TF-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heat Shrink Tubing</td>
			<td style="width:171px;">DigiKey</td>
			<td>A332B-4-ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Zapit Accelerator</td>
			<td style="width:171px;">DVA</td>
			<td style="width:341px;">SKU ZA/ZAA</td>
			<td>Use with standard Super Glue.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Microelectrode AC Amplifier 1800</td>
			<td style="width:171px;">AM Systems</td>
			<td style="width:341px;">700000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">MP-285 Motorized Micromanipulator</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:341px;">MP-285</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">4-channel Digital Oscilloscopes</td>
			<td style="width:171px;">Tektronix</td>
			<td style="width:341px;">TDS2000C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Powered Speakers</td>
			<td style="width:171px;">Harman</td>
			<td>Model JBL&#160;Duet</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Manual Manipulator</td>
			<td style="width:171px;">Scientifica</td>
			<td style="width:341px;">LBM-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">800 &#181;m Fiber Optic Patch Cable</td>
			<td style="width:171px;">ThorLabs</td>
			<td style="width:341px;">FC/PC BFL37-800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Power Meter</td>
			<td style="width:171px;">ThorLabs</td>
			<td>PM100D (Power Meter), S121C (Standard Power Sensor)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">475 nm Cree XLamp XP-E</td>
			<td>DigiKey</td>
			<td>XPEBLU-L1-R250-00Y01DKR-ND</td>
			<td>LED power and efficiency are continually increasing, so we recommend checking for the latest products (www.cree.com).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arduino UNO</td>
			<td style="width:171px;">DigiKey</td>
			<td>1050-1024-ND</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

We here share our strategy for obtaining single-unit recordings from genetically-classified inhibitory interneurons in the anesthetized mouse cortex, using an optogenetic method developed by Lima et al.1. Table 1 details the suggested anesthetic cocktail, Ketamine-Medetomidine-Acepromazine (&#8220;KMA&#8221;). Figure 1 depicts a tungsten microelectrode, prepared for recording. Figure 2 contains a circuit diagram for a simple LED control unit. ...

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A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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Neuroscience

19.0K Views.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

Video: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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 ...