GoKawiilAll experimental procedures were conducted according to the Institutional Animal Care and Use Committee (IACUC) at Howard Hughes Medical Institute (HHMI) Janelia. Data analysis and simulations were performed in Python using pytorch and numpy, and figures were made using matplotlib and jupyter-notebooks61,62,63,64,65.
Data acquisition
Animals
All experimental procedures were conducted according to IACUC ethics approval received from the IACUC board at HHMI Janelia Research Campus. We performed 18 recordings in cortex in: (1) 12 mice bred to express jGCaMP8s66 in excitatory neurons: TetO-jGCaMP8s × Camk2a-tTA mice (available as JAX 037717 and JAX 007004); (2) 3 mice bred to express jGCaMP8s66 in the somas of excitatory neurons: riboL1–jGCaMP8s × Slc17a7-Cre (similar to JAX 039267 without IRES; and JAX 037512) and (3) 3 mice bred to express tdTomato in inter-neurons VGAT-CRE × Ai14 (JAX 016962; JAX 007914) with injections of a dual virus Thy1s:TTA (AAV9, 1.64 × 1013 vector genomes ml−1) and TRE3G:RiboL1–jGCaMP8s (AAV9, 2.45 × 1013 vector genomes ml−1) as in ref. 67, see also ref. 68. We also performed eight recordings in hippocampal CA1 in six mice bred to express GCaMP6f in excitatory neurons: Thy1-GCaMP6f GP5.17 mice (JAX 025393)69, as well as in two wild-type C57 mice (JAX 000664) with injections of the same RiboL1-jGCaMP8s virus combination described above. These mice were male and female, and ranged from 2 to 12 months of age. Mice were housed in reverse light cycle, and were pair-housed with their siblings before and after surgery. Holding rooms are set to a temperature of 70 °F ± 2 °F, and humidity of 50% relative humidity ± 20%.
Surgical procedures
Surgeries were performed in adult mice (P35–P125) following procedures outlined in refs. 70,71. In brief, mice were anaesthetized with isoflurane while a craniotomy was performed. Marcaine (no more than 8 mg kg−1) was injected subcutaneously beneath the incision area, and warmed fluids plus 5% dextrose and buprenorphine 0.1 mg kg−1 (systemic analgesic) were administered subcutaneously along with dexamethasone 7 mg kg−1 by the intramuscular route. In the canula implants, the same total dexamethasone dose was administered tapered over 3 days: 4 mg kg−1 on the first day, 2 mg kg−1 on the second day and 1 mg kg−1 on the third day.
For the visual cortical windows (which included the posterior parietal cortex), measurements were taken to determine the bregma–lambda distance and location of a 4-mm circular window over the V1 cortex, as far lateral and caudal as possible without compromising the stability of the implant. For the sensorimotor windows, the craniotomy was centred at −0.75 mm anteroposterior (AP) and 2.2 mm mediolateral (ML) from bregma. A 4- and 5-mm double window was placed into the craniotomy so that the 4-mm window replaced the previously removed bone piece and the 5-mm window lay over the edge of the bone. For the hippocampal windows, the craniotomy was centred at 1.8 mm AP and 2.0 mm ML from bregma. Cortex was aspirated and a 3-mm glass coverslip attached to a stainless-steel was implanted over the dorsal CA1 region. CA1 surgeries were similar to those described in ref. 71.
After surgery, ketoprofen (5 mg kg−1) was administered subcutaneously and the animal allowed to recover on heat. The mice were monitored for pain or distress and ketoprofen 5 mg kg−1 was administered for 2 days following surgery.
Imaging acquisition
We used a custom-built 2p mesoscope72 to record neural activity, and ScanImage73 for data acquisition. We used a custom online Z-correction module (now in ScanImage), to correct for Z and XY drift online during the recording. As described in ref. 70, for the visual area and hippocampal recordings, we used an upgrade of the mesoscope that allowed us to approximately double the number of recorded neurons using temporal multiplexing74.
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