An immunohistochemical study of the ontogeny of the neuroendocrine system in the chicken oesophagus

 The ontogenesis and distribution of serotonin-, chromogranin A-, chromogranin B-, galanin-, neurotensin-, bombesin- and neuropeptide Y-immunoreactive elements were studied in the chicken oesophagus during pre- and post-hatching life. Unlike positive nerve elements, that were present in pre- and post-hatching life, positive endocrine cells were observed only during embryonic life in the oesophageal epithelium. The first endocrine cells, immunoreactive for serotonin and chromogranins, appeared on day 12, in the cervical and thoracic portions of the oesophagus. At the same age, but only in its distal portion, a few bombesin- and neurotensin-immunoreactive cells also appeared. The number of the endocrine cells progressively increased, reaching a maximum on day 15. They then decreased, with a cranio-caudal progression, until they disappeared a few days after hatching. Almost all the serotonin-immunoreactive cells but only a subpopulation of bombesin- and neurotensin-immunoreactive cells colocalized chromogranins. About half of this subpopulation also colocalized serotonin. All these cells reacted positively with Grimelius argyrophile stain. The mucosa of the crop never contained positive endocrine cells. Positive nervous elements appeared first in the wall of the terminal oesophagus and only one or two days later in the proximal oesophagus including the crop. Nervous elements immunoreactive for galanin first appeared from days 6 to 7, for neurotensin from days 7 to 8, for neuropeptide Y from 13 to 15 and for bombesin from 15 to 18. At day 15 galanin-immunoreactive ganglionic cells and fibres occupied both the myenteric and submucous plexus and galanin-positive nerve fibres could be seen throughout the oesophageal wall from the adventitia to a thin subepithelial network. Neurotensin- and neuropeptide Y-immunopositive ganglionic cells and fibres, by contrast, invariably occupied the muscular and submucous layers. Scattered bombesin-immunoreactive ganglionic cells were observed only in the myenteric plexus. The number of positive nerve elements progressively increased until some weeks after birth. Density and intensity were always much higher for galanin and neurotensin than for neuropeptide Y and bombesin. Accepted: 5 November 1997     

Development of the ultrastructural features of neuropeptide Y-immunoreactive neurons in the rat visual cortex

Summary Immunohistochemical studies have localized neuropeptide Y into a small population of non-pyramidal neurons in the mammalian cerebral cortex. In the rat, these cells are distributed in layers II–VI and are characterized at the ultrastructural level by an abundance of cytoplasm containing a plethora of organelles, most conspicuous of which are cisternae of granular endoplasmic reticulum stacked in parallel arrays. In the present study, we used electron microscopic immunocytochemistry to examine the ultrastructural development of neuropeptide Y-labelled neurons in the rat visual cortex from birth, when they first appear in this cortical area, until postnatal day 32. At birth and in the subsequent few days, neuropeptide Y neurons, found exclusively in layers V and VI, often show a deeply infolded nucleus and little cytoplasm containing few organelles. At the end of the first postnatal week, labelled cells are still restricted to layers V and VI and display immature features. However, at this stage, cells often show irregularly enlarged proximal dendrites filled with organelles. During the second postnatal week, neuropeptide Y-immunoreactive cell bodies appear for the first time in layers II and III, and at the end of this week they have a distribution similar to that observed in the adult. Labelled cells are overall more differentiated than at earlier ages showing some of the ultrastructural features which distinguish them in the adult. No differences in maturation are evident between immunoreactive neurons located in the superficial layers and those in the deep layers, suggesting that the neuropeptide Y neurons in the more superficial layers express the peptide after having completed their migration and have acquired their characteristic ultrastructural features. Maturation proceeds during the third postnatal week. At the end of this stage, neuropeptide Y-containing cells acquire their mature nuclear and cytoplasmic features and an adult complement of synapses.     

Ontogeny of ( -Ala)-deltorphin I-like immunoreactive neurones in foetal rat brain

Foetal rat brain from embryonic day (ED) 12–22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of ( -Ala²)-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15–17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.     

Ontogeny of (D-Ala(2))-deltorphin I-like immunoreactive neurones in foetal rat brain

Foetal rat brain from embryonic day (ED) 12-22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of (D-Ala(2))-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15-17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.     

The development of neuropeptide Y-immunoreactive neurons in a model of pure cortical culture.

