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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 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.
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 interneurons1-3. Interneurons release γ-aminobutyric acid (GABA) fro....
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
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.......
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.......
The authors wish to thank Ina Wolter for her excellent technical assistance. VGAT-Venus transgenic rats were generated by Drs. Y. Yanagawa, M. Hirabayashi and Y. Kawaguchi in National Institute for Physiological Sciences, Okazaki, Japan, using pCS2-Venus provided by Dr. A. Miyawaki.
....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 |
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