The mouse visual system consists of several visual cortical areas thought to be specialized for different visual features and/or tasks. Previous studies have revealed differences between primary visual cortex (V1) and other higher visual areas, namely, anterolateral (AL) and posteromedial (PM), and their tuning preferences for spatial and temporal frequency. However, these differences have primarily been characterized using methods that are biased toward superficial layers of cortex, such as two‐photon calcium imaging. Fewer studies have investigated cell types in deeper layers of these areas and their tuning preferences. Because superficial versus deep‐layer neurons and different types of deep‐layer neurons are known to have different feedforward and feedback inputs and outputs, comparing the tuning preferences of these groups is important for understanding cortical visual information processing. In this study, we used extracellular electrophysiology and two‐photon calcium imaging targeted toward two different layer 5 cell classes to characterize their tuning properties in V1, AL, and PM. We find that deep‐layer neurons, similar to superficial layer neurons, are also specialized for different spatial and temporal frequencies, with the strongest differences between AL and V1, and AL and PM, but not V1 and PM. However, we note that the deep‐layer neuron populations preferred a larger range of SFs and TFs compared to previous studies. We also find that extratelencephalically projecting layer 5 neurons are more direction selective than intratelencephalically projecting layer 5 neurons.

Retrograde monosynaptic tracing using glycoprotein-deleted rabies virus is an important component of the toolkit for investigation of neural circuit structure and connectivity. It allows for the identification of first-order presynaptic connections to cell populations of interest across both the central and peripheral nervous system, helping to decipher the complex connectivity patterns of neural networks that give rise to brain function. Despite its utility, the efficiency with which genetically modified rabies virus spreads retrogradely across synapses remains uncertain. While past studies have revealed conditions that can increase or decrease the numbers of presynaptic cells labeled, it is unknown what proportion of total inputs to a starter cell of interest are labeled. It is also unknown whether synapses that are more proximal or distal to the cell body are labeled with different efficiencies. Here we use a new rabies virus construct that allows for the simultaneous labeling of pre and postsynaptic specializations to quantify efficiency of spread at the synaptic level in mouse primary visual cortex. We demonstrate that with typical conditions about 40% of first-order presynaptic excitatory inputs are labeled. We show that using matched tracing conditions there is similar efficiency of spread from excitatory or inhibitory starter cell types. Furthermore, we find no difference in the efficiency of labeling of excitatory inputs to postsynaptic sites at different subcellular locations.