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.

Retrograde transport of sodium selenite and intracellular injection of micro-ruby: a combined method to describe the morphology of zinc-rich neurones.

Zinc is found in synaptic vesicles in a large number of glutamatergic systems. Its involvement in neurotransmission and neurological disorders has been suggested. There are methods for tracing these circuits, but they do not fill the dendritic tree. In this study, extracellular selenite injections in vivo were combined with intracellular injection of fluorochromes in fixed tissue to reveal the morphology of these zinc-rich neurones. Intraperitoneal and intracerebral injections of sodium selenite alone or intracerebral injections of selenite combined with bisbenzimide were made in the visual cortex of the rat in order to locate the somata of zinc-rich neurones. After 24 h of retrograde transport, animals were killed and fluorescent markers were injected intracellularly into fixed slices to show neuronal morphology: (a) Lucifer Yellow (LY) followed by biocytin, (b) LY coupled to biocytin or (c) micro-ruby (MR) (dextranamines bound to rhodamine and biotin). Double-labelled somata (selenite+fluorochrome) were plotted. Details of the dendritic morphology were then revealed by incubation in avidin-biotin complex and development in 3,3'-diaminobenzidine and H(2)O(2). Camera lucida drawings showed that zinc-rich neurones in layers II-III involved in cortico-cortical visual projections were typical pyramidal neurones. This technique is noteworthy for its analysis of the morphology (and connections) of zinc-rich neurones.

Zinc-rich neurones in the rat visual cortex give rise to two laminar segregated systems of connections.

Zinc-rich synaptic boutons in the neocortex arise from the neocortex itself. However, the precise organisation of these circuits is not known. Therefore, the laminar and areal pattern of zinc-rich cortico-cortical connections between visual areas was studied by retrograde tracing using intracerebral injections of sodium selenite. This tracer was injected in supragranular and infragranular layers in various cortical visual areas in order to precipitate zinc in the synaptic boutons, which was retrogradely transported to neuronal somata. Supragranular injections led to retrogradely labelled neurones in layer II-III, ipsilaterally and contralaterally. Neurones often appeared in groups or clusters. Infragranular injections labelled neurones in layers II-III, VI and, to a lesser extent, in layer V, both ipsilaterally and contralaterally. Neurones in layer VI formed a wide continuous band. Concerning the connections between visual (=occipital) areas, injections in occipital area 2, lateral part (Oc2L), rendered the largest number of retrogradely labelled neurones, which were located in occipital area 1 (Oc1), occipital area 2, medial part (Oc2M) and outside the visual cortex. Callosal zinc-rich projections were dense in the homotopic area but sparse in Oc1 and temporal cortex. Injections in Oc1 rendered moderate numbers of labelled neurones in occipital areas, in both hemispheres. Injections in Oc2M labelled moderate numbers of neurones in occipital areas in both hemispheres and in the frontal and cingulate cortices. These results indicate that zinc-rich cortico-cortical connections are organised into two segregated systems arising from either supragranular or infragranular neurones. In addition, in the visual cortex, zinc-rich systems appear to converge on Oc2L. Zinc-rich connections appear as an extensive, highly organised association system.