Slow transport of freely movable cytoskeletal components shown by beading partition of nerve fibers in the cat

To account for the transport in nerve fibers of tubulin and neurofilament proteins in slow component a, the Structural Hypothesis holds that these proteins are assembled into microtubules and neurofilaments in the cell bodies and the cytoskeletal organelles then moved down in the fibers as part of an interconnected matrix at a uniform rate of about 1 mm/day. The Unitary Hypothesis, on the other hand, considers these proteins to be carried down within the fibers as soluble components or as freely movable small polymers or subunits turning over locally in the stationary cytoskeleton. To differentiate between the two hypotheses, cat L7 dorsal roots were taken at times from 7 to 25 days after their L7 dorsal root ganglia were injected with [3H]leucine to assess the labeling of the cytoskeleton by the use of beading and autoradiography. Beading was induced by a mild stretch and after fast-freezing and freeze-substitution of the roots for histological preparation, the beads were seen in the fibers as a series of expanded regions alternating with constrictions. In the constrictions the cytoskeleton was compacted into an area as small as 5% that of the normal axon, with the axoplasmic fluid and displaceable (freely movable) components squeezed from the constrictions into the adjoining expansions. Roots taken after 7 and 14 days, times consistent with slow component a downflow, were assessed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their content of tubulin and neurofilament proteins shown to constitute 40-50% of all the labeled proteins present. In autoradiographs of dorsal roots taken at those times, numerous grains due to radioactivity were located over the non-constricted regions of the fibers. Few or no grains were present over the constrictions after 7 days. The findings are in accord with the labeled tubulins and neurofilament proteins being present in soluble form in the fibers and expressed from the constrictions into the expansions of the beaded fibers. In contrast, a number of fibers in roots taken at 14-20 days after injection showed somewhat higher grain densities over the constrictions, and more so after 25 days, indicating uptake of labeled subunits into the cytoskeletal organelles at later times. The results are consistent with the downflow of tubulin and neurofilament proteins as soluble components which drop off in the axon to turn over locally in their respective cytoskeletal organelles.     

Inhibition of axoplasmic transport in the developing visual system of the rat-II, Quantitative analysis of alterations in transport of tritiated proline or fucose

Developmental alterations in the amount of tritiated proline and fucose incorporated by retinal neurons and transported within axons of the optic nerve to the dorsal lateral geniculate nucleus and superior colliculus were measured at 1, 5, 10 and 15 days postnatal using quantitative autoradiography and liquid scintillation analysis. The amount of axon transport inhibition induced by introacular injections of colchicine (10-4 M-5 X 10-3M) and xylocaine (10-3 M-10-1 M) was also determined by this methodology. Grain counts of retinal autoradiograms obtained from animals of all ages employed in this study demonstrated that [3H] proline is rapidly incorporated by all retinal neurons but becomes increasingly concentrated within the inner nuclear, ganglion cell and optic fiber layers between 2h and 2 days after injection. [3H] fucose is preferentially taken up and concentrated within the plexiform and sensory element layers. Intraocular colchicine administered 24 h prior to isotope injection exhibited no significant effect on incorporation but depressed the amount of activity in the layer of optic fibers. Comparison of the effects of colchicine and xylocaine on axon transport of [3H] proline injected at 3 days of age revealed dose-dependent suppression of transport occurring up to six hr after isotope injection; however, with longer survival periods the effects of xylocaine were no longer significant whereas colchicine maintained suppression of axon transport to 20% of normal for periods of up to 10 days. Additionally, the rate and quantity of 3H] proline transported to the dorsal lateral geniculate nucleus and tectum of 1-15 day old animals was found to be inversely proportional to the age of the animal. Maximally-effective concentrations of colchicine, determined for each age level examined by the previous study to be highest compatible with the viability of the retino-fugal projection, 34 also reduced transport to from 20-40% of normal, being most effective in animals of 1-10 days of age. Autoradiographs of the optic disc demonstrated a colchicine-induced suppression of the amount of label in the exiting optic fibers and similar preparations of the tectum revealed an elimination of activity in the stratum griseum superficialis and stratum opticum which are normally heavily labeled following intraocular [3H] proline or fucose. Following [3H] fucose injection at 10 days of age, isotope appears in the contralateral visual cortex between 3 and 10 days later, indicating a substantial amount of transneuronal transfer of label which appeared to occur in concert with periods of maximal synaptogenesis. Injection of colchicine reduced the amount of label in the cortex to background levels. Concomitantly, autoradiographs of the dorsal lateral geniculate nucleus revealed cytoplasmic labeling of geniculate neurons in control animals but this was eliminated following colchicine injection...     

