W. M. Cowan

A study of subcortical afferents to the hippocampal formation in the rat

Abstract The distribution of neurons in diencephalon and brainstem which project upon the hippocampal formation has been analyzed in adult rats by the injection of horseradish peroxidase into different parts of the hippocampus and dentate gyrus and the related retrohippocampal structures, including the subicular complex and the entorhinal cortex. Any large injection of horseradish peroxidase inlo the hippocampal region results in the retrograde labeling of some neurons in each of the following structures: in the thalamus—the nucleus reuniens, the parataenial and paraventricular nuclei, the anterodorsal and antermedial nuclei, and the laterodorsal and lateral posterior nuclei; in the hypothalamus, septum and preoptic region—the medial septal nucleus and the diagonal band of Broca, the substantia innominata, the lateral preoptic area, the magnocellular preoptic nucleus, and the anterior amygdaloid area, the dorsomedial hypothalamic nucleus, the lateral and posterior hypothalamic areas, the ventral premammillary nucleus, the supramammillary region, and parts of the tuberomammillary and lateral and medial mammillary nuclei: in the brainstem: the ventral tegmental area, the substantia nigra, the interpeduncular and interfascicular nuclei, the dorsal and median nuclei of the raphe, the dorsal and laterodorsal tegmental nuclei, the locus coeruleus, the central gray, and certain of the tegmental reticular fields. It is clear that these afferents to the hippocampal formation do not comprise a single, homogeneous system, and that their terminations within the hippocampal region are not restricted to a particular topographic level (i.e. septal, intermediate or temporal).

The cellular localization of the two brain specific proteins, S-100 and 14-3-2.

Abstract An attempt has been made to localize, at a cellular level, the two brain specific proteins, S-100 and 14-3-2, by determining their relative concentrations in thalamic nuclei undergoing retrograde cell degeneration after appropriate lesions of the cerebral cortex. As the neuronal degeneration progresses the concentration of the 14-3-2 protein declines so that 6 weeks postoperatively it is only approximately 40% of its control level. On the other hand, there is a slight, but statistically significant, increase in the level of the S-100 protein between the 1st and 4th postoperative weeks which appears to be correlated with the early gliosis in the affected nuclei. On this basis it is suggested that 14-3-2 is primarily a neuronal protein and that the S-100 protein is largely, if not exclusively, confined to glial cells.

The time of origin of neurons in Ammon's horn and the associated retrohippocampal fields

SummaryThe time of origin of the neurons in the hilus of the dentate gyrus, in the regio superior and regio inferior of Ammons horn, and in the following retrohippocampal fields — the subiculum, presubiculum, parasubiculum, medial and lateral entorhinal areas and the perirhinal cortex, has been determined in the rat, by the technique of 3H-thymidine autoradiography. In each field the cells are generated over a limited period of about five days, with the majority being formed in a 24–48 h period. As in the neocortex, the cells in the various hippocampal fields are generated in a distinct “inside-out” sequence with respect to the ependymal zone in which they arise. In addition there are two distinct gradients along the transverse, or dentato-rhinal axis, of the formation. Thus the neurons in the hilar region of the dentate gyrus (including field CA4) tend to arise earlier than the pyramidal cells in the regio inferior, and these in turn, are on average, generated earlier than those in the regio superior or in the subiculum. In the retrohippocampal region there is a comparable gradient extending medially from the perirhinal cortex to the presubiculum, the cells in the lateral entorhinal area being formed, on average, earlier than those in the medial entorhinal cortex, and these, in turn, are generated earlier than those in the para- or presubiculum. There is no evidence for a dorso-ventral (or septotemporal gradient) in any part of the hippocampal formation like that found in the dentate gyrus. Cell counts indicate that there are over 320,000 pyramidal cells in the regio superior (field CA1) and about 150,000 in the regio inferior (fields CA2 and CA3).

