Regressive events in neurogenesis.

The development of most regions of the vertebrate nervous system includes a distinct phase of neuronal degeneration during which a substantial proportion of the neurons initially generated die. This degeneration primarily adjusts the magnitude of each neuronal population to the size or functional needs of its projection field, but in the process it seems also to eliminate many neurons whose axons have grown to either the wrong target or an inappropriate region within the target area. In addition, many connections that are initially formed are later eliminated without the death of the parent cell. In most cases such process elimination results in the removal of terminal axonal branches and hence serves as a mechanism to "fine-tune" neuronal wiring. However, there are now also several examples of the large-scale elimination of early-formed pathways as a result of the selective degeneration of long axon collaterals. Thus, far from being relatively minor aspects of neural development, these regressive phenomena are now recognized as playing a major role in determining the form of the mature nervous system.

The corticospinal tract attains a normal configuration in the absence of myelin: observations in jimpy mutant mice.

The corticospinal projection was examined in dysmyelinated, jimpy mice and in unaffected littermates following cortical injections of either wheat germ agglutinin conjugated to horseradish peroxidase or biocytin. Corticospinal axons in both phenotypes traverse the medulla within a well-defined pyramidal tract, decussate within several fascicles at the spinomedullary junction, and extend down the spinal cord in a compact bundle in the ventral-most part of the dorsal funiculus. Very few labeled fibers are seen separated from the main bundle. This normal configuration of the corticospinal tract is attained despite the virtual absence of CNS myelin in jimpy mice. It seems unlikely then that the myelin normally present in fiber bundles adjacent to this relatively late emerging projection can significantly influence pathway selection during its development.

The morphology of the hippocampus and dentate gyrus in normal and reeler mice.

The morphology of the hippocampus and dentate gyrus in normal and reeler mice has been studied in Nissl, myelin, Golgi, Timm's sulfide silver and gold chloride-sublimate preparations. It is evident from both cell-and fiber-stained sections that despite the obvious defect in the positioning of the hippocampal pyramidal and dentate granule cells in the reeler mouse within the radial dimension, the hippocampal formation as a whole shows a normal and consistent progression of cytoarchitectonic fields along its transverse axis, and a normal and consistent progression of changes in the structure of the hippocampus and dentate gyrus along their longitudinal axes. Thus, at least in these structures, the reeler gene seems to exert its effect only in the radial dimension. Cell counts in the area dentata indicate that the number of dentate granule cells in the reeler mouse is reduced compared to that found in normal or heterozygous animals. Although it has been known for some time that the number of granule cells in the reeler cerebellar cortex is markedly reduced, this appears to be the first evidence for a reduction in cell number in a forebrain structure. All the major cell types normally found in the hippocampus and the dentate gyrus are recognizable in Golgi-stained preparations from the brains of reeler mutants. However, in both regions there are a number of abnormalities in the appearance of the cells which seem to be related to the cellular ectopia. Thus, whereas most of the pyramidal and granule cells which attain a normal position in the mutant usually have normal, or near-normal dendritic arbors, the dendrites of nearly all ectopic cells are severely distorted, both in their orientation and general configuration. In preparations stained by the Timm's sulfide silver technique it is evident that the general lamination pattern seen in normal mice is retained in the reeler hippocampus and dentate gyrus despite the gross malpositioning of many of the relevant neurons. However, although the overall laminar arrangement is preserved, there are some fairly consistent abnormalities; for example, the normal trilaminar staining pattern seen in the stratum moleculare of the dentate gyrus is replaced in the reeler by a bilaminar pattern. In gold chloride-sublimate impregnated preparations there is no obvious alignment of the astrocytes in the stratum moleculare of the dentate gyrus in either normal or reeler mice. Moreover, the distribution of the astrocytes within this zone is fairly normal in the reeler mouse, although, in general, these cells appear to be more consistently stellate in form than in normal animals.