Radioautographic study of the axonal transport of proteins into the sensory nerve endings of avian mechanoreceptors

The axonal transport of proteins to the nerve endings of Herbst and Grandry sensory receptors has been investigated by electron-microscope radioautography. Soon after the injection of [3H]leucine into the trigeminal ganglia of young ducks, labeled proteins are conveyed along the suborbital sensory nerves to the sensory nerve endings at rates of at least 200-280 mm/day. Most of these rapidly transported proteins accumulate in areas containing vesicles of various kinds and along the axolemmal region. Later, the bulk of labeled proteins migrate along the axons at rates of about 15 mm/day and are distributed mainly to the mitochondria. A small portion of labeled material is transferred to the adjoining modified Schwann and specialized Grandry receptor cells. It is concluded that the transport of proteins from sensory ganglia to sensory nerve endings of mechanoreceptors is conveyed at fast and intermediate rates and is mainly used for the renewal of vesicles, axolemmal constituents and mitochondria.     

Ontogeny of enkephalin-like immunoreactivity in the rat hippocampus

The postnatal development of leucine5-enkephalin-like immunoreactivity within the hippocampal formation of the rat has been analyzed using immunocytochemical techniques. From the day of birth to postnatal day three, no intrinsic hippocampal elements exhibit immunoreactivity although labeled axons are found within the fimbria, within the alveus, and in the vicinity of the angular bundle. On postnatal day 4, a few immunoreactive hippocampal neurons can be seen in stratum radiatum of the region CA3 and by postnatal day 8, within the hilus, strata pyramidale and oriens of regio superior, and the subiculum. There is a dramatic increase in the incidence of immunoreactive perikarya between postnatal days 8 and 10 in all fields as well as the appearance of labeled neurons in CA1 stratum pyramidale and stratum granulosum of the dentate gyrus. Two days after the first appearance of immunoreactive perikarya, intensely immunoreactive neurons, labeled much more extensively than is ever seen in the adult, are encountered in each subfield of the hippocampus. The spatio-temporal order in both the emergence of perikaryal immunoreactivity and the transient appearance of intensely immunoreactive neurons follows that of neurogenesis, with immunoreactivity developing 12-14 days after the peak period of last cell division for a given hippocampal region. The incidence of immunoreactive perikarya in the dentate gyrus was quantified in rat pups ranging from postnatal days 8 to 19. The appearance of labeled neurons followed the spatio-temporal gradients that have been described for neurogenesis in this region as well. Immunoreactive perikarya emerged in the suprapyramidal stratum granulosum prior to their emergence in the infrapyramidal zone and in the temporal pole of the dentate earlier than in the mid-dorsoventral dentate. The lateral perforant path and mossy fiber axons, seen to exhibit enkephalin-like immunoreactivity in the adult hippocampal formation, differ in their relative maturity at the age immunoreactivity first appears. Immunoreactivity appears as early as postnatal day 4 in the lateral perforant path, an age at which these axons are just growing into their target field while it is not found within the mossy fibers until after postnatal day 10, an age at which mossy fiber bouton elaboration is well advanced and physiologically competent mossy fiber synapses with the regio inferior pyramidal cells have been established. The latter observation indicates that enkephalin is not necessary for synaptic transmission at the mossy fiber synapse.     

