Connectional specification of regenerating entorhinal projection neuron classes cannot be overridden by altered target availability in postnatal organotypic slice co-culture.

Layer II neurons of the entorhinal cortex project to the dentate gyrus and field CA3 and also send collaterals to the subiculum; layer III neurons project to the subiculum and field CA1, but not to the dentate gyrus; and layer IV neurons project to the perirhinal cortex. We have previously shown that these specific differences between the projections of the layer II and III neurons are maintained and can regenerate in organotypic slice culture. In the present experiments we have confronted Postnatal Day 7 (P7) rat entorhinal cortex with P7 tissue selected from restricted parts of the overall entorhinal projection field. (1) When entorhinal slices were co-cultured with target slices containing only dentate gyrus, extracellular uptake of biotin dextran from crystals placed on the dentate gyrus retrogradely labeled neurons in layer II, and those of layer III were not labeled. (2) When entorhinal slices were co-cultured with target slices that contained only the subiculum and the hippocampal field CA1 (but not dentate gyrus), neurons in both layers II and III of the entorhinal area were labeled. (3) When entorhinal slices were co-cultured with target slices containing the perirhinal area and no hippocampal or dentate tissue, the neurons of entorhinal layer IV were labeled, but (4) when co-cultured with control target slices taken from the rostral parietal neocortex, no entorhinal neurons were labeled. Thus the exclusive relationship of layer II entorhinal neurons to the dentate gyrus has already been established by 1 week of age and is maintained by the regenerating entorhinal axons. Layer III entorhinal neurons cannot be induced to project to the dentate gyrus even when deprived of their own target, and layer IV neurons are specified to project to the perirhinal area and will not project to any part of the hippocampal complex or to the rostral parietal cortex. Thus, deprivation of the normal target tissue and presentation of an incorrect target tissue (even when it is the correct target for one of the other classes of entorhinal neurons) are not sufficient to override the specificity of the entorhinal projection neurons.