The development of the hippocampus and dentate gyrus in normal and reeler mice.

The histogenesis, the time of origin and the pattern of migration of the cells in the hippocampus and dentate gyrus, have been studied in normal and reeler mice. The earliest indication of a defect in the reeler hippocampus is seen on the fifteenth embryonic day (E15) which is at least 24 hours after the first indication of a defect in the neocortex. It is not until E18, that the dentate gyrus shows signs of its incipient abnormality. It appears then, that in both the hippocampus and the dentate gyrus the gene defect first manifests itself at the stage at which the definitive cellular layers are assembled. Experiments involving the injection of 3H-thymidine (3H-TdR) at different developmental stages have confirmed that the site and rate of cellular proliferation in the reeler hippocampus and dentate gyrus are normal, as is the initial pattern of cell migration. However, in the reeler dentate gyrus, most postnatal cell proliferation occurs ectopically and in the hippocampus the normal "inside-out" sequence of neurogenesis is reversed, the earliest pyramidal cells generated coming to lie superficially within the stratum pyramidale and the later formed cells being added at progressively deeper levels. There is no discernible gradient in the time of origin of the granule cells in the radial dimension of the reeler dentate gyrus, whereas there is an obvious "outside-in" gradient in the normal animal. The characteristic gradients in cell proliferation seen in the transverse and longitudinal dimensions of the normal dentate gyrus are, however, also evident in the reeler mouse. Taken together, these observations suggest that the reeler gene exerts its effect on neuronal position only in the radial dimension, and does so at a stage of development subsequent to the proliferation and initial migration of the relevant neurons. Timm's sulfide silver preparations indicate that the characteristic staining patterns seen in the dentate gyrus and hippocampus appear at the same time, and mature at the same rate in normal and reeler mice.

The transient corticospinal projection from the occipital cortex during the postnatal development of the rat.

The transient occipital cortical component of the pyramidal tract which we previously had identified during the postnatal development of the rat (Stanfield et al., '82) has been studied with anterograde as well as retrograde techniques. A continuous band of retrogradely labeled layer V neurons which spans the entire cortex including the occipital cortex is seen following injections of the fluorescent marker Fast Blue into the pyramidal decussation during the first postnatal week. No labeled cells are found in the occipital cortex following similar injections made on postnatal day 20 (P20), although such injections label many neurons in the more rostral cortical fields. However, if the Fast Blue injection is made on P2 and the animal is allowed to survive until P25 a large number of Fast Blue-labeled layer V neurons is found in the occipital cortex, even though an acute, second injection of the retrograde tracer Nuclear Yellow made into the pyramidal decussation shortly before the animal is killed results in no occipital cortical labeling. When Fast Blue injections confined to the mid- or upper-cervical spinal cord are made on P4 and the animals are killed on P9, again many retrogradely labeled neurons are found in the occipital cortex. Further, when injections of 3H-proline or wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) confined to the occipital cortex are made during the first 2 postnatal weeks, anterogradely transported label is seen within the pyramidal tract. At all stages examined the amount of such label and its caudal extent are less than that seen following similar injections into the parietal or frontal cortex. The greatest extent of the labeled occipital cortical fibers is reached at about the end of the first postnatal week and the number of these fibers seems to peak at about this same time. At this stage many of these labeled axons extend for a considerable distance down the spinal cord with some reaching as far caudal as lower lumbar levels, and at this stage some of these labeled occipital corticospinal fibers enter into the spinal gray. Over the next week the number of occipital cortical fibers in the pyramidal tract rapidly decreases and by P17 occipital cortical injections of 3H-proline or WGA-HRP result in virtually no transported label caudal to the pons.(ABSTRACT TRUNCATED AT 400 WORDS)

The organization of certain afferents to the hippocampus and dentate gyrus in normal and reeler mice.