The development of neuropeptide Y-immunoreactive neurons in the rat brain cerebral cortex was studied in a model of a pure cortical culture. In this model, development of neurons devoid of any afferents from other brain structures could be observed. Since mutual interactions between neuropeptide Y and catecholamines have been postulated, such a pure cortical culture offers a possibility of studying the development of neuropeptide Y neurons devoid of any brainstem monoaminergic afferents. A tissue dissected from 16-day-old rat fetuses and cultivated in a dissociated culture for 14 days was examined immunohistochemically for the presence of neuropeptide Y-immunoreactive neurons. Three main types of neuropeptide Y-immunoreactive neurons were found: unipolar, bipolar and multipolar. Cell processes and terminal varicose fibres were also observed. The results obtained indicate that neuropeptide Y-immunoreactive neurons and fibres may develop in a pure culture of the rat cerebral cortex without the influence of any other structures.     

Transient populations of presumptive macrophages in the brain of the developing hamster, as indicated by endocytosis of blood-borne horseradish peroxidase

During postnatal development, clusters of cells associated with the mononuclear phagocytic system appear within the white matter of rodents and cats. We studied the distribution and morphology of these cells in the hamster's brain during the first 2 weeks after birth. In animals of different ages, horseradish peroxidase was injected into the heart. After 3-6 h survival, the animals were perfused with aldehydes and had their brains removed, cut and reacted. In another series, fixed brain sections from horseradish peroxidase-injected and non-injected animals were reacted for a non-specific esterase expressed by monocytes and macrophages. The horseradish peroxidase reaction-product was seen throughout the nervous tissue at the first postnatal day, appearing more concentrated in certain brain sectors from postnatal day 3 through 10, to finally become restricted to a few regions at postnatal day 16. Horseradish peroxidase-labeled cells appeared in increasing numbers from postnatal day 1 to 8, decreasing thereafter to disappear completely at postnatal day 16. Some labeled cells were roundish or elliptical with few, if any, processes; others had several clearly detectable processes. Horseradish peroxidase-labelled cells formed clusters within the dorsal subventricular zone, dorsal cortical white matter, corpus callosum and several other prosencephalic fiber tracts. The morphology of esterase-reactive cells was less clearly outlined but their distribution and relative density correlated with those of horseradish peroxidase-labeled cells. Also, many horseradish peroxidase-labeled cells were esterase-positive in most clusters. We conclude that (1) some cells in the developing brain selectively endocytose and accumulate blood-borne horseradish peroxidase in their cytoplasm, (2) these cells do not appear to be neurons but a particular cell type associated to the mononuclear phagocytic system and (3) they cluster transiently in particular sectors of the cortical and subcortical white matter during the first 2 weeks after birth.     

Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development

Summary The postnatal maturation of astrocytes in the rat visual cortex was analysed by immunostaining the astroglial proteins vimentin and glial fibrillary acidic protein with poly- and monoclonal antibodies. Vimentin immunoreactivity was present in the visual cortex up to the third postnatal week, whereas immunolabelling first disappeared in the cortical layers and then in the white matter. In the early postnatal period, vimentin antibodies labelled radial glial fibres. After the first postnatal week staining of radial glial fibres gradually disappeared and vimentin immunoreactivity was localized in a few protoplasmic astrocytes in the grey matter and fibrous astrocytes in the white matter. The development of glial fibrillary acidic protein-positive astrocytes was not fully complete until postnatal day 50. Glial fibrillary acidic protein-positive radial glial fibres were present after birth and disappeared towards the end of the third postnatal week. Staining of astrocytes in the white matter and in cortical layers I and VI reached an adult density at postnatal days 8 and 20, respectively. A progressively later development of glial fibrillary acidic protein-positive astrocytes was observed in cortical layers II–V which was completed between postnatal days 47 and 50. In the adult rat visual cortex glial fibrillary acidic protein-positive astrocytes were especially dense in layers I and VI, moderate in layers II/III and V and nearly absent in layer IV and lower layer III. The time course of the loss of vimentin and the gradual appearance of glial fibrillary acidic protein immunoreactivity in the visual cortex is considered as an index of astrocytic maturation and the spatiotemporal sequence of this maturation pattern is discussed in terms of reciprocal neuron-astrocyte interactions during brain development.     

