Simultaneous Voltron (1.0) imaging and whole-cell patch-clamp recordings of somatosensory cortex layer 1 interneurons in vivo

Posted on 12.04.2021 - 19:00 by Marton Rozsa

Membrane potential recordings were made from twelve Voltron 1-expressing layer 1 neurons that were simultaneously imaged with a fast sCMOS camera (400Hz or 1kHz). Mice were under isoflurane anesthesia and imaged in darkness. Multiple movies were recorded for each cell. Metadata is included as .json files. The collection contains a sample script demonstrating how to use the data. Please cite this collection using the DOI to cite any of these datasets.

In vivo whole-cell recordings were made in NDNF-Cre mice (JAX 28536) (4 females, 3 males; 54-160 days old at the time of the first surgery). In these mice Cre recombinase is expressed in NDNF-positive layer 1 interneurons. AAV2/1-syn-FLEX-Voltron-ST (Abdelfattah et al 2019) (titer: 2E12 GC/ml) was injected at 6-10 injection sites 200 µm deep in the somatosensory cortex (30 nl each; injection rate, 1 nl/s) (Liu et al 2020). Headbars and cranial windows (2.5 mm diameter) were implanted centered on 1.6 mm lateral, 1.2 mm posterior from lambda (Daie et al 2021). To prepare the JF dye for injection, 100 nanomoles of lyophilized JF525 were dissolved in 20 µl of DMSO, 20 µl Pluronic F-127 (20% w/v in DMSO), and 60 µl of PBS (final dye concentration 1 μM). 33-71 days after the first surgery, mice were anesthetized with 2-3% isoflurane and 100 μl of the dye solution was injected into the retro-orbital sinus of the right eye using a 30 gauge needle. One day later we removed the cranial window and performed durotomy (Goldey et al 2014). The craniotomy was filled with 10-15 μl of 1% agarose, then a D-shaped coverslip was secured on top to suppress brain motion, but leaving access to the brain on the lateral side of the craniotomy.

Micropipettes (3–6MΩ) were filled with (in mM): 126 K-gluconate, 4 KCl, 4 ATP-Mg, 0.3 GTP-Na2, 10 HEPES, 10 creatine phosphate (pH 7.25; 300 mOsm). Whole-cell recordings were obtained from somata of layer 1 interneurons (20-100 μm deep) visualized with infrared oblique illumination (Szucs et al 2009). An infrared LED (Osram SFH 4550, 850 nm) was attached to the imaging objective with a custom-printed holder and illuminated the craniotomy 30-45° from normal. Warm saline (35-37°C) was perfused onto the craniotomy to maintain physiological temperature. Recordings were in current clamp mode. Signals were filtered at 20 kHz (Multiclamp 700B, Axon Instruments) and digitized at 100 kHz using acq4 (Campagnola et al 2014). A wide-field fluorescence microscope equipped with a water immersion objective (40X, NA 0.8, Olympus) was used for imaging. Illumination was delivered using a 525 nm LED (Mightex, LCS-0525-60-22); intensity at the sample, <20 mW/mm^2. A custom filter set (517/20 nm (excitation, Semrock FF01-517/20), 537LP nm (emission, Chroma RET537lp), and a 532LP dichroic mirror (Chroma, RT532rdc)) was used for fluorescence imaging of Voltron525. Images were collected using a sCMOS camera (Hamamatsu Orca Flash 4.0 v3). The pixel size was 0.5 µm/pixel.


Rozsa, Marton; Singh, Amrita; Svoboda, Karel (2021): Simultaneous Voltron (1.0) imaging and whole-cell patch-clamp recordings of somatosensory cortex layer 1 interneurons in vivo. Janelia Research Campus. Collection.
Select your citation style and then place your mouse over the citation text to select it.




need help?