Comparing neuronal microcircuits across different brain regions, species and individuals can reveal common and divergent principles of network computation. Simultaneous patch-clamp recordings from multiple neurons offer the highest temporal and subthreshold resolution to analyse local synaptic connectivity. However, its establishment is technically complex and the experimental performance is limited by high failure rates, long experimental times and small sample sizes. We introduce an in vitro multipatch setup with an automated pipette pressure and cleaning system facilitating recordings of up to 10 neurons simultaneously and sequential patching of additional neurons. We present hardware and software solutions that increase the usability, speed and data throughput of multipatch experiments which allowed probing of 150 synaptic connections between 17 neurons in one human cortical slice and screening of over 600 connections in tissue from a single patient. This method will facilitate the systematic analysis of microcircuits and allow unprecedented assessment of inter-individual variability.

8Comparing neuronal microcircuits across different brain regions, species and individuals can 9 reveal common and divergent principles of network computation. Simultaneous patch-clamp 10

Computation within cortical microcircuits is determined by functional properties of the neurons and their synaptic interactions. While heterogeneity of inhibitory interneurons is well established, the anatomical, physiological, and molecular differentiation of excitatory pyramidal neurons is not fully resolved. To identify functional subtypes within the pyramidal neuron population, we focused on human layer 2-3 cortex which greatly expanded during evolution. We performed multi-neuron patch-clamp recordings in brain slices from the temporal cortex of 22 epilepsy patients. We characterized the electrophysiological properties of up to 80 pyramidal neurons per patient, enabling us to assess inter- and intra-individual functional variability. Hierarchical clustering of the high-dimensional parameter space yielded functionally distinct clusters of pyramidal neurons which were present across individuals. This may represent a generic organizational principle converging with previously described transcriptomic heterogeneity. We further observed substantial heterogeneity in physiological parameters with intra-individual variability being severalfold larger than inter-individual variability. The phenotypic variability within and across pyramidal neuron subtypes has important implications for the computational capacity of the cortical microcircuit.