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

Summary

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.

Abstract

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.

Introduction

Hippocampal neuronal circuits have long been the subject of intense scrutiny, with respect to both anatomy and physiology, due to their essential role in learning and memory as well as spatial navigation in both humans and rodents. Equally, the prominent, but simple laminar organization of the hippocampus makes this region a favored subject of studies addressing structural and functional properties of cortical networks.

Hippocampal circuits are comprised of excitatory principal cells (>80%) and a smaller (10-20%), but highly diverse cohort of inhibitory interneurons^1-3. Interneurons release γ-aminobutyric acid (GABA) fro....

Protocol

Ethics Statement: All procedures and animal maintenance were performed in accordance with Institutional guidelines, the German Animal Welfare Act, the European Council Directive 86/609/EEC regarding the protection of animals, and guidelines from local authorities (Berlin, T-0215/11)

1. Preparation of Acute-hippocampal Slices

1. Take a transgenic rat (17 to 24 day old), expressing the fluorescent Venus/YFP protein under the vGAT promoter, which labels the majority of cortical inhibitory interneurons¹³. Decapitate the rat. Rapidly dissect the brain (<40 sec) into semifrozen, carbogenated (95% O₂/5% CO_2<....

Results

Provided that slice quality is appreciably good, recording from both CA1 PCs and FS-INs can be achieved with minimal difficulty. The transgenic rat line expressing Venus / YFP under the vGAT promoter¹³ does not unequivocally identify FS-INs, or indeed BCs. However recordings from INs in and around str. pyramidale, where the density of FS-INs is typically high¹, results in a high probability of selecting FS-INs (Figure 2B). FS-INs can be distinguished by their characteristic.......

Discussion

We describe a method which combines electrophysiological and neuroanatomical techniques to functionally characterize morphologically- and neurochemically-identified neurons in vitro; in particular the diverse types of cortical inhibitory INs. Key aspects of the procedure are: (1) pre-selection of potential INs; (2) intracellular recording and neuron visualization; and finally (3) morphological and immunocytochemical analysis of recorded INs. Although this study has addressed PV-INs in particular, the described protocol c.......

Materials

Name	Company	Catalog Number	Comments
Name	Company	Catalog Number	Comments
Transgenic vGAT-venus rats	-	-	see Uematsu et al., 2008
Venus (515 nm) goggles	BLS Ltd., Hungary	-	-
Dissection tools	i.e. FST	-	For brain removal
Vibratome	Leica	VT1200S	Or other high end vibratome with minimal vertical oscillation
Slice holding chambers	-	-	Custom-made in lab
Upright IR-DIC microscope	Olympus, Japan	BX50WI	With micromanipulator system; i.e. Luigs and Neumann, Kleindiek etc.
CCD camera	Till Photonics	VX55
505 nm LED system	Cairn Research	OptiLED system	Or mercury lamp or other LED system i.e. CooLED.
Multiclamp 700B	Axon Instruments		Alternatively 2x Axopatch 200B amplifiers
WinWCP acquisition software	John Dempster, Strathclyde University	-	Any quality acquisition software could be used, i.e. EPHUS, pClamp, Igor etc.
Electrode Puller	Sutter	P-97	Used with box-filament
Borosilicate pipette glass	Hilgenberg, Germany	1405020	2 mm outer, 1 mm inner diameter, no filament
Peristaltic pump	Gilson	Minipuls	Other pumps or gravity feed could be used instead
Digital Thermometer	-	-	Custom made
Digital Manometer	Supertech, Hungary	-
Isolated constant voltage stimulator	Digitimer, Cambridge	DS-2A	-
Biocytin	Invitrogen	B1592	Otherwise known as ε-Biotinoyl-L-Lysine
DL-AP5(V) disodium salt	Abcam Biochemicals	ab120271
DNQX disodium salt	Abcam Biochemicals	ab120169	Alternatively NBQX or CNQX
Gabazine (SR95531)	Abcam Biochemicals	ab120042	Alternatively bicuculline methiodide
R-Baclofen	Abcam Biochemicals	ab120325
CGP-55,845 hydrochloride	Tocris	1248
Streptavidin 647	Invitrogen	S32357
anti-PV mouse monoclonal antibody	Swant, Switzerland	235	Working concentration 1:5000-1:10,000
anti-mouse secondary antibody	Invitrogen	A11030	If using Venus or GFP rodent using a red-channel (i.e. 546 nm) is advisable.
Normal Goat Serum	Vector Labs	S-1000
Microscopy slides	-	-	Any high quality brand
Glass coverslips	-	-	Usually 22 x 22 mm
Agar spacers	-	-	Agar block, cut on vibratome to 300 μm
Laser scanning confocal microscope	Olympus, Japan	Fluoview FV1000	Or other comparable system
Fiji (Fiji is just ImageJ)	http://fiji.sc/Fiji	-	See Schindelin et al., 2012