GoKawiilAnimal care and use
Experimental procedures that involved the use of mice were all conducted with approved protocols in accordance with NIH (US National Institutes of Health) guidelines. They were also approved by the Allen Institute for Brain Science Institutional Animal Care and Use Committee.
Mice were housed with five or less mice per cage and were maintained on a 12-h light–dark cycle, in a humidity-controlled and temperature-controlled room with water and food available ad libitum.
Transgenic mice and sparse labelling
Transgenic driver and reporter mice used in Patch-seq and WNM studies are listed in Supplementary Table 1 (Patch-seq only) and Supplementary Table 2. Characterization of the expression pattern of many of the transgenic mouse lines can be found in the AIBS Transgenic Characterization database (http://connectivity.brain-map.org/transgenic/search/basic)51. Many of the brains used for WNM studies were described in a previous article28. Additional brains were sparsely and robustly labelled for WNMs studies using Supernova virus, which was provided as a gift by M. Luo as pAAV-TRE-fDIO-GFP-IRES-tTA (Addgene plasmid #118026; http://n2t.net/addgene:118026; RRID: Addgene 118026), and variants.
Tissue processing and slicing procedure
For preparation of acute brain slices, adult male and female mice (postnatal day 45 (P45)–P70 of age) were first fully anaesthetized by 5% isoflurane inhalation. Intracardiac perfusion was then performed with 25–50 ml of ice-cold cutting artificial cerebrospinal fluid (ACSF; 0.5 mM calcium chloride (dehydrate), 25 mM d-glucose, 20 mM HEPES buffer, 10 mM magnesium sulfate, 1.25 mM sodium phosphate monobasic monohydrate, 3 mM myo-inositol, 12 mM N-acetyl-l-cysteine, 96 mM N-methyl-d-glucamine chloride, 2.5 mM potassium chloride, 25 mM sodium bicarbonate, 5 mM sodium l-ascorbate, 3 mM sodium pyruvate, 0.01 mM taurine and 2 mM thiourea (pH 7.3), which had been continuously bubbling with a mixture of 95% O 2 –5% CO 2 ). Sections (350 µm) were sliced on a vibrating microtome (Compresstome VF-300 vibrating microtome, Precisionary Instruments or VT1200S Vibratome, Leica Biosystems), either coronally or at a 17° angle from the coronal plane. For the VIS, this latter slice angle helps to maximize the integrity of neuronal processes. To optimize registration to the CCFv3, a block-face image was collected before each section was cut (Mako G125B PoE camera with custom integrated software). Immediately after slicing, brain slices were placed in warm (34 °C) oxygenated cutting ACSF for 10 min, then allowed to further recover in holding ACSF (2 mM calcium chloride (dehydrate), 25 mM d-glucose, 20 mM HEPES buffer, 2 mM magnesium sulfate, 1.25 mM sodium phosphate monobasic monohydrate, 3 mM myo-inositol, 12.3 mM N-acetyl-l-cysteine, 84 mM sodium chloride, 2.5 mM potassium chloride, 25 mM sodium bicarbonate, 5 mM sodium l-ascorbate, 3 mM sodium pyruvate, 0.01 mM taurine and 2 mM thiourea (pH 7.3)), bubbling with a mixture of 95% O 2 –5% CO 2 at room temperature until transferred to the microscope for recordings.
Patch-clamp recording
Slices were bathed in warm (34 °C) recording ACSF (2 mM calcium chloride (dehydrate), 12.5 mM d-glucose, 1 mM magnesium sulfate, 1.25 mM sodium phosphate monobasic monohydrate, 2.5 mM potassium chloride, 26 mM sodium bicarbonate and 126 mM sodium chloride (pH 7.3) and continuously bubbled with 95% O 2 –5% CO 2 . The bath solution contained blockers of fast glutamatergic (1 mM kynurenic acid) and GABAergic synaptic transmission (0.1 mM picrotoxin). Thick-walled borosilicate glass (G150F-3, Warner Instruments) electrodes were manufactured (Narishige PC-10) with a resistance of 4–5 MΩ. Before recording, the electrodes were filled with approximately 1.0–1.5 µl of internal solution with biocytin (110 mM potassium gluconate, 10.0 mM HEPES, 0.2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid, 4 mM potassium chloride, 0.3 mM guanosine 5′-triphosphate sodium salt hydrate, 10 mM phosphocreatine disodium salt hydrate, 1 mM adenosine 5′-triphosphate magnesium salt, 20 µg ml−1 glycogen, 0.5 U µl−1 RNAse inhibitor (2313A, Takara) and 0.5% biocytin (B4261, Sigma), pH 7.3). The pipette was mounted on a Multiclamp 700B amplifier headstage (Molecular Devices) fixed to a micromanipulator (PatchStar, Scientifica).
Electrophysiology signals were recorded using an ITC-18 Data Acquisition Interface (HEKA). Commands were generated, signals processed and amplifier metadata were acquired using MIES (https://github.com/AllenInstitute/MIES/), written in Igor Pro (Wavemetrics). Data were filtered (Bessel) at 10 kHz and digitized at 50 kHz. Data were reported uncorrected for the measured liquid junction potential of −14 mV between the electrode and bath solutions. Before data collection, all surfaces, equipment and materials were thoroughly cleaned in the following manner: a wipe down with DNA away (Thermo Scientific), RNAse Zap (Sigma-Aldrich) and finally with nuclease-free water.
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