The effect of inclusion criteria on the functional properties reported in mouse visual cortex

Neurophysiology studies require the use of inclusion criteria to identify neurons responsive to the experimental stimuli. Five recent studies used calcium imaging to measure the preferred tuning properties of layer 2/3 pyramidal neurons in mouse visual areas. These five studies employed different inclusion criteria and report different, sometimes conflicting results. Here, we examine how different inclusion criteria can impact reported tuning properties, modifying inclusion criteria to select different sub-populations from the same dataset of almost 10,000 layer 2/3 neurons from the Allen Brain Observatory. The choice of inclusion criteria greatly affected the mean tuning properties of the resulting sub-populations; indeed, the differences in mean tuning due to inclusion criteria were often of comparable magnitude to the differences between studies. In particular, the mean preferred temporal frequencies of visual areas changed markedly with inclusion criteria, such that the rank ordering of visual areas based on their temporal frequency preferences changed with the percentage of neurons included. It has been suggested that differences in temporal frequency tuning support a hierarchy of mouse visual areas. These results demonstrate that our understanding of the functional organization of the mouse visual cortex obtained from previous experiments critically depends on the inclusion criteria used.