Summary

Ein Leitfaden zur<em> In vivo</em> Single-Unit-Aufnahme von Optogenetically Identifizierte kortikalen inhibitorischen Inter

Published: November 07, 2014
doi:

Summary

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.

Abstract

Eine große Herausforderung in der Neurophysiologie war es, die Reaktionseigenschaften und Funktion der zahlreichen hemmenden Zelltypen in der Großhirnrinde zu charakterisieren. Wir teilen hier unsere Strategie für den Erhalt von stabilen, gut isolierten Single-Unit-Aufnahmen von identifizierten hemmenden Inter im narkotisierten Maus Kortex mit Hilfe eines von Lima und Kollegen 1 entwickelten Methode. Die Aufnahmen werden in Mäusen, Channelrhodopsin-2 (ChR2) in bestimmten neuronalen Subpopulationen durchgeführt. Mitglieder der Population werden durch ihre Reaktion auf einen kurzen Blitz aus blauem Licht identifiziert. Diese Technik – genannt "PINP" oder Photostimulation gestützte Identifizierung von neuronalen Populationen – Verwendbar mit Standard extrazellulären Kontrollgerät durchgeführt werden. Es kann als kostengünstige und leicht zugängliche Alternative zu Calcium Imaging oder visuell geführte Patch dienen, zum Zwecke der Ausrichtung der extrazellulären Aufnahmen genetisch identifizierten Zellen. Hehe wir bieten eine Reihe von Leitlinien für die Optimierung der Verfahren in der täglichen Praxis. Wir verfeinert unsere Strategie speziell für Targeting Parvalbumin-positive (PV +) Zellen, haben aber festgestellt, dass es für andere Intern Typen sowie wie Somatostatin-exprimierenden (SOM +) und Calretinin-exprimierenden (CR +) Inter.

Introduction

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 “PINP” – 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.

Protocol

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 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…

Representative Results

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 (“KMA”). Figure 1 depicts a tungsten microelectrode, prepared for recording. Figure 2 contains a circuit diagram for a simple LED control unit. …

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

References

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Cite This Article
Moore, A. K., Wehr, M. A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons. J. Vis. Exp. (93), e51757, doi:10.3791/51757 (2014).

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