The rat renal nerves during development

The prenatal and postnatal development of the innervation of the rat kidney has been investigated using immunocytochemical methods. The efferent innervation was studied using dopamine-beta-hydroxylase and neuropeptide Y antibodies. Calcitonin gene related peptide and substance P antibodies were used to investigate the afferent innervation. Kidneys from embryos of 14 to 20 days, from newborn rats, and from animals of 4, 10, 12, 21, 38, 60, and 90 days of age were studied. Slices of whole kidneys were analyzed, and frozen sections were used to investigate the location of the nerves in more detail. Both afferent and efferent nerves are observed inside the kidney by embryonic day 16. At birth, the afferent nerves are found (1) forming a rich plexus in the renal pelvis; (2) associated with the renal vasculature as far as the interlobular arteries (cortical radial arteries) and (3) in the corticomedullary connective tissue. The efferent innervation appears, at birth, to extend to the interlobular arteries and to the afferent arterioles of the perihilar juxtamedullary nephrons. The efferent innervation increases rapidly during the following days, and by postnatal day 21 a distribution of the innervation similar to that of the adult is observed. While the afferent innervation reaches the major target regions of the kidney by birth, the efferent does most of its expansion into the kidney postnatally. Afferent and efferent fibers are found, extrarenally and intrarenally, in the same nerve bundles. This proximity between afferent and efferent fibers may represent anatomical bases for their interaction in the adult as well as during development.Supported by U.S. Public Health Service Grant Rol 18340 from the National Institute of Health     

Neurogenesis and development of callosal and intracortical connections in the hamster

The developmental time-course of callosal and ipsilateral corticocortical projections was studied in embryonic and postnatal hamsters, from the time of neurogenesis until the appearance of adult patterns. Callosal neurogenesis was determined by combining the incorporation of [3H]thymidine injected on specific embryonic days with retrograde labelling of callosal neurons in the adult animal. The development of both callosal and corticocortical projections was studied by the transport of wheat germ agglutinin conjugated to horseradish peroxidase. Despite a significant radial disperson of postmigratory neurons born on the same day, it was found that the birthdates of callosally-projecting neurons in the frontal cortex were not restricted to a short period of time, but extended between embryonic days 13 and 15. This period covers the neurogenesis of cells in cortical layers III-V. Elongation of callosal axons (and possibly also of corticocortical fibres) started a couple of days before birth in the frontal cortex, and continued through the first postnatal days. After a "waiting period" of a few days, axons from both sets of projections were seen innervating restricted target sectors of the cortex. The zones of origin of these projections were initially exuberant, but were subsequently trimmed to overlap completely with the corresponding terminal fields. It is concluded that callosal and ipsilateral corticocortical projections undergo similar sequences of ontogenetic stages, suggesting that the development of neocortical connectivity as a whole may be governed by one and the same set of rules.     

The olfactory marker protein in the olfactory system of the mouse during development

The olfactory marker protein, a protein specific to the olfactory sensory neurons, has been studied in mouse during embryogenesis and in the postnatal period up to 30 days, with the unlabeled antibody enzyme method of immunohistochemistry. Olfactory neurons, which are morphologically detectable in 10-day-old embryos, do not contain olfactory marker protein. The protein is present in the olfactory neuroepithelium at embryonic day 14 and its appearance coincides with the establishment of sensory synapses in the olfactory bulb. Neurons containing the protein increase in number up to 30 days after birth. At 15 days of embryonic life, immunostaining was observed in sensory axons at the rostral tip of the olfactory bulb, and by embryonic day 17 a plexus of stained fibres has covered the bulbar surface. Between embryonic day 15 and postnatal day 1, olfactory axons have been observed to reach the mitral cell layer. In the vomeronasal system the olfactory marker protein is present at later stages and both the receptors' perikarya and their axons and axon terminals in the accessory olfactory bulb show a lower level of staining than the olfactory system proper.This study of the olfactory marker protein has allowed us to correlate its appearance with significant developmental phenomena.     

Apolipoprotein D gene expression in the rat brain and light and electron microscopic immunocytochemistry of apolipoprotein D expression in the cerebellum of neonatal, immature and adult rats

Apolipoprotein D gene and protein expression were investigated in the rat brain and cerebellum, respectively, during development. Apolipoprotein D gene expression was first observed in embryonic day 12 rat brain, with a moderate increase in apolipoprotein D messenger RNA levels towards the later part (embryonic days 15-17) of gestation. In the postnatal rat brain, a marked induction of apolipoprotein D messenger RNA occurred at postnatal day 10, with progressively higher levels of apolipoprotein D messenger RNA observed up to postnatal day 20. Somewhat lower, but none the less high, levels of apolipoprotein D messenger RNA continued to be present in brains of adult animals. In the immature cerebellum (day 3 up to one- to two-week-old rats), there were many densely labeled apolipoprotein D-immunoreactive cells that had features of oligodendrocyte precursors. Purkinje neurons showed apolipoprotein D immunoreactivity in one- to two-week-old animals, after which there appeared to be some decrease in staining. Oligodendrocytes in the cerebella of two-week-old animals were strongly apolipoprotein D positive, with immunoreactivity declining in older animals. These results reveal a maturation-associated induction of apolipoprotein D gene expression in the rat brain, and expression of apolipoprotein D in glial (immature oligodendrocyte) cells in the immature cerebellum, followed by specific expression of apolipoprotein D in Purkinje neurons.     

