250µm Coronal Slices of Somatosensory ‘barrel’ Cortex Prepared using 7000smz

Abstract

(from Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior, Magno et al, 2021).

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Cortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers—due to a defect in precursor cell proliferation during embryogenesis—extra parval-bumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network matura-tion. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

Methods

Brain Slice Preparation

The brain was removed and submerged in ice-cold slicing solution containing125 mM NaCl, 2.5 mM KCl, 2.5 mM MgCl2, 1.25 mM NaH2PO4, 0.5 mM CaCl2,25mMD-glucose, and 26 mM NaHCO3, saturated with 95% O2and 5% CO2, pH 7.4. In some cases, CaCl2 was reduced to 0.5 mM, and MgCl2increased to 4 mM. Coronal slices(250mm thick) containing somatosensory ‘barrel’ cortex were prepared using a vibratome (Leica VT12000S or Campden 7000smz). Before recording, slices were allowed to recover in recording solution (as above, but with 2 mM CaCl2 and 1 mM MgCl2) for 30 minutes at 32–34C and thereafter at room temperature for 30 minutes. To prepare slices from older (P60) ‘early cKO’ and WT littermate mice, the same methods were used but the slicing solution contained 120 mM K-gluconate, 15 mM KCl, 20 mM HEPES, 25 mM D-glucose, 4 mM Na-pyruvate, 0.05 mM EGTA and 10 mM Na-ascorbate saturated with 100% O2, pH adjusted to 7.4 with KOH. Individual slices were transferred to a submerged chamber on the stage of an upright microscope (Scientifica SliceScope or Olympus BX51WI) and perfused with recording solution at 2 ml/min. Pyramidal cells of the S1 barrel field were visualized using 40x or 60x water immersion objectives.