Brent B. Stanfield

Evidence that the early postnatal restriction of the cells of origin of the callosal projection is due to the elimination of axonal collaterals rather than to the death of neurons

By using two fluorescent dyes that are retrogradely transported along axons, we have been able to demonstrate that many of the neurons in the parietal region of the rat cerebral cortex that can be labeled from the contralateral hemisphere early in postnatal development, persist well beyond the period when the callosal projection normally becomes restricted. This indicates that the major factor in the progressive restriction of the callosal projection is the withdrawal or degeneration of axon collaterals, rather than the selective death of many of the cells that initially project to the opposite side.

On the numbers of neurons on fields CA1 and CA3 of the hippocampus of Sprague-Dawley and Wistar rats

In a previous study it was found that there are significant differences in the numbers of granule cells in the dentate gyrus of adult Sprague-Dawley and Wistar rats and also that the continued postnatal addition of new cells to the dentate gyrus has quite different consequences in the two strains. We have now extended these observations to the two major cytoarchitectonic fields of the hippocampus (the regio superior or field CA1; and the regio inferior or field CA3). The mean number of pyramidal neurons in field CA1 of 1-month-old Sprague-Dawley rats is 420,000 (+/- 60,000 S.E.), while Wistar rats at the same age have 320,000 (+/- 20,000). The numbers of neurons in field CA3 in the two strains are: 330,000 (+/- 30,000) and 210,000 (+/- 20,000), respectively. Whether these strain differences reflect specific differences in the neural organization of the hippocampal formation in the two strains, or are related to more general differences in total body weight or brain weight, is unknown. Since during the first two days postnatally we estimate that there are between 358,000 and 491,000 cells in field CA1 of Sprague-Dawley rats, it would seem that there is no significant naturally-occurring neuronal death in this hippocampal field. This may be due to the extensive collateral projections of the hippocampal pyramidal neurons.

Occipital cortical neurons with transient pyramidal tract axons extend and maintain collaterals to subcortical but not intracortical targets

During the early postnatal development of the rat large numbers of pyramidal tract neurons are present in layer V of the occipital cortex, but by the end of the third postnatal week the distribution of pyramidal tract neurons becomes restricted to the more rostral cortical areas. This restriction is brought about by selective collateral elimination rather than by cell death. We have found, by using retrogradely transported fluorescent dyes as either short-term or long-term markers, that occipital cortical neurons which had transiently extended pyramidal tract axons maintain subcortical axonal connections to either the superior colliculus or the pons, and, at least in the case of the corticotectal projection, that the maintained collateral is present prior to the elimination of the transient pyramidal tract collateral. Further, it appears that at no time during postnatal development do the occipital pyramidal tract neurons form either callosal or ipsilateral cortico-cortical collaterals. Thus in the early postnatal occipital cortex the neurons which project through the pyramidal tract constitute a population of cells which is separate from neurons which make cortico-cortical connections, but which largely overlaps with the population of corticotectal and corticopontine neurons.

A transient pyramidal tract projection from the visual cortex in the hamster and its removal by selective collateral elimination

During the early postnatal development of the neocortex in rats there is an axonal projection from the occipital cortex (which includes the visual cortex) to the spinal cord which is subsequently completely removed through a process of selective collateral elimination. In order to determine whether a similar phenomenon occurs during the development of the hamster cortex, we have injected the retrogradely transported fluorescent dye Fast Blue (FB) into the pyramidal decussation of hamsters at various ages. In adult hamsters such an injection results in a band of labeled neurons confined to layer V and to about the rostral two-thirds of the neocortex; no labeled cells are seen in the occipital cortex. However, a similar FB injection made during the first postnatal week results after a 4-day survival in a continuous band of FB-labeled layer V neurons spread throughout the tangential extent of the neocortex, including the occipital cortex. A similar continuous band of FB labeled layer V neurons is seen throughout the tangential extent of the neocortex including the occipital region in hamsters injected during the first postnatal week but allowed to survive until the fourth week (i.e., after the restriction of the widespread neonatal pattern has occurred). Injections of the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase made into the occipital cortex, or for comparison, into more rostral cortical regions in hamsters ranging in age from neonates to adults, reveal that the extension of pyramidal tract axons is staggered along the anterioposterior axis of the cortex such that axons originating from the posterior regions lag behind those arising from more rostral areas. The transient occipital projection appears to reach a maximum around the end of the first postnatal week: a large number of labeled occipital axons is seen in the medullary pyramidal tract, and some of these can be followed through the pyramidal decussation and into the dorsal funiculus of the spinal cord. Injections into the occipital cortex on P16 label only a few fibers in the medullary pyramidal tract, and none is labeled in hamsters injected as adults.(ABSTRACT TRUNCATED AT 400 WORDS)

The sprouting of septal afferents to the dentate gyrus after lesions of the entorhinal cortex in adult rats

The projection of the septum to the dentate gyrus has been demonstrated autoradiographically and the pattern of acetylcholinesterase (AChE) staining in the dentate gyrus has been mapped histochemically, in a series of normal young adult rats and in a group of animals in which the entorhinal cortex had been ablated or its efferents to the dentate gyrus interrupted, some weeks earlier. It is clear from this material that the normal disposition of the septal projection to the dentate gyrus differs significantly from the pattern of AChE staining; however, in the denervated region of the molecular layer in the experimental animals there is a marked increase in the density of the septal projection which precisely coincides with the zone of intensification of AChE staining. It follows from this that although the distribution AChE does not accurately reflect the organization of the septo-dentate projection in normal animals, the intensification of AChE staining provides a good indication of the reorganization which occurs in this pathway following entorhinal deafferentation.