Chronological changes in prosaposin in the developing rat brain

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.     

Asynchrony in the expression of guanosine 3':5'-phosphate-dependent protein kinase by clusters of Purkinje cells during the perinatal development of rat cerebellum

The early maturation of Purkinje cells was studied by immunocytochemistry in the rat cerebellum. The antiserum against guanosine 3':5'-phosphate-dependent protein kinase used in this study has been shown previously to label specifically all Purkinje cells in the adult rat. Immunoreactive Purkinje cells are first observed at embryonic day 17, 2 days after the end of proliferation of this neuronal population. At this time, most of the labeled cells are situated in the subventricular zone, although some immunoreactive Purkinje cells have already reached the cortex. Between embryonic day 17 and birth, four clusters of immunoreactive Purkinje cells appear in each hemicerebellum. Their time course and their pathways of migration to the cortex were followed. The immunoreactive clusters are tailed by a fibre-like immunostained material. The pattern of the migrating clusters at embryonic day 19 is very similar to the pattern of the corticonuclear projection observed at birth. From comparison between sections of embryos processed either for immunocytochemistry or Cresyl Violet staining, it appears that all the Purkinje cells are not immunoreactive. Positive and negative clusters of Purkinje cells are sharply delineated, their cells never mix. Immunopositive and negative clusters of Purkinje cells coexist until postnatal day 3. However, from birth onwards, negative clusters begin progressively in a caudorostral sequence to express guanosine 3':5'-phosphate-dependent protein kinase and rapidly attain the same level of immunoreactivity as previously labeled clusters. From postnatal day 5 all the Purkinje cells are immunoreactive. It is concluded that a compartmentalization of the cerebellar cortex is present very early and is evidenced by differences in the biochemical maturation of Purkinje cells. The axons of Purkinje cells reach the deep nuclei, following the same pathways as the clusters of Purkinje cells migrating to the cortex. Therefore, the mechanisms regulating the selection of the migratory routes followed by each Purkinje cell cluster are essential for the achievement of the topography of the corticonuclear projection. The level of protein kinase immunoreactivity cannot be taken as an index of the overall maturation of Purkinje cells, because it does not always coincide with the expression of other makers of biochemical and morphological differentiation of these neurons. During the early establishment of the cerebellar maps, an asynchrony in the expression of parts of the same genotype in the Purkinje cells may help in the establishment of ordered connections.     

Ontogeny of neurotensin-containing neuron system of the rat: immunohistochemical analysis--II. Lower brain stem

The ontogeny of the neurotensin neuron system in the lower brain stem of the rat was investigated by means of indirect immunofluorescent method. Neurotensin-like immunoreactivity-containing cells first appear in the primordium of the n. tractus solitarii, n. tractus spinalis nervi trigemini, reticular formation just medial to the latter nucleus, n. reticularis parvocellularis, n. laterodorsalis tegmenti, and midbrain reticular formation of the fetus at gestational day 17. At gestional day 18, neurotensin-immunoreactive cells newly appear in the n. raphe dorsalis. Between gestational day 19 and postnatal day 7, the animals show a remarkable increase in number of immunoreactive cells and fibers in various lower brain stem areas except for n. tractus spinalis nervi trigemini and n. tractus solitarii. Moreover, during this stage, neurotensin-immunoreactive cells located in the n. prepositus hypoglossi and n. vestibularis lateralis appear for the first time at birth and postnatal day 5, respectively. Since postnatal day 7, although the majority of immunoreactive cells located in the lower brain stem decrease in number as the rats grow, immunoreactive cells in the n. tractus spinalis nervi trigemini, on the contrary, increase in number from after birth until postnatal day 10, and maintain more or less their immunoreactivity even in the adult rat. In addition, neurotensin-immunoreactive cells in the nucleus of the solitary tract increase in number during the fetal period, reach the maximum content at postnatal day 7-10, and maintain their immunoreactivity even in the adult rats. Thus, the present study demonstrated that neurotensin-like immunoreactive structures appear at a very early ontogenetical stage, suggesting that neurotensin plays an important role in the development of the lower brain stem of the rat. In addition, the present study further showed that neurotensin-immuno-reactivity shows various fluctuations during the ontogeny, suggesting multiple functions of neurotensin in the central nervous system.     

