Connections of layer III pyramidal neurons to corticospinal neurons of layer V and corticothalamic neurons of layer VI in the rat primary motor cortex were examined in brain slices by combining intracellular staining with Golgi-like retrograde labeling of corticofugal neurons. Forty layer III pyramidal neurons stained intracellularly were of the regular-spiking type, showed immunoreactivity for glutaminase, and emitted axon collaterals arborizing locally in layers II/III and/or V. Nine of them were reconstructed for morphologic analysis; 15.2% or 3.8% of varicosities of axon collaterals of the reconstructed neurons were apposed to dendrites of corticospinal or corticothalamic neurons, respectively. By confocal laser scanning and electron microscopy, some of these appositions were revealed to make synapses. These findings suggest that corticospinal neurons receive information from the superficial cortical layers four times more frequently than corticothalamic neurons. The connections were further examined by intracellular recording of excitatory postsynaptic potential (EPSP) that were evoked in layer V and layer VI pyramidal neurons by stimulation of layer II/III. EPSPs evoked in layer V pyramidal neurons showed short and constant onset latencies, suggesting their monosynaptic nature. In contrast, most EPSPs evoked in layer VI pyramidal neurons had long onset latencies, showed double-shock facilitation of onset latency, and were largely suppressed by an N-methyl-D-aspartic acid receptor blocker, suggesting that they were polysynaptic. The results suggest that information from the superficial cortical layers is transferred directly and efficiently to corticospinal neurons in layer V and thereby exerts an important influence on cortical motor output. Corticothalamic neurons are, in contrast, considered relatively independent of, or indirectly related to, information processing of the superficial cortical layers.

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