Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: An electron microscope study

The development of the auditory nerve endings and their target cells in nucleus magnocellularis was studied by electron microscopy of perfusion-fixed brains from embryonic day 12 to hatching. Embryonic days 12–13: somatic processes extend from the perikaryon. The cytoplasm of the soma and processes contains free ribosomes, mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus and an eccentric, heterochromatic nucleus. Small profiles of auditory nerve fibers containing round, clear vesicles make specialized contacts, including some synapses, on distal somatic processes but rarely on proximal somatic processes or on the soma. The postsynaptic zones contain a flocculent matrix. Days 15–17: somatic processes disappear and occasional attachment plaques are seen between cell bodies. The nucleus appears euchromatic. Cytoplasmic organelles form a dense matrix indicative of intense metabolic activity. Somatic spines are evident. The afferent axons form large, vesiculated profiles located, increasingly, on the cell body and somatic spines, with many points of synaptic contact. Opposite each ending a band of amorphous, flocculent material fills the postsynaptic cytoplasm. Embryonic day 18-hatching: the somatic cytoplasm becomes less dense; stacks of rough endoplasmic reticulum start to condense. Afferent axon terminals mature, especially the synaptic membrane complex and associated densities. The postsynaptic flocculent material diminishes in extent until it is found associated only with somatic spines.The ultrastructural observations on the maturation of nucleus magnocellularis closely corroborate and extend previous results with the Golgi methods. Developing auditory nerve fibers initially synapse on the distal parts of the somatic processes of the immature cells. As the somatic processes disappear or retract, axonal endings move to the soma and develop into large axosomatic end-bulbs. Possibly, the somatic processes as they retract drag the auditory nerve endings to the cell body. The findings also suggest a role of the transiently appearing, flocculent material of the postsynaptic regions in the formation of synapses.     

The development of innervation patterns in the avian cochlea

The sequence of developmental events leading to the innervation of the cochlea and the differentiation of its receptor cells has been studied in chick embryos with Golgi methods. We describe the morphogenesis of cochlear ganglion cell peripheral processes from their appearance in early embryos to the formation of their mature endings on hair cells in the basilar papilla (organ of Corti) of prehatching chicks. In the stage of peripheral fiber outgrowth, embryonic days 3-5, the fibers emerge from the ganglion cell bodies and grow, in a uniform fashion, toward the undifferentiated receptor epithelium of the otocyst. In the stage of the invasion of the otocyst by the peripheral fibers, embryonic days 6-7, some fibers enter the epithelium directly after reaching it, others enter after traveling some distance longitudinally beneath its basal lamina. The invading fibers appear to encounter resistance at the basal lamina, but, once within the epithelium, at embryonic days 8-9, they form a surfeit of branches in columnar zones oriented radially toward the surface. In early synaptogenesis (embryonic days 8-9) hair cells first become apparent. They differentiate from primitive epithelial cells. These cells withdraw their basal processes, which appear to accompany the growing fibers into the superficial epithelium. At embryonic days 11-13, the stage of mid-synaptogenesis, the fibers develop large, bulbous, preterminal and terminal swellings, which are located below the bases of the hair cells; the surplus branches atrophy or withdraw. Efferent axons are first seen in the epithelium at this time. In late synaptogenesis (embryonic days 14-17), the preterminal swellings disappear and the endings transform into mature foot-shapes at the bases of the hair cells. These morphological changes during the development of the peripheral endings are comparable to those of cochlear axons in nucleus magnocellularis (cochlear nucleus). During mid-synaptogenesis, when the ganglion cells develop swellings in the periphery, their central axons ramify extensively. Late in synaptogenesis, while the peripheral swellings disappear, there is a corresponding condensation of the central terminals to form the end-bulbs of Held. Thus, specific connections of the cochlear ganglion cells and their target cells in the ear and brain may result from two sequential developmental phases: (1) loosely organized and overabundant initial growth of branches from the fibers entering their target tissue; (2) reorganization of these fibers with the disappearance or resorption of the surplus branches during the transformation of their