A note on the method of retrograde transport of horseradish peroxidase as a tool in studies of afferent cerebellar connections,particularly those from the inferior olive; with comments on the orthograde transport in Purkinje cell axons

1. Injections of horseradish peroxidase (HRP) suspension were made in the cerebellar cortex of cats (most often the paramedian lobule). The staining of the cerebellar cortex and the ensuing labeling of neurons in the inferior olive were studied in experiments with variations of concentration of HRP, amounts of fluid injected, survival time and age of the animals. Light microscopical studies were supplemented with electron microscopical observations. The folded cerebellar cortex offers particular difficulties with regard to obtaining a predictable extent of stained tissue, and the spreading of the fluid within the cortex shows great variations even with the same amounts and concentrations of HRP suspension. Diffusion of fluid appears to occur most easily within the molecular layer. Often there are unstained parts of folia between stained parts. Staining of the cortex is barely visible after 7 days, but appreciable shrinking of the stained area does not appear to occur until after 4 days. 2. The first signs of labeling of olivary neurons are seen after 5-10 hours, after 7 days there are no labeled cells. The rate of retrograde transport in olivocerebellar fibers is calculated to be between 50 and 100 mm/day. Labeling of cells appears to require staining of the molecular layer of its projection areas in the cerebellum. 3. For studies of the olivocerebellar projection survival times of 2-3 days and injections of 0.5 mul of a 50% HRP suspension seem in general to be well suited. Best results are obtained with animals weighing 1-3 kg. There is a clearcut correlation between the site of staining of the cortex of a particular part of the cerebellum and the site(s) and extension of olivary area(s) containing labeled cells. 4. Anterograde transport in axons of Purkinje cells has been observed. Electron microscopically the axons of these fibers contain HRP labeled tubules and vesicles as do their terminal boutons in the nuclei. 5. In cases where the injected fluid has spread to the cerebellar nuclei, localized parts contain neurons which are labeled as are the cells in the injected cerebellar cortex.     

Projections of auditory nerve in the cat as seen by anterograde transport methods.

Ascending projections of the auditory nerve of the cat were studied by autoradiographic methods after injection of labeled precursors into the cochlea. In animals in which [³H]leucine was injected, transport to the cochlear nucleus was observed after survival periods of 2–7 days, but there was no evidence of transport to more central structures in the auditory pathways. In these cases the pattern of labeling around cell bodies depended on the type of cell and on the location of the cell within subdivisions of the cochlear nucleus. For example, there was dense perisomatic labeling around spherical cells in the anteroventral cochlear nucleus and around octopus cells in the posteroventral cochlear nucleus. Some other types of cells in subdivisions of both anteroventral and posteroventral cochlear nucleus had little perisomatic labeling. In the dorsal cochlear nucleus, the most densely labeled area consisted of a band just central to the layer of fusiform cells. Labeled fibers were not found peripheral to this band in the granular layer, molecular layer and peripheral part of the layer of fusiform cells. In the dorsal nucleus there was no evidence of dense perisomatic labeling such as was found around some cells in the ventral divisions of the cochlear nucleus. Following injections of a mixture of [³H]proline and [³H]fucose into the cochlea, labeling was seen in the superior olivary complex. Transneuronal transport was suggested as the explanation for this finding because of (1) the choice of the labeled precursors, (2) the length of the survival periods (14 or 20 days), and (3) the evidence that isotope was within neurons in the cochlear nucleus.We concluded that under appropriate conditions, unique patterns of silver grains are found around different types of cells in the various subdivisions of the cochlear nucleus, apparently because fibers and terminals of the auditory nerve become saturated with the radioisotope. As a result, a highly detailed survey of projections of the auditory nerve to each subdivision of the cochlear nucleus is revealed at the light-microscopic level. Under different experimental conditions labeled fibers are found in the superior olivary complex, but these conditions are those most conducive to transneuronal transport and so such experiments cannot be used as evidence of projection of auditory nerve fibers beyond the cochlear nucleus.     

Axonal transport of adrenaline, noradrenaline and phenylethanolamine-N-methyl transferase (PNMT) in sympathetic neurons of the cod, Gadus morhua.