The organization of certain of the major afferents to the hippocampus and dentate gyrus has been studied in normal and reeler mutant mice using the autoradiographic and the anterograde degeneration methods. The distribution of the hippocampal and dentate afferents which arise in the medial and lateral parts of the entorhinal cortex and the hippocampus of both sides, has been found to be generally similar to that previously described in the rat, but there are a few minor differences that are discussed in the text. Despite the marked ectopia of many of the neurons in the hippocampal formation in the reeler mouse, the principal afferents to the hippocampus and the dentate gyrus maintain many of the features seen in normal mice. In particular, they maintain a normal radial sequence and a characteristic laminated and complementary arrangement. However, there are a number of significant differences in their distribution; for example, in the reeler mouse, the entorhinal afferents occupy the entire radial extent of the stratum moleculare of the dentate gyrus, whereas in normal mice they are restricted to the outer four-fifths of this layer. Furthermore, in the mutant the commissural and associational afferents to the dentate gyrus do not occupy the inner one-fifth of the molecular layer (as they do in normal animals) but rather are spread throughout the zone containing granule cells, which includes both the poorly-defined stratum granulosum and most of the hilar region of the dentate gyrus. Some of the developmental and functional implications of these and other abnormalities in the organization of the afferents to the hippocampus and dentate gyrus are discussed.

The distribution of hippocampal and spinal projecting cells in the locus coeruleus of tottering mice.

Wheat germ agglutinin conjugated to horseradish peroxidase and Fast Blue were used as retrograde tracers to examine the distribution of coeruleohippocampal and coeruleospinal somata within the locus coeruleus of normal and tottering mutant mice. The distributions of these projection neuron populations in normal mice are similar to what has been found in other species, and the distributions of these projection neurons in tottering mice are indistinguishable from those in normal mice, in spite of the norepinephrine hyperinnervation of certain locus coeruleus targets, including the hippocampus, in the tottering mutant. These observations lend support to the notion that the defect in tottering acts fairly directly on mechanisms involved in the development of locus coeruleus axonal arbors within certain target regions.

Induction of somatic sensory inputs to the lateral geniculate nucleus in congenitally blind mice and in phenotypically normal mice.

The incidence of aberrant innervation of the lateral geniculate nucleus by ascending somatic sensory axons was examined following injections of wheat germ agglutinin conjugated with horseradish peroxidase into the dorsal column nuclei of adult mice which were: (1) normal; (2) normal, but bilaterally enucleated on the day of birth; (3) normal, but received a large unilateral lesion of the rostral cortex on the day of birth; (4) normal, bilaterally enucleated, as well as unilaterally lesioned in the rostral cortex on the day of birth; (5) homozygous for an ocular retardation mutation (orj/orj); or (6) homozygous for the orj mutation and received a large unilateral lesion of the rostral cerebral cortex on the day of birth. In the phenotypically normal animals which were untreated, no somatic sensory inputs enter into the dorsal lateral geniculate nucleus. A few labeled axons enter into and arborize within the dorsal lateral geniculate nucleus in normal animals which received bilateral enucleations or unilateral rostral cortical lesions on the day of birth. However, in congenitally blind animals and in phenotypically normal animals which received bilateral enucleations as well as unilateral rostral cortical lesions on the day of birth, a significant number of labeled axons enter into and arborize within the dorsal lateral geniculate nucleus. Among all these experimental groups, the densest innervation of the lateral geniculate nucleus occurred in congenitally blind animals which received rostral cortical lesions on the day of birth. In these, robust arborizations of labeled somatic sensory axons occupy a substantial extent of the lateral geniculate nucleus. These results not only demonstrate that ascending somatic sensory axons can be rerouted to the lateral geniculate nucleus, but also indicate that the ability of a thalamic afferent pathway to undergo extensive reorganization and to innervate inappropriate thalamic targets following early perturbations is not unique to the retinal projection (in which this has previously been demonstrated), and may be a more general characteristic of the major thalamic afferent systems.

