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-iCre1...

<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. B., Sahani, M., Linden, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-dependent+temporal+response+properties+in+core+auditory+cortex.">Depth-dependent temporal response properties in core auditory cortex.</a> J Neurosci. 31, 12837-12848 (2011).</li><li>Povysheva, N. V., Zaitsev, A. V., Gonzalez-Burgos, G., Lewis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+heterogeneity+of+fast-spiking+interneurons:+chandelier+versus+basket+cells.">Electrophysiological heterogeneity of fast-spiking interneurons: chandelier versus basket cells.</a> PLoS One. 8, 70553 (2013).</li></ol>

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. ...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

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 Aufrufe.  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. 