The axonal transport of adrenaline, noradrenaline and phenylethanolamine-N-methyl transferase (PNMT) has been studied in vivo in sympathetic neurons of the splanchnic nerve in the cod, Gadus morhua. Adrenaline and noradrenaline are transported at a mean axonal transport rate of 16 mm/day. After correction for a non-mobile fraction of adrenaline and noradrenaline, which does not contribute to the amine accumulation proximal to a ligature, a maximal rate of transport was calculated to about 45 mm/day for both amines. The increased level of catecholamines in front of a ligature could be depleted by reserpine treatment, which strongly suggests that both amines are stored in granules. PNMT is transported at a slow rate of 2 mm/day. The subcellular distribution of the cod PNMT was exclusively non-particular. The cod PNMT was further characterized by studying substrate specificity, temperature and pH optima. It is concluded that adrenaline and noradrenaline, stored in granules, are transported in a proximo-distal direction at a high rate compared to PNMT.     

Axonal transport and axon sprouting in the adult rat dentate gyrus: An autoradiographic study

In response to an entorhinal lesion, the commissural and associational afferents to the dentate gyrus have been shown to expand beyond their normal terminal zone into the area denervated by the entorhinal lesion. The present study has investigated the axonal transport of [³H]-labeled proteins in the commissural and associational projections following an entorhinal lesion. Injections of [³H]proline, [³H]leucine or [³H]fucose were given in the vicinity of the commissural and associational cells of origin before, immediately subsequent to, or at 5 to 15 days after the entorhinal lesion. The disposition of previously- or newly-synthesized proteins was examined in the commissural and associational terminal field at different times after an entorhinal lesion by light-microscopic autoradiography. The expanded commissural and associational zone was labeled whenever injections were given on, or before, the day of the entorhinal lesion and the width of the expanded zone increased with the time after the lesion. However, in contrast to the expanded associational projection, the expanded commissural projection was usually not labeled when the injections were made near the time that afferent reactivity commences (approximately 5 days after the lesion) and the animals were killed 2–4 days later. The expanded commissural projection was labeled, however, if injections were made at a later stage of afferent reactivity and the animals were killed 1 day later.The selective labeling of the expanding associational zone, when animals receive injections near the period of afferent reactivity and are killed 2–4 days later, appears to be due to the ability of labeled, slowly transported material to mark selectively the sprouting associational axons. The failure to label the expanding commissural zone in this experimental situation, is ascribed to the failure of labeled, fast-axonally transported materials to mark the sprouting commissural axons.Pre-labeling the normal commissural/associational projections several days in advance of the entorhinal lesion did not alter the subsequent labeling pattern of the expanding commissural/associational zone. Thus, proteins present in cells at the time of the lesion participate in the formation of axonal sprouts.     

Sequences in antibody molecules important for receptor-mediated transport into the chicken egg yolk.

Large quantities of antibodies are transported into the yolk of the chicken's egg. We have identified several regions within the antibody molecule important for its uptake into the egg yolk. An intact Fc and hinge region but not the Fc-associated carbohydrate are required for transport. Our data suggest that the C(H)2/C(H)3 interface is recognized by the receptor responsible for immunoglobulin (Ig) transport. At this interface, residues 251-254 form an exposed loop on the surface of C(H)2. Chicken IgY (cIgY) has the sequence LYIS and human IgG (hIgG) has the sequence LMIS at these positions; mutation of MIS to glycines results in an IgG that is not transported. A second site important for transport is at positions 429-432 within C(H)3. All transported antibodies have the sequence HEAL, whereas, murine IgG2b (mIgG2b) with the sequence HEGL and cIgA with the sequence HDGI fail to be transported. hIgA has the HEAL sequence and is transported.     

