Studies were undertaken to determine whether neurons of the subplate layer represent a transient or stable population of cells in developing neocortex of rat. The first set of studies sought to determine the fraction of subplate neurons that is lost during early postnatal development. The optical dissector method was used to analyze fluorescently stained material in animals the age of postnatal day 0 (P0) to P40. These results demonstrate a reduction of slightly less than half of the total number of subplate neurons from P0 to P40. Counts of labeled cells in littermates at varied ages after [(3)H]thymidine or BRDU treatment on gestational day 14 (G14 - birthdate of occipital subplate neurons) or G18 (birthdate of layers III-IV neurons) demonstrate loss of approximately 50% of neurons in the subplate layer between P0 and P40, somewhat greater than the loss of neurons from cortical layers III-IV. The second set of studies investigated whether subplate neurons display cellular atrophy during postnatal development. Analysis of subplate neurons injected intracellularly with Lucifer yellow in fixed slice preparations indicates no reduction in soma size, number of dendrites, or extent of dendritic fields of subplate neurons taken from animals age P0 to P60. The third set of studies investigated whether functional markers of subplate neurons are reduced during postnatal development. Analysis of tissue stained histochemically for cytochrome oxidase or acetylcholinesterase, or stained immunocytochemically for GABA, somatostatin, or neuropeptide Y, demonstrate a remarkable loss of expression of staining patterns from late gestational ages to P20. These data demonstrate that, although subplate neurons seem not to be a transient population of cells in the usual sense of being eliminated by cell death or structural atrophy, the loss of histochemical and immunocytochemical markers indicates that they may be a functionally transient population of cells.

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In order to examine the effects of activity on spine production and/or maintenance in the cerebral cortex, we have compared the number of dendritic spines on pyramidal neurons in slices of P0 mouse somatosensory cortex maintained in organotypic slice cultures under conditions that altered basal levels of spontaneous electrical activity. Cultures chronically exposed to 100 microM picrotoxin (PTX) for 14 days exhibited significantly elevated levels of electrical activity when compared to neurons in control cultures. Pyramidal neurons raised in the presence of PTX showed significantly higher densities of dendritic spines on primary apical, secondary apical, and secondary basal dendrites when compared to control cultures. The PTX-induced increase in spine density was dose dependent and appeared to saturate at 100 microM. Cultures exhibiting little or no spontaneous activity, as a result of growth in a combination of PTX and tetrodotoxin (TTX), showed significantly fewer dendritic spines compared to cultures maintained in PTX alone. These results demonstrate that the density of spines on layers V and VI pyramidal neurons can be modulated by growth conditions that alter the levels of spontaneous electrical activity.

To examine the contribution of local versus extrinsic influences on postnatal development of cortical neurons, we compared the maturation of deep (infragranular) layer neurons in isolated slices of neocortex grown in organotypic culture to a similar population of neurons developing in vivo. All slice cultures were prepared from sensorimotor cortices of newborn mice (P0) and neurons in these cultures were examined at daily intervals during the first 9 days in vitro (DIV). The maturational state of neurons developing in vivo over this same time period was assessed in acute slices prepared from animals of equivalent postnatal age, P1-P9. Electrophysiological recordings were obtained from neurons in both cultured and acute slices, using Lucifer yellow filled whole-cell recording electrodes, enabling subsequent morphometric analysis of the labeled cells. We report significant changes in both cellular morphology and electrical membrane properties of these deep layer cortical neurons during the first week in culture. Morphological maturation over this time period was characterized by a two- to three-fold increase in cell body size and total process length, and an increase in dendritic complexity. In this same population of cells a three-fold decrease in input resistance and changes in the action potential waveform, including a two-fold decrease in the AP duration, also occur. The degree of morphological and electrophysiological differentiation of individual neurons was highly correlated across developmental ages, suggesting that the maturational state of a cell is reflected in both cellular morphology and intrinsic membrane properties. A remarkably similar pattern of neuronal maturation was observed in neurons in layers V, VI/SP examined in acute slices prepared from animals between P1-P9. Because our culture system preserves many aspects of the local cortical environment while eliminating normal extrinsic influences (including thalamic, brainstem, and callosal connections), our findings argue that this early phase of neuronal differentiation, including the rate and extent of dendritic growth and development of AP waveform, results from instructive and/or permissive local influences, and appears to proceed independently of the many normally present extrinsic factors.

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