Postnatal reorganization of cortical projections: the role of collateral elimination

Recent studies of the development of callosal and pyramidal tract projections suggest that there is a widespread reorganization of cortical projections shortly after birth and that this reorganization is largely brought about by the selective elimination of certain early-formed axonal collaterals. Evidence for this derives mainly from experiments involving retrograde-labeling procedures using horseradish peroxidase (HRP) or fluorescent dyes. These experiments indicate that both callosal and pyramidal tract neurons are more widely distributed in the tangential plane of the cortex during the immediate postnatal period than they are in the adult and that they achieve their adult distribution during the first few weeks of postnatal life. Long-term labeling with certain fluorescent dyes which are not degraded within neurons and apparently are not injurious to the labeled cells make it clear that the restriction of the distribution of these populations of projection neurons is largely, if not exclusively, due to the elimination of certain early-formed axonal collaterals.

The mode of termination of the hypothalamic projection to the dentate gyrus: An EM autoradiographic study

It has been suggested, on electrophysiological grounds, that the projection from the hypothalamus to the dentate gyrus constitutes a long-axon, monosynaptic, inhibitory pathway. To clarify the mode of termination of this projection we have examined in EM autoradiographs the distribution and form of labeled synapses in the rat dentate gyrus following the injection of [3H]proline of high specific activity into the supramammillary region of the hypothalamus. As suggested by previous light microscopic studies the hypothalamo-dentate projection has been found to terminate in a narrow zone that extends from about the superficial half of the layer of granule cell somata to the inner one-fifth of the overlying molecular layer. Within this zone more than 80% of the silver grains observed were associated with vesicle-containing profiles, most of which could be identified as forming Type I, asymmetric synapses upon large dendritic shafts. A smaller number of labeled synapses was found upon granule cell somata or upon the sessile dendritic spines that occur on the proximal parts of the granule cell dendrites. Since all of the labeled synapses showed distinct asymmetric membrane specializations and contained spheroidal vesicles it is difficult to reconcile these morphological findings with the view that the hypothalamic afferents to the dentate granule cells are inhibitory.

Observations on the development of certain ascending inputs to the thalamus in rats. I. Postnatal development.

We have studied the postnatal development of the major ascending afferents to the thalamus in postnatal rats using tetramethylbenzidine histochemistry following wheat germ agglutinin-conjugated horseradish peroxidase injections into either the dorsal column nuclei, the deep cerebellar nuclei, or the inferior colliculus. By the day of birth, the efferents from each of these regions have already entered, and arborized extensively within, their appropriate thalamic relay nuclei. However, the overall distribution of each of these ascending afferent systems differs dramatically from that seen in mature rats. In neonatal rats, a substantial proportion of the ascending axons extend beyond the thalamus and often enter the internal capsule, some bypassing the thalamus altogether. In addition, some of the axons which enter and arborize within the thalamus extend beyond their appropriate terminal field into adjoining thalamic nuclei. Retrograde tracing experiments utilizing Fast blue indicate that the cells of origin of these overshooting axons are distributed similarly to the cells of origin of the definitive thalamic afferents. These early erroneous projections are all subsequently eliminated and the characteristically restricted adult distribution of each afferent system is evident by P30. These results indicate that developmental overgrowths and targeting errors of thalamic afferent fibers are not unique to the visual system (where they have been documented previously), but may be a general feature in the development of these pathways.

An EM autoradiographic study of the hypothalamo-hippocampal projection

Previous studies have shown that in many different mammals there is a small but distinct projection from the supramammillary region in the caudal hypothalamus to the junctional region between the regio superior and regio inferior of the hippocampus. We have analyzed the mode of termination of this hypothalamo-hippocampal projection in the rat by electron microscopic (EM) autoradiography following injections of [3H]proline into the caudal hypothalamus. The projection is confined to the regio inferior where it is centered over the subicular end of field CA3, but also spans the adjoining region, field CA2. In our material the highest densities of labeling have been seen over the deeper part of the pyramidal cell layer and in the adjacent stratum oriens but, in addition, above background levels of labeling have been found superficial to the pyramidal cell layer in the stratum lucidum and the deeper part of the stratum radiatum. Most of the labeled synapses appear to be on the perikarya and primary dendrites of the hippocampal pyramidal cells, but some axo-spinous contacts have also been seen. All the labeled boutons contained clear, spheroidal synaptic vesicles and made asymmetric, Type I, contacts with their targets.

Evidence that selective collateral elimination during postnatal development results in a restriction in the distribution of locus coeruleus neurons which project to the spinal cord in rats

Experiments utilizing retrogradely transported fluorescent tracers in rats reveal that coeruleospinal cells are present throughout the locus coeruleus just after birth, but are confined to its ventral portion by the end of the fourth postnatal week. This change in distribution is not brought about by cell death, since neurons retrogradely labeled through their spinal axon following an injection of tracer shortly after birth are still present in the dorsal locus coeruleus even if the animal is not killed until the end of the fourth postnatal week. Thus the dorsal coeruleospinal neurons in newborn rats do not die but rather lose their spinal collateral.