Axonal transport of adrenaline,noradrenaline and phenylethanolamine-N-methyl transferase (PNMT) in sympathetic neurons of the cod,Gadus morhua

The axonal transport of adrenaline, noradrenaline and phenylethanolamine-N-methyl transferase (PNMT) has been studied in vivo in sympathetic neurons of the splanchnic nerve in the cod, Gadus morhua. Adrenaline and noradrenaline are transported at a mean axonal transport rate of 16 mm/day. After correction for a non-mobile fraction of adrenaline and noradrenaline, which does not contribute to the amine accumulation proximal to a ligature, a maximal rate of transport was calculated to about 45 mm/day for both amines. The increased level of catecholamines in front of a ligature could be depleted by reserpine treatment, which strongly suggests that both amines are stored in granules. PNMT is transported at a slow rate of 2 mm/day. The subcellular distribution of the cod PNMT was exclusively non-particular. The cod PNMT was further characterized by studying substrate specificity, temperature and pH optima. It is concluded that adrenaline and noradrenaline, stored in granules, are transported in a proximo-distal direction at a high rate compared to PNMT.     

Role of transthyretin in posttraumatic regeneration of the sciatic nerve in mouse

Although thyroid hormones are known to have a significant influence on the development of nervous system, the absence of changes in the brain of mice deficient in transthyretin--a protein providing thyroid hormone transport across the blood-brain and blood-nerve barrier--remains unexplained. Therefore, the aim of this study was to determine the effect of transthyretin on the formation of myelinated and unmyelinated nerve fibers in sciatic nerve of mice. The myelinated and unmyelinated nerve fibers were counted in sciatic nerve of 3-months-old normal and transthyretin-knockout (transthyretin(-/-)) mice 15 and 30 days after nerve crushing. No differences were detected in the number of myelinated and unmyelinated nerve fibers in intact control (wild-type) animals group vs. transthyretin(-/-) mice. By days 15 and 30 after nerve crushing the number of myelinated nerve fibers was diminished by 54.7 and 71.8%, respectively, in transthyretin(-/-) mice, as compared to that in control animals. The number of unmyelinated nerve fibers at day 15 after the injury was not different in transthyretin(-/-) and control mice, however, by day 30 the number of these fibers in control group was found to increase significantly, exceeding that one in transthyretin(-/-) mice by 27.9%. These results indicate the important contribution of transthyretin, as a thyroxin carrier protein, to the process of posttraumatic regeneration of sciatic nerve. The absence of changes in nerve fiber numbers in transthyretin-knockout mice in postembryonic period suggests the presence of transthyretin-independent mechanism of thyroxin transport into the peripheral nervous system.     

Neurons in the rat subiculum with transient postmamillary collaterals during development maintain projections to the mamillary complex

Summary We recently found that in developing rats large numbers of fibers extending through the fornix initially grow well past the mamillary bodies and into the mesencephalic and pontine tegmentum (Stanfield et al. 1987). This postmamillary component of the fornix is essentially completely eliminated during the first few postnatal weeks, although the cells of origin of this projection within the subicular complex of the hippocampal region persist. To determine if the subicular neurons which transiently extend postmamillary axons maintain a projection to the mamillary complex after the elimination of the postmamillary component of the fornix, we employed a delayed, double-retrograde labeling paradigm. The cells of origin of the postmamillary component of the fornix in rat pups were labeled with an injection of fast blue (FB) into the mesencephalic/pontine tegmentum, and, in the same animals, but at a stage after the elimination of the postmamillary component of the fornix, the cells of origin of the definitive fornix were labeled with an injection of diamidino yellow (DY) aimed at the mamillary complex. In these cases the FB labeled neurons within the subicular complex can be DY labeled as well, indicating that subicular neurons which transiently extend postmamillary axons maintain projections to the definitive targets of the fornix within the caudal hypothalamus.Abbreviations FB fast blue - DY diamidino yellow dihydrochloride - UV ultraviolet     

