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Synaptic Physiology Experimental Methods

The Synaptic Physiology Dataset was generated with a standardized, large-scale approach using in vitro multipatch electrophysiology. This allowed us to explore connectivity among a diverse set of neuronal subclasses. Below is a workflow describing how this dataset was acquired; more detailed information can be found in our publications Seeman, Campagnola et al. 2018 and Campagnola, Seeman et al. 2022. Once data was collected it was processed through our Analysis Workflow.


Transgenic Driver Pvalb-FlpO Sst-FlpO Vip-FlpO


Layer 4


Layer 5 ET


Layer 5 IT


Layer 6 ET


Cell class targeting

Cre-/FlpO- transgenic breeding drove fluorescent reporter expression in two cell subclasses within the same mouse enabling targeted recording between them. Human excitatory cells were identified by morphology and cortical depth.

More on transgenic mouse lines

Acute cortical slices

Slices from adult (P40-P60) mouse primary visual cortex or human (18-75 years) frontotemporal cortex were prepared and held in artificial cerebrospinal fluid (aCSF) warmed to 32oC. The aCSF typically contained physiological levels (1.3 mM) of calcium.


In each slice, up to eight neurons were recorded simultaneously, allowing for probing of up to 56 potential synaptic connections. Cells were recorded in voltage and current clamp mode at two holding potentials to identify excitatory and inhibitory connections.

Experimental protocol

A variety of stimuli were delivered to each recorded cell in turn to characterize the strength, kinetics, and short-term plasticity of identified synapses. Synaptic stimuli consisted of trains of eight pulses at frequencies ranging from 10 - 200 Hz, followed by four pulses with a variable delay. Additional stimuli were delivered to measure intrinsic cell features.

More on the experimental protocol

Morphological annotation

Recorded cells were filled with biocytin, and slices were fixed and stained. Layer boundaries were identified from DAPI staining. Biocytin-filled cells received an annotated layer as well as general morphologic characterization, including spiny-ness and axon and dendrite length.

More on morphological annotation