Excessive intra- and supragranular mossy fibers in the dentate gyrus of tottering (tg/tg) mice.

In Timm's sulfide silver preparations, intragranular and supragranular mossy fiber staining is found to be much more prevalent in the temporal dentate gyrus of the spontaneous epileptic mouse, tottering, than at matching levels in unaffected littermate controls. This aberrant distribution of mossy fibers may be due to the spontaneous seizures affecting this mutant.

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.

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.

Differential sprouting responses in axonal fiber systems in the dentate gyrus following lesions of the perforant path in WLDs mutant mice.

Axons in both peripheral nerves and central fiber pathways undergo very slow Wallerian degeneration in Wlds mutant mice. It has recently been shown that in Wlds mutant mice there is a delay in the intensification of acetylcholinesterase histochemical staining in the molecular layer of the dentate gyrus following lesions of the entorhinal cortex. Thus, it appears that delayed post-lesion reactive sprouting is associated with the delayed degeneration of cut central axons in this mutant. We have studied the time course of changes in the septohippocampal and the hippocampal commissural projections following interruption of perforant path in Wlds mutant mice and in normal (C57BL/6J) mice using the anterograde tracer, wheat germ agglutinin conjugated horseradish peroxidase. In normal mice, changes in the distribution of labeled septal and commissural axons indicative of sprouting are seen in the dentate molecular layer as early as 3 days post-lesion. The earliest survival time at which similar changes are found in Wlds mutant mice is seven days post-lesion, when an increase in the density of labeled septal axons begins in the outer molecular layer. The delay in the sprouting of commissural axons in the mutant is even longer. Changes in the distribution of labeled commissural axons in the dentate gyrus of Wlds mutant mice are first seen 12 days post-lesion. These results confirm that post-lesion reactive axonal sprouting can be delayed in the central nervous system of Wlds mutant mice. In addition, our results indicate that the extent of this delay may differ among axonal fiber systems. These findings are consistent with the notion that various central axonal systems may respond differentially to sprouting cues and are reminiscent of differences found in the regenerating response exhibited by sensory and motor axons in the Wlds mutant after peripheral nerve cuts.

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.

Antibody to a soluble protein purified from brain selectively labels layer V corticofugal projection neurons in rat neocortex.

An antibody to a soluble protein (protein 36) isolated and purified from rat brain labels the cell bodies and processes of pyramidal cells within layer V of the rat neocortex. We have used the fluorescent retrograde axonal tracer, Fast blue, in combination with FITC immunocytochemistry to determine the projection sites of the cortical neurons detected by this antibody. Retrogradely labeled pyramidal tract neurons and corticotectal neurons are labeled with the protein 36 antibody, but the callosally projecting neurons within layer V are not. Thus within the neocortex the antibody to protein 36 may selectively detect a particular class of neuron, the corticofugal projection neurons of layer V.

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)

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.

On the numbers of neurons in 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.

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.

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.

The physiological identification of pyramidal tract neurons within transplants in the rostral cortex taken from the occipital cortex during development.

Axons from neurons in the occipital cortex transiently extend to the pyramidal tract (PT) during the early postnatal development of rats. Normally, these axons are eliminated by the end of the third postnatal week. However, if a portion of fetal occipital cortex is transplanted to the parietofrontal region in newborn hosts then some neurons in the transplant will extend pyramidal tract axons and maintain them. Intracortical microstimulation and electrophysiological recording techniques were used to identify the physiological characteristics of the transplanted pyramidal tract cells and to determine if motor effects could be elicited from the occipital transplant. Microstimulation of the transplant did not reliably evoke movement but the low density and disarray of PT cells within the transplant might account for this. Recording from within the transplant revealed that the overall cell activity was depressed. We were able to identify neurons within the transplant which responded antidromically to stimulation of the pyramidal tract, indicating that their axons have the capacity to conduct impulses and are therefore likely to have developed some viable connections. The functional significance of such projections remains uncertain.