An optogenetics- and imaging-assisted simultaneous multiple patch-clamp recording system for decoding complex neural circuits

Abstract

Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4–8 simultaneously recorded neurons and/or 10–30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy–based, optogenetics- and imaging-assisted, stable, simultaneous quadruple–viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3–4 d.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…While bioelectronic interfaces are already widely used for deep‐brain stimulation, pacemakers and cochlear implants, existing electrodes are often physically large, have limited resolution, and frequently inducing adverse physiological responses. Similarly, the patch‐clamp, the current gold‐standard in single‐cell electrophysiology, is effective, but complex to implement for any more than a few cells at a time . Better neural interfaces are required to understand the role of peripheral nerves on diseases such as diabetes and liver disease, neurological conditions such as epilepsy and Parkinson's disease, and in the development of brain–machine interfaces …”

BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method

“…Simultaneous multiple whole-cell recordings were obtained from nearby infected/transfected and noninfected/nontransfected CA1 pyramidal neurons or LA large pyramidal-like neurons under visual guidance with fluorescence and transmitted light illumination (Lim et al 2014;Wang et al 2015b) using up to four Axopatch-200B or Axoclamp 2B amplifiers (Axon Instruments). Bath solution (29°C ± 1.5°C), unless otherwise stated, contained 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl 2 , 4 mM MgCl 2 , 26 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 11 mM glucose, 0.1 mM picrotoxin (PTX), and 0.002 mM 2-chloroadenosine (pH 7.4) and was gassed with 5% CO 2 /95% O 2 .…”

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

“…Using simultaneous multiple patch clamp recordings and/or multiple two-photon imaging techniques (Wang et al 2015), we found that, in sharp contrast to the other Ttype calcium channels (i.e., Ca V 3.1 and Ca V 3.3 channels), Ca V 3.2 channels did not contribute to either modulation of membrane properties or production of lowthreshold calcium spikes in central neurons. Instead, functional Ca V 3.2 channels primarily incorporated into synapses by replacing existing synaptic Ca V 3.2 channels and served to control the strength of NMDA transmission.…”

“…Using our recently developed stable multiple patch clamp recording system (Jiang et al 2013;Lee et al 2014b;Wang et al 2015), simultaneous and/or sequential multiple whole-cell recordings were obtained from two to five nearby infected and noninfected TRN neurons or cortical L5 or hippocampal CA1 pyramidal neurons under visual guidance using fluorescence and transmitted light illumination. For cultured slices, bath solution (29°C ± 1.5°C) contained 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl 2 , 4 mM MgCl 2 , 26 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 11 mM glucose, 0.1 mM picrotoxin (PTX), 0.01 mM bicuculline, and 0.002 mM 2-chloroadenosine (pH 7.4) gassed with 5% CO 2 /95% O 2 .…”

“…Two-photon imaging and electrophysiology recordings were simultaneously performed using a custom-built microscope operated by a custom-written IGOR Pro 6 program (WaveMetrics) (Wang et al 2015). To minimize the Ca 2+ signal variances resulting from the different location of synapses and loading time of indicators (Sabatini and Svoboda 2000;Yasuda et al 2003), neighboring expressing and nonexpressing CA1 neuron pairs were broken in simultaneously to load 200 μM green calcium-sensitive indicator Fluo-5F and 20 μM red calcium-insensitive indicator Alexa 594.…”