Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development

The rat retrosplenial cortex is part of a heavily interconnected limbic circuit, considered to have an important role in spatial memory. Interestingly, the granular retrosplenial cortex has an exceptionally distinct system of dendritic bundles, originating from callosally projecting pyramidal neurons in layer II. These can be detected as early as postnatal day 5; and, although their functional significance remains to be elucidated, the existence of these bundles makes the granular retrosplenial cortex an attractive model system for a wide range of development and functional investigations. Here, we report four results concerning the development of modularity in the granular retrosplenial cortex in rats as investigated by neurochemical markers associated to cortico-cortical and thalamo-cortical connections. Emphasis is placed on zinc, an activity-related substance associated with glutamatergic, non-thalamic terminations. 1) Zinc shows a transient strong expression during early postnatal development, but later than the appearance of the upper layer bundles (at postnatal day 5). By postnatal day 11 to postnatal day 15 staining for zinc achieved its most complex pattern; such that layer I had an elaborate organization both in the tangential and radial dimensions. Three sublaminae were distinguished (layers Ia-c): a superficial, thin tier (Ia) with patchy, moderate staining which periodically intruded into the underlying layer Ib ("funnel" modules), a middle band of variable width and light staining (Ib), and a deep, thin band with heavy and patchy staining (Ic) which, at rostral levels, spread upward into layer Ib (as "dome-like" modules). 2) At postnatal day 15, immunohistochemical methods showed that layers Ia, b zinc-funnels were co-localized with glutamate receptor subunits 2/3, GABA receptor type A alpha1 subunit and the thalamo-cortical marker, vesicular glutamate transporter 2. Layer Ic and the zinc dome-like modules were co-labeled for the cortico-cortical marker, vesicular glutamate transporter 1 and calretinin. 3) The spatial coincidence between zinc funnels in layers Ia, b and vesicular glutamate transporter 2 was further investigated by electron microscopy, which demonstrated co-localization of zinc and vesicular glutamate transporter 2 in synaptic boutons. The unusual co-localization of zinc and thalamo-cortical terminations was confirmed by retrograde transport of zinc to neurones in the anterodorsal thalamic nucleus at postnatal day 9 and postnatal day 13, and can thus be considered a transient zinc expression in thalamo-cortical boutons. This was not observed at postnatal day 28 or later. 4) After postnatal day 18, zinc staining started to fade in all layers. Before postnatal day 21, the heavy staining for zinc in the domes had completely disappeared. Zinc staining in layer Ia and the funnels virtually disappeared after postnatal day 28. A transient expression of zinc is reported in at least one other cortical area (layer IV of barrel cortex from postnatal day 5 to postnatal day 14, maximal at postnatal days 9-11). We conclude that the transient expression of zinc can occur in both limbic and sensory areas, and that down-regulation of zinc in cortical modules might be related to synaptic plasticity and remodeling during development.