Axoclamp 2a

For confirming optical imaging results, single- and multi-unit responses to visual stimulation were recorded extracellularly to define the minimum spatial response field (RF), preferred orientation and direction of motion and ocularity using hand-held stimulation at random locations within the mapped area. To this end, glass electrodes (8–10 μm of tip diameter) were filled with 0.5–1 M NaCl (~5 MΩ). The pipettes were advanced perpendicularly to the cortical surface using a manual micro-drive fitted with a digital depth meter (10 μm accuracy, Sylvac SA, Crissier, Switzerland). Along each electrode track single and multi-units were recorded at several depths (300–2000 µm) from the cortical surface. To stabilize recordings, after advancing the electrode into the cortex, the chamber was filled with 3 % agar and covered with low-melting point paraffin (~43 °C, Merck, Darmstadt, Germany). Signals were amplified by AxoClamp-2A (Axon Instruments, Foster City, Canada) and filtered (0.3–10 kHz). Spike activity was monitored using a window discriminator (World Precision Instruments Inc., Sarasota, FL, USA) coupled to an audio monitor. The recorded units had large minimum discharge field (average: 9.5° ± 6.8° of diagonal length, N = 71 from six animals). In some cases RF had large size (maximum, 51°), especially at depths corresponding to layers 5/6.

Action potentials of iPSC-CMs were obtained with microelectrodes (50–70 MΩ) filled with 2.7 M KCl and 10 mM HEPES and recorded at 35°C using Clampex9.2 software (Axon Instruments, Axoclamp-2A, San Jose, CA, United States). For each iPSC-CM measured, the lowest membrane potential (referred to as diastolic potential), threshold potential, peak spike potential, and action potential duration were averaged from 5 action potentials per cell. Action potential duration (APD₉₀) was defined as the time (ms) from the start of the action potential until membrane potential returned to 10% of its peak height.

To image Ca changes we used a MiCAM ULTIMA (SciMedia) imaging and data-acquisition system, which uses a CMOS (complementary metal-oxide-semiconductor) sensor with 100 × 100 imaging elements, combined with a MVX10 macro-zoom microscope (Olympus). The acquisition rate ranged between 100 and 400 Hz. Exposed cortex was illuminated using epi-illumination with led light (480 nm) and appropriate filters and dichroic mirror. A post hoc filter based on the ECG recordings was used to subtract changes in signal induced by heart beat movements. Patch glass pipettes (5–7 MΩ) were used for single unit recordings from the cerebellar cortex. Recordings were made using an AxoClamp 2A (Axon Instruments, Union City, CA, United States) amplifier and sampled by a National Instruments board at rates of either 10 or 20 kHz (after being low pass filtered at 3 or 10 kHz).

Prior to recording, the slice containing the VMH was transferred into a recording solution containing 127 mM NaCl, 1.8 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM MgSO₄, 26 mM NaHCO₃, 2.5 mM glucose, and 0.005 mg/L phenol red. Patch pipettes were filled with 130 mM K-gluconate, 10 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 0.5 mM EGTA, 2 mM K₂ATP, 0.5 mM NaGTP (pipette resistance 7 Ω). PACAP cells were visualized using a fluorescent microscope (Olympus BX50WI, Japan). Current-clamp recordings were amplified using AXOCLAMP-2A (Axon Instruments, CA, USA) and acquired via a CED 1401 mk1 A/D interface, controlled by Spike2 software (version 6, Cambridge Electronic Design Limited, Cambridge, UK). Data analysis was carried out using Spike2 software and plotted using Prism (version 7, GraphPad Software, La Jolla California USA). Data are represented as mean ± SEM. Firing frequency is normalized against baseline firing frequency.