Patch-clamp recording pipettes (resistance: 4–9 MΩ) were made from borosilicate glass capillaries (1.5 outer diameter × 0.86 inner diameter; Harvard Apparatus) and back-filled with internal solution of the following composition: 120 mM K-gluconate, 10 mM KCl, 10 mM Na2-phosphocreatine, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na-GTP, with pH adjusted to 7.3 and osmolality to 300–305 mOsm. Biocytin (5 mg/ml; Iris Biotech) was added to the internal solution in order to recover cell morphology. Acute slices were moved to the recording setup and visualized using infrared differential interference contrast optics aided by a ×20/1.0 NA water immersion objective (Zeiss Axio Examiner D1, Carl Zeiss). Electrophysiological recordings were performed at 35°C and slices superfused with oxygenated recording ACSF containing 126 mM NaCl, 3 mM KCl, 1.2 mM Na2HPO4, 10 mM D-glucose, 26 mM NaHCO3, 1.5 mM MgCl2, and 1.6 mM CaCl2. LVb in both LEC and MEC was identified through the presence of the densely packed small cells, and LVb neurons labeled with GFP were selected for recording. LVa neurons were selected for recording on the basis of their large soma size and the fact that they are sparsely distributed directly superficial to the smaller neurons of LVb. Gigaohm resistance seals were acquired for all cells before rupturing the membrane to enter whole-cell mode. Pipette capacitance compensation was performed prior to entering whole-cell configuration, and bridge balance adjustments were carried out at the start of current-clamp recordings. Data acquisition was performed by Patchmaster (Heka Elektronik) controlling an EPC 10 Quadro USB amplifier (Heka Elektronik). Acquired data were low-pass Bessel filtered at 15.34 kHz (for whole-cell current-clamp recording) or 4 kHz (for whole-cell voltage-clamp recording) and digitized at 10 kHz. No correction was made for the liquid junction potential (13 mV as measured experimentally). Data were discarded if the resting membrane potential was ≥ −57 mV or/and the series resistance was ≥40 MΩ.

Intrinsic membrane properties were measured from membrane voltage responses to step injections of hyperpolarizing and depolarizing current (1 s duration, −200 pA to 200 pA, 20 pA increments). Acquired data were exported to text file with MATLAB (MathWorks) and were analyzed with Clampfit (Molecular Devices). The following electrophysiological parameters analyzed were defined as follows:

Resting membrane potential (Vrest; mV): membrane potential measured with no current applied (I = 0 mode).

Input resistance (Mohm): resistance measured from Ohm’s law from the peak of voltage responses to hyperpolarizing current injections (−40 pA injection).

Time constant (ms): the time it took the voltage deflection to reach 63% of peak of voltage response at hyperpolarizing current injections (−40 pA injection).

Sag ratio (steady-state/peak): measured from voltage responses to hyperpolarizing current injections with peaks at −90 ± 5 mV, as the ratio between the voltage at steady state and the voltage at the peak.

AP threshold (mV): the membrane potential where the rise of the AP was 20 mV/ms.

AP peak (mV): voltage difference from AP threshold to peak.

AP half-width (ms): duration of the AP at half-amplitude from AP threshold.

AP maximum rate of rise (mV/ms): maximal voltage slope during the upstroke of the AP.

Fast AHP (in mV): the peak of AHP in a time window of 6 ms after the membrane potential reached 0 mV during the repolarization phase of AP.

Medium AHP (mV): the peak of AHP in a time window of 200 ms after fast AHP.

AP frequency after 200 pA inj. (Hz): frequency of APs evoked with +200 pA of 1-s-long current injection.

Adaptation: measured from trains of 10 ± 1 APs as [1 -(Last/First)], where Last and First are, respectively, the frequencies of the last and first interspike interval.

DAP: depolarizing voltage deflection after AP. DAP was defined based on previous studies (Canto and Witter, 2012a; Canto and Witter, 2012b; Hamam et al., 2000; Hamam et al., 2002).

Optogenetic stimulation and patch-clamp data analysis oChIEF+ fibers were photostimulated with a 473 nm laser controlled by a UGA-42 GEO point scanning system (Rapp OptoElectronic) and delivered through a ×20/1.0 NA WI objective (Carl Zeiss Axio Examiner.D1). Laser pulses had a beam diameter of 36 μm and a duration of 1 ms. The tissue was illuminated with individual pulses at a rate of 1 Hz in a 4 × 5 grid pattern. The grid was positioned such as to allow for the light stimulation to cover across layer I to Va. Laser power was fixed to an intensity that evokes inward currents (EPSCs) but not action potentials. The voltage- or current traces from individual stimulation spots were averaged over 4–6 individual sweeps to create an average response for each point in the 4 × 5 grid. The stimulation point that showed the largest voltage response was used for further analysis. Deflections of the average voltage trace exceeding 10 SDs (±10 SDs) of the baseline were classified as synaptic responses. Postsynaptic potentials were calculated as the difference between the peak of the evoked synaptic potential and the baseline potential measured before stimulus onset. The latency of optical activation was defined as the time interval between light onset and the point where the voltage trace exceeded 10% amplitude. Data analysis was performed using MATLAB. Only slices with at least one successful synaptic response to photostimulation were included in the analysis. There were no outliers that were removed from the data.

All data presented in the figures are shown as mean ± standard errors. Prism software was used for data analysis (GraphPad software), and one-way ANOVA with Bonferroni’s multiple comparison test was used to compare the electrophysiological properties and voltage responses between each cell types. To analyze mediolateral gradient in LVb-to-LVa connectivity of MEC, we used Pearson correlation coefficient. A principal component analysis based on the 12 electrophysiological properties (Figure 2—source data 1) was conducted in MATLAB. For this purpose, all variables were normalized to a standard deviation of 1.

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