Development of striatal patch/matrix organization in organotypic co-cultures of perinatal striatum, cortex and substantia nigra

Organotypic cultures of fetal or early postnatal striatum were used to assess striatal patch formation and maintenance in the presence or absence of dopaminergic and glutamatergic influences. Vibratome-cut slices of the striatum prepared from embryonic day 19 to postnatal day 4 rat pups were maintained in static culture on clear membrane inserts in Dulbecco's modified Eagle's medium/F12 (1:1) with 20% horse serum. Some were co-cultured with embryonic day 12-16 ventral mesencephalon and/or embryonic day 19 to postnatal day 4 cortex, which produced a dense dopaminergic innervation and a modest cortical innervation. Donors of striatal and cortical tissue were previously injected with bromo-deoxyuridine (BrdU) on embryonic days 13 and 14 in order to label striatal neurons destined to populate the patch compartment of the striatum. Patches of BrdU-immunoreactive cells were maintained in organotypic cultures of late prenatal (embryonic days 20-22) or early postnatal striatum in the absence of nigral dopaminergic or cortical glutamatergic influences. In slices taken from embryonic day 19 fetuses prior to the time of in vivo patch formation, patches were observed to form after 10 days in vitro, in 39% of nigral-striatal co-cultures compared to 6% of striatal slices cultured alone or in the presence of cortex only. Patches of dopaminergic fibers, revealed by tyrosine hydroxylase immunoreactivity, were observed in the majority of nigral-striatal co-cultures. Immunostaining for the AMPA-type glutamate receptor GluR1 revealed a dense patch distribution in nearly all cultures, which developed in embryonic day 19 cultures after at least six days in vitro.These findings indicate that striatal patch/matrix organization is maintained in organotypic culture, and can be induced to form in vitro in striatal slices removed from fetuses prior to the time of in vivo patch formation. Furthermore, dopaminergic innervation from co-cultured pieces of ventral mesencephalon enhances patch formation in organotypic cultures.     

The ontogeny of brain neurotensin receptors studied by autoradiography

Using in vitro receptor autoradiographic techniques we have analysed the pre- and postnatal development of neurotensin receptors in the rat brain. Receptors were labeled with [3H] or [125I]neurotensin in mounted tissue sections from animals of ages gestational day 14 until the postnatal day 21 as well as young adult animals. Very low densities of neurotensin receptors were visualized on gestational days 14 and 15. Between gestational days 16 and 18 a marked increase in the density of neurotensin receptors was seen in the developing neocortex. Densities in other brain areas, particularly the midbrain and brainstem were much lower than cortical densities. The density of neurotensin receptors in the cortex increased through the last part of the gestation and early postnatal life until it peaked at the end of the first postnatal week. After that, neurotensin receptor binding decreased dramatically reaching lower densities seen in the adult animal at the end of the third postnatal week. Development of neurotensin receptors in other brain areas followed very different time patterns. Neurotensin receptors in the midbrain were seen first at gestational day 18 and increased slowly with development to reach adult levels at about the second week of postnatal life. Neurotensin receptors in the hippocampal formation demonstrated postnatal development; they were detected at postnatal day 5 and showed a developmental peak around the second week. These patterns were seen with both 3H- and 125I-labeled neurotensin, thus excluding possible differential quenching artifacts. These clear differential regional ontogenetic patterns for neurotensin receptors are the main findings of these experiments. The very high densities present in the cortex even in fetal stages suggest that neurotensin could play a role in the development of the brain.     