猫内脏大神经一级传入纤维在脊髓灰质和薄束核中的分布

本文共用猫14只,取1～1.5mg HRP溶于7～10μl蒸馏水中,注入一侧的腹腔神经节或内脏大神经中,采用TMB成色法,观察跨神经节传递的一级内脏传人纤维在中枢神经系的分布。标记的一级内脏感觉纤维经后根进入脊髓后,绝大多数先行于背外侧束(或Lissauer束)中,少数进入后索上行。自背外侧束间断地发出内、外侧投射纤维,包绕着后角的内、外侧缘。外侧投射纤维在数量上比内侧的多,止于Ⅰ、Ⅴ、Ⅶ层和中央管周围。进入中间外侧核的纤维,再沿颅尾方向分开纵行,与交感节前细胞的纵向树突紧密平行排列。内侧投射纤维主要止于中央管周围区域。行于后索的纤维,止于闩平面以下薄束核的腹外侧部。     

Anatomical correlations of intrinsic axon repair after partial optic nerve crush in rats.

About 15% of retinal ganglion cells survive diffuse axonal injury of the optic nerve in adult rats. Following initial blindness, discrimination of visual stimuli in behavioral tests recovers within three weeks. To investigate the mechanisms promoting this functional recovery the axonal transport and the neurofilaments were studied. Intraocularly applied MiniRuby is transported until the place of crush and accumulated in enlarged axon terminals. Three weeks after lesion the anterograde transport of MiniRuby recovers distal to the place of crush. At the same point in time the retrograde transport of surviving retinal ganglion cells is restored which was visualized by horseradish peroxidase injected into the superior colliculus. The heavy neurofilament was stained immunohistochemically and analyzed statistically up to three weeks after optic nerve crush. The stained filaments in the axon fibers of retinal ganglion cells appear wavelike and/or fragmented up to day 8, but first signs of heavy neurofilament restitution in the fibers of the optic nerve are seen at day 12 after axonal injury. Because these results cannot be explained by longlasting axon regeneration, the present results provide convincing evidence for intrinsic axon repair soon after diffuse axonal injury that correlates in time with recovery of vision.     

The axoplasmic transport of 4S RNA within the sciatic nerve of the chicken

Following the introduction of [³H]uridine into the adult chicken spinal cord, a preferential labelling of 4S RNA with respect to rRNA was detected in the sciatic nerve at 2 and 4 days post-injection. No such proferential incorporation was observed within the spinal cord at the times examined nor in the sciatic nerve at 1 day post-injection. The results suggest that about 40% of the radioactivity in the sciatic nerve RNA after 4 days is due to tRNA transported from the spinal cord. Autoradiographic studies have shown that about 40% of radioactivity labelled RNA within the chicken sciatic nerve at 7–9 days post- injection is intraaxonal. It is concluded that RNA, but probably not rRNA, is transported within the axoplasm of the chicken sciatic nerve at approx. 20–25 mm/day.     

Development of a hybrid artificial liver using a polyurethane foam/hepatocyte-spheroid packed-bed module

Primary dog hepatocytes spontaneously formed spheroids in the pores of polyurethane foam (PUF) within 1-2 days of stationary culture. The spheroids, about 100-150 microm in diameter, partly attached to the surface and immobilized inside these pores. The lidocaine disappearance rate decreased to about 4 microg/10(5) viable cells/day for 10 days, while in the PUF/spheroid culture the rate was maintained at almost the initial level of 8 microg/10(5) viable cells/day for 10 days. Then, two scales of PUF packed-bed modules were designed. A small module (PUF volume; 14.5 cm3) was used for in vitro culture to investigate optimum culture conditions, and a large module (PUF volume; 300 cm3) was designed for dog experiments. Hepatocytes inoculated in these modules also formed spheroids and maintained almost the same activity of albumin secretion rate (111 microg/cm3 PUF/day in the small module and 87.7 microg/cm3 PUF/day in the large module). These results indicate that the PUF packed-bed module containing hepatocyte-spheroids is promising as a hybrid artificial liver.     

