There exist two major types of striatum-targeting neocortical neurons, specifically, intratelencephalic (IT) neurons and pyramidal-tract (PT) neurons. Regarding their striatal projections, it was once suggested that IT axons are extended whereas PT axons are primarily focal. However, subsequent study with an increased number of well-stained extended axons concluded that such an apparent distinction was spurious due to limited sample size. Recent work using genetically labeled neurons reintroduced the differential spatial extent of the striatal projections of IT and PT neurons through population-level analyses, complemented by observations of single axons. However, quantitative IT vs. PT comparison of a large number of axons remained to be conducted. We analyzed the data of axonal end-points of 161 IT neurons and 33 PT neurons in the MouseLight database (http://ml-neuronbrowser.janelia.org/). The number of axonal end-points in the ipsilateral striatum exhibits roughly monotonically decreasing distributions in both neuron types. Excluding neurons with no ipsilateral end-point, the distributions of the logarithm of the number of ipsilateral end-points are considerably overlapped between IT and PT neurons, although the proportion of neurons having more than 50 ipsilateral end-points is somewhat larger in IT neurons than in PT neurons. Looking at more details, among IT subpopulations in the secondary motor area (MOs), layer 5 neurons and bilateral striatum-targeting layer 2/3 neurons, but not contralateral striatum-non-targeting layer 2/3 neurons, have a larger number of ipsilateral end-points than MOs PT neurons. We also found that IT ipsilateral striatal axonal end-points are on average more widely distributed than PT end-points, especially in the medial-lateral direction. These results indicate that IT and PT striatal axons differ in the frequencies and spatial extent of end-points while there are wide varieties within each neuron type.

In the neocortex, both layer 2/3 and layer 5 contain corticocortical pyramidal cells projecting to other cortices. We previously found that among L5 pyramidal cells of the secondary motor cortex (M2), not only intratelencephalic projection cells but also pyramidal tract cells innervate ipsilateral cortices and that the two subtypes are different in corticocortical projection diversity and axonal laminar distributions. Layer 2/3 houses intratelencephalically projecting pyramidal cells that also innervate multiple ipsilateral and contralateral cortices. However, it remained unclear whether layer 2/3 pyramidal cells can be divided into projection subtypes each with distinct innervation to specific targets. In the present study we show that layer 2 pyramidal cells are organized into subcircuits on the basis of corticocortical projection targets. Layer 2 corticocortical cells of the same projection subtype were monosynaptically connected. Between the contralaterally and ipsilaterally projecting corticocortical cells, the monosynaptic connection was more common from the former to the latter. We also found that ipsilaterally and contralaterally projecting corticocortical cell subtypes differed in their morphological and physiological characteristics. Our results suggest that layer 2 transfers separate outputs from M2 to individual cortices and that its subcircuits are hierarchically organized to form the discrete corticocortical outputs. NEW & NOTEWORTHY Pyramidal cell subtypes and their dependent subcircuits are well characterized in cortical layer 5, but much less is understood for layer 2/3. We demonstrate that in layer 2 of the rat secondary motor cortex, ipsilaterally and contralaterally projecting corticocortical cells are largely segregated. These layer 2 cell subtypes differ in dendrite morphological and intrinsic electrophysiological properties, and form subtype-dependent connections. Our results suggest that layer 2 pyramidal cells form distinct subcircuits to provide discrete corticocortical outputs.

Feedback projections from the secondary motor cortex (M2) to the primary motor and sensory cortices are essential for behavior selection and sensory perception. Intratelencephalic (IT) cells in layer 5 (L5) contribute feedback projections to diverse cortical areas. Here we show that L5 IT cells participating in feedback connections to layer 1 (L1) exhibit distinct projection patterns, genetic profiles, and electrophysiological properties relative to other L5 IT cells. An analysis of the MouseLight database found that L5 IT cells preferentially targeting L1 project broadly to more cortical regions, including the perirhinal and auditory cortices, and innervate a larger volume of striatum than the other L5 IT cells. We found experimentally that in upper L5 (L5a), ER81 (ETV1) was found more often in L1-preferring IT cells, and in IT cells projecting to perirhinal/auditory regions than those projecting to primary motor or somatosensory regions. The perirhinal region-projecting L5a IT cells were synaptically connected to each other and displayed lower input resistance than contra-M2 projecting IT cells including L1-preferring and nonpreferring cells. Our findings suggest that M2-L5a IT L1-preferring cells exhibit stronger ER81 expression and broader cortical/striatal projection fields than do cells that do not preferentially target L1.