Slice preparation
All housing of animals and procedures were approved by the University of
Michigan Institutional Animal Care and Use Committee. Multiple mouse
lines were used in this study, including PV-IRES-Cre (Jackson
Laboratories, 008069), CaMKII-Cre (Jackson Laboratories, 005359), Ai32
(Jackson Laboratories, 024109), Ai14 (Jackson Laboratories, 007914),
PV-IRES-Cre x Ai14 (crossed in house), PV-IRES-Cre x Ai32 (crossed in
house), CaMKII-Cre x Ai32 (crossed in house), and NTSR1-Cre (MMRRC,
030648-UCD). A total of 167 recordings are included in this study from
the following mouse lines: PV-IRES-Cre (55), CaMKII-Cre (15), Ai32 (3),
PV-IRES-Cre x Ai14 (3), PV-IRES-Cre x Ai32 (64), CaMKII-Cre x Ai32 (20),
and NTSR1-Cre (7). Mice of both sexes between the ages of P21-31 and
P62-63 were included in the experiments. No differences in cell type
properties were observed across mouse lines, while both age and sex were
explicitly analyzed in terms of their relationship to cell type
properties (see results).
Mice underwent deep isoflurane anesthesia before decapitation. Brains
were removed within one minute of decapitation and placed in an ice-cold
high-sucrose slicing solution that had been saturated with carbogen gas
for at least 30 minutes prior to use. Coronal slices (300um) were cut
using a Leica 1200 VT vibratome. Slices were allowed to rest in the
slicing solution for about 2 minutes before being placed in a
carbogen-saturated high-magnesium artificial CSF (ACSF) solution to
incubate at body temperature (32°C) for 20 minutes. The entire bubbling
bath was then removed from the heater, allowing the slices to gradually
cool to room temperature. Slices rested an additional 30 minutes at room
temperature before use.
Slices were submerged in a recording chamber with a constant flow of
ACSF containing 126 mM NaCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 3 mM KCl, 10
mM dextrose, 1.20 mM CaCL2, and 1 mM MgSO4. Recordings were done between
29-31°C with an ACSF flow rate of 2 mL per minute. All recordings were
done within 8 hours of slicing to ensure reputable health of the cells.
Patch pipettes with 2-3 um diameter and resistances of 3-6 MΩ were
filled with a potassium gluconate internal solution containing 130 mM
K-gluconate, 2 mM NaCl, 4 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 0.3 mM
GTP-Tris, 14 mM phosphocreatine-Tris, and 4 mM ATP-Mg (pH 7.25,
~290 mOsm).