Effect of nerve length and temperature on the induction of action potentials in denervated slow muscle fibres of the frog

A. Pyriformis and extensor longus digiti IV muscles of Rana temporaria were denervated by cutting the sciatic or peroneal nerve at various distances from the muscles. Slow fibres were identified by their membrane time constants, and examined for their ability to produce action potentials in response to intracellularly applied current pulses. B. The slow muscle fibres acquired the ability to generate action potentials several days after denervation. The duration of this latent period depended on the length of the peripheral nerve stump, and on the temperature at which the frogs were kept after the operation. C. At 18 degrees C the latent period increased by 0.36 days per mm of sciatic nerve stump. At 11.5 degrees C the corresponding value was 0.7 days/mm. The effect of length of the peroneal nerve was smaller than that of the sciatic nerve. D. It is suggested that the peripheral nerve stump serves as a reservoir of 'trophic' material which is transported towards the slow fibres at a rate of 2.8 mm/day (at 18 degrees C) and seems to block the formation of Na channels. The Q10 value of this transport system would be 2.7.     

Cell migrations to the isocortex in the rat

Cells that took up tritiated thymidine (H-3T) at various periods of intrauterine and early infant life in the periventricular proliferative zone and migrated to form the isocortex in the rat were tracked autoradiographically in series of stages to characterize their movements. Cells labeled at any stage soon separated themselves into cohorts, some continuing to proliferate, others migrating at once, and still others delaying before migrating. Migratory cells moved to the developing cortex along the curved and oblique paths of the pallial fibers, whose basic plan was established by the early thalamocortical fibers. Magnitude of speed was 15 to 30 μ per hour. The primitive neural cells that originated on each of the fourteenth to eighteenth intrauterine days first reached the cortex in about 48 hours, others took two or three days longer. Migrations originating on the nineteenth to twenty-first days continued into the week after birth; as the primitive cells approached the cortex, however, they differentiated into young neurons, and traveled perpendicularly to its outer part. The first cohort of twentieth day labeled cells reached their intracortical destinations in about three days, the last in about ten days. The isocortex was formed essentially from within outward. The first neuroglia destined for the isocortex arose on the twenty-first intrauterine day.     

Transient direct connection of vestibular mossy fibers to the vestibulocerebellar Purkinje cells in early postnatal development of kittens

Postnatal development of mossy fiber afferents from the vestibular and the visual system to the vestibulocerebellum was studied electrophysiologically and morphologically. In kittens anesthetized with pentobarbital sodium and N2O plus halothane, extracellular simple and complex spikes of Purkinje cells were recorded in the flocculus, nodulus and uvula. In the flocculus, stimulation of the VIIIth, but not the optic nerve, evoked simple spike responses with a latency of 16 ms at the day of birth which decreased to 5 ms by day 15 (short latency group). On the other hand, another group of simple spike responses with much longer latencies (50-80 ms) began to be elicited on day 7 via both the optic and VIIIth nerves. The latency decreased to 24 ms by day 15 and 10 ms on day 30. These latencies further shortened with development to the adult latency value (3-5 ms). Simple spike responses of the short latency group were also evoked in the nodulus and uvula from the VIIIth nerve with a slightly longer latency than that in the flocculus (23 ms on day 3 and 12 ms on day 17). Because of the immaturity of granule cells in early postnatal days, short latency simple spike responses from the VIIIth nerve suggested the direct synaptic connection of vestibular mossy fibers with Purkinje cells. Horseradish peroxidase was injected into the white matter of the flocculus, nodulus and uvula in slice preparations. Mossy fibers labeled with horseradish peroxidase showed fine branches extending to reach Purkinje cell somata from mossy swellings in the internal granular layer during days 2-20. Electron microscopy showed that the labeled mossy fibers made intimate contacts with Purkinje cell somata and the terminals contained many round synaptic vesicles. Pre and postsynaptic densities were occasionally found. After day 20, direct mossy fiber connections with Purkinje cells could not be observed. During days 7-20, these direct connections, as well as mossy fiber-granule cell connections could be observed. It was demonstrated that during early postnatal development, vestibular mossy fibers temporarily make direct contact with Purkinje cells, through which impulses could be transmitted to elicit simple spikes in Purkinje cells.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.