Studies on the development of the chick optic tectum. IV. An autoradiographic study of the development of retino-tectal connections

The normal pattern of innervation of the optic tectum has been studied autoradiographically in a series of chick embryos which were injected intraocularly with [3-H]proline at intervals between the 6th and 21st days of incubation. From the distribution of the radioactively labeled proteins transported in the rapid phase of axonal flow, it is evident that retinal fibers first enter the tectum late on the 6th day of incubation and then spread across its surface from its rostrolateral aspect to its caudo-dorso-medial pole during the ensuing 6 days. At the 9th day, when the fibers have grown across the surface of the rostral half of the tectum, there is no indication that the terminal portions of the axons have left the stratum opticum to enter the outer layers of the stratum grieseum et fibrosum superficiale. The first suggestion of such an invasion of the stratum grieseum et fibrosum superficiale is found at day 10, when labeled fibers can be seen in this stratum, over a restricted, oval, area near the center of the tectum. Over the course of the next 2 days the region of the stratum grieseum et fibrosum superficiale occupied by retinal fibers expands, more-or-less concentrically, until by day 14 some labeled fibers are seen in the outer part of the stratum, throughout the tectum. Concurrent with the ingrowth of optic nerve fibers the final cytoarchitectonic differentiation of the outer layers of the tectum occurs so that by day 18 the autoradiographs show a pattern of labeling of layers a-f of the stratum griseum et fibrosum superficiale similar to that seen in mature, post-hatched chicks. Since the region of the tectum which is first innervated by retinal fibers corresponds to the projection field of the region around the upper end of the choroid fissure where the first ganglion cells are generated, it would appear that the axons of the first-formed ganglion cells grow over the rostral surface of the tectum before establishing the first retino-tectal synapses near the central portion of the tectum. Subsequently, as the ganglion cell population grows concentrically from around the area centralis, there is a parallel expansion of the region in which retino-tectal synapses are being formed in the tectum.

Evidence for an input to the molecular layer and the stratum granulosum of the dentate gyrus from the supramammillary region of the hypothalamus.

SummaryInjections of a mixture of tritiated amino acids were made into the posterior hypothalamus in a series of rats and cats. In every case in which the injection involved a significant proportion of the cells in the supramammillary region, labeled fibers could be followed to the dentate gyrus, the anterior hippocampal rudiment and the induseum griseum of both sides. In the dentate gyrus the hypothalamic afferents terminate in a narrow band in the outer half of the stratum granulosum and the inner 20 μm or so, of the stratum moleculare, immediately deep to the zone of termination of the associational and commissural afferents. As judged by silver grain counts across the width of the zone of labeled terminals, the projection to the ipsilateral side is several times as heavy as that to the contralateral side, and although it involves the entire septo-temporal (= rostro-caudal) extent of the gyrus on both sides, the projection to the suprapyramidal (inner) blade of the dentate gyrus is approximately twice as heavy as that to the infrapyramidal (outer) blade.

Changes in the concentrations of the two brain specific proteins, s-100 and 14-3-2, during the development of the avian optic tectum.

Summary The concentrations of the two brain specific proteins, 14-3-2 which is predominantly neuronal, and S-100 which is glial, have been determined in the optic tectum of a series of chick embryos between the 3rd day of incubation and the 9th week post-hatching. Both proteins first appear during the 4th day of incubation at about the time the first differentiated cells can be recognized morphologically. The level of the two proteins shows little increase during the ensuing 8 days at which time cell proliferation ceases, but between about the 17th day of incubation and the 2nd week after hatching the concentration of both proteins increases more-or-less exponentially. Thereafter the rate of increase declines rapidly, but there is a slow continuing increase for some weeks until adult levels are reached. Although the changes in concentration of the two proteins show a close parallelism, the concentration of 14-3-2 is approximately 100 times greater than that of the S-100 protein at each stage in development.

Observations on the transport of radioactively labeled proteins in the visual system of the chick

Abstract The transport of proteins synthesized in the somata of retinal ganglion cells of 3–5-week-old chicks has been studied autoradiographically and by liquid scintillation counting following the intravitreal injection of [3H]leucine or [3H]proline. Most of the transported proteins move with a velocity of 1.5–2 mm/day and are distributed throughout the extent of the optic nerve and to the various diencephalic and mesencephalic structures to which it sends fibers. A relatively small proportion of the transported material has a rate of approximately 200 mm/day and is preferentially (but not exclusively) distributed to the region of the axon terminals. The differential distribution of the two components is readily demonstrated by grain counts in autoradiographs of the optic tectum: the rapid phase clearly labels the outer laminae of the stratum griseum et fibrosum superficiale in which the retinal fibers terminate very heavily, whereas the arrival of the slow phase results in an increasing grain density over the optic nerve fibers in the stratum opticum. We have seen no evidence of a specific transsynaptic movement of label in this system, nor do the terminals of the centrifugal fibers to the retina appear to be able to incorporate significant amounts of label for retrograde transport to the isthmo-optic nucleus. Preliminary observations indicate that the autoradiographic method for tracing connections can be used successfully to map the developing retino-tectal projection.

The organization of the fimbria, dorsal fornix and ventral hippocampal commissure in the rat

SummaryThe morphological organization of the two principal efferent pathways of the hippocampal formation — the fimbria and the dorsal fornix — and of the interhippocampal fibers that cross in the ventral hippocampal commissure, has been studied autoradiographically following injections of 3H-amino acids into the relevant cytoarchitectonic fields. To a considerable extent the rostrally-directed fibers from each cytoarchitectonic field remain topographically segregated within the fimbria or dorsal fornix, their locations being determined by the relative levels at which they join these bundles. Thus, the fibers from the subicular region tend to be aggregated along the ependymal border of the fimbria while those from the hilar region of the dentate gyrus are medially-placed along its pial margin. Similarly, the efferents from the septal and temporal parts of the various fields are distributed along the proximo-distal dimension of the fimbria, from its base (adjacent to field CA3) to its tip. The dorsal fornix contains fibers from the septal (dorsal) part of the subiculum and field CA1; although there appears to be some overlap, the subicular efferents tend to be more medially placed while those from CA1 are further lateral. The topographic organization of the fibers in the fimbria is reflected in the arrangement of the crossed components in the hippocampal commissure. Those arising at more temporal levels cross most rostrally while those from the septal pole of field CA3 cross in the caudal part of the commissure; those from the hilar region of the dentate gyrus cross most ventrally, while those from the more lateral parts of field CA3 (CA3b and CA3a) cross at successively more dorsal levels.We estimate that the fimbria contains about 900,000 fibers of which approximately 700,000 are myelinated (with internal diameters ranging between 0.1 and 2.8μm; mean 0.78 μm) and just over 200,000 are unmyelinated (with diameters between 0.1 and 0.8 μm; mean 0.24 μm).

The development of the retino-tectal projection in Rana pipiens.

Abstract The development of the retino-tectal projection in Rana pipiens has been studied by the intraocular injection of small amounts of [ 3 H]proline at late embryonic and at several larval stages. After survival periods varying from 1–24 hr the distribution of the radioactively labeled proteins in the axons of the retinal ganglion cells was studied autoradiographically. It is evident from the appearance of labeled proteins in the optic nerve and chiasm at late embryonic and early larval stages that there is a rapid phase of axonal transport at these stages and that some fraction of the materials transported in this phase are distributed to the tips of the growing axons. The first retinal fibers reach the contralateral optic tectum at embryonic Stage 22 ; at this stage they are confined to the rostrolateral portion of the tectum where the first tectal neurons are generated. At successively later stages the fibers appear to grow across the surface of the tectum in a general rostrolateral to caudomedial direction, reaching the dorsal part of the mid-tectum at larval Stage II and the lateral part of its caudal third by Stage V . However, it is not until relatively late larval stages ( XVIII ) that the fibers reach the caudomedial region of the tectum, and it is only at the time of metamorphosis ( Stage XXV ) that the retinal projection appears to cover the entire tectum.

An automated system for counting silver grains in autoradiographs

Abstract A system, incorporating a microscope, a television camera, and a small digital computer, has been developed for automated counting of silver grains in autoradiographs. The system has been designed specifically for analysis of experiments using the autoradiographic method of demonstrating axonal connections, although it should be adaptable to other uses. The system is capable of counting grains over stained sections up to a density of 300–500 grains/1000 sq. μm, depending on the grain size. Throughout most of this range, the accuracy of the counts is better than ± 10%. The counting algorithm is based upon the direct recognition of individual grains rather than on photometric measurements. This makes the counts relatively independent of grain size or shape, and permits accurate counts even though the grains occupy a layer of the emulsion thicker than the depth of focus of the objective. This is achieved by collating the counts obtained at different planes of focus on a grain-to-grain basis. Several automatic features, notably automatic focusing and stage movement, permit the counting of large areas of a section with minimal operator intervention. The system also includes features for the convenient identification of morphological landmarks in the section, and their correlation with the count data. There are several options available to the user which influence the accuracy and spatial resolutions of the output data and conversely the time required to count a large area. Because of these options the time required to count a 1 sq. mm area can vary from 2 to 8 h.