Developmental changes in enzyme activities and in morphology of rat cortex mitochondria

Development of mitochondria in rat brain cortex was investigated in terms of mitochondrial respiratory enzyme activities, and structural and numerical developments of mitochondria. Measurements of succinate-O2 and NADH-O2 oxidoreductase activities of mitochondria resulted in simultaneous changes of activities in postnatal rat. Both oxidoreductase activities were still low at 0-5 days old, increased until 15 days, decreased slightly at 21 days and drastically in adult mitochondria. In morphological study, the cross-sectional area of mitochondrion per cell increased gradually until 21 days old, but decreased drastically in adult. The area of a mitochondrion at 5 days increased about 1.5-fold in comparison with that at 0 days, and maintained at 15 and 21 days. However, the values of area of one mitochondrion from 10 days and adult are about half of a maximum value (21 days). Numbers of mitochondrion per cell were still low at 0-5 days, and high constantly (about twice) at 10-21 days. These findings suggest that the organelle division of mitochondria may be carried out at 5-10 days postnatal. The number of adult rat mitochondria decreased slightly. The small and undeveloped mitochondria were observed at 0 day postnatal by use of transmission electron microscopy (TEM). However, during development from 5 days postnatal, larger and elongated mitochondria were observed, and the maximal complexity of structure of cristae is observed at 15 days and 21 days by TEM. In adult cortex, the small mitochondria were also observed with compact and dense cristae. Our results indicate that the changes of activities of mitochondrial respiratory enzymes in rat cortex is good correlated with the structural maturation of mitochondria.     

Monoclonal antibodies reveal molecular differences between terminal fields in the rat dentate gyrus.

We have derived a number of monoclonal antibodies which detect molecular differences correlating with the afferent inputs to the molecular layer of the adult rat hippocampal dentate gyrus. One group, dubbed OM-1 to OM-4, strongly stain the outer zone of the molecular layer, which receives its major innervation from the ipsilateral entorhinal cortex. A second group, IM-1 and IM-2, show a complementary pattern and preferentially stain the inner molecular layer, which receives inputs from the ipsilateral and contralateral hippocampus. These antigens are not, however, restricted to these layers, being found outside the hippocampus in several other areas of neuropil in the adult brain. In the developing brain the IM-1 antigen appears ubiquitously from the earliest age studied, embryonic day 12. Within the dentate gyrus, its restriction to the inner terminal field of the molecular layer only occurs during the second postnatal week. In contrast, OM staining appears only sparsely and late in the prenatal brain, appearing in developing cortical white matter between embryonic days 18 and 20. The outer dentate molecular layer becomes OM-positive from birth onwards, corresponding to the time of arrival of entorhinal axons during the first postnatal week. These two groups of monoclonal antibodies recognize a number of different glycoproteins. Ultrastructural immunohistochemistry shows they are cell surface molecules, and as such may be involved in the recognition events required for the establishment of specific patterns of neuronal connectivity.     

The formation and growth of the cortical layers in the cerebellum of the opossum

The development of the cerebellar cortex in the opossum was analyzed in Nissl-stained sections using qualitative and quantitative methods. The young of the opossum are born 12-13 days after conception and mature for approximately 85 days in an external pouch providing an excellent model for embryological studies. Qualitative observations of cerebellar growth were made from birth to postnatal day (PN) 19. At birth the opossum cerebellar anlage can be divided into two layers, a ventricular layer and an intermediate layer; histologically his is comparable to the rat cerebellar anlage at embryonic day 13 (Altman and Bayer 1978) and the human cerebellar anlage prior to the seventh embryonic week (Rakic and Sidman 1970). By PN 3 the cerebellar anlage consists of five layers: the ventricular layer, the ventral intermediate layer, the acellular layer, the dorsal intermediate layer and the marginal layer. The external granular layer begins migrating over the dorsal surface of the cerebellum at PN 5. The immature Purkinje cell layer is first seen at PN 12 and is subsequently arranged as four clusters between PN 12 and PN 22. At PN 19 the opossum cerebellum is comparable to the rat cerebellum at birth (Korneliussen 1968c). A quantitative analysis of cerebellar growth was performed between PN 17 and PN 77 using vermal sections. The area and thickness of each of the cortical layers was determined from five vermal sagittal sections using two methods; a Zeiss Videoplan and a point counting system. The external granular layer increases in area from PN 17 to PN 75, however its maximal width is achieved between PN 19 and PN 33. The persistence of the EGL until after PN 105 suggests that synaptic contacts between granule cell axons and Purkinje cells may continue to form after PN 77 when the Purkinje cell is mature based on Golgi and fine structural features (Laxson and King 1983). Between PN 17 and PN 77 the area of the molecular layer and the area of the internal granular layer increase at a more rapid rate than the other cerebellar layers. The maturation of the cerebellum in the opossum is a lengthy process lasting approximately 77 days in comparison to rodent cerebellar growth which requires about 25 days (Korneliussen 1968c). Also, the entire process of cortical lamination occurs after birth while the opossum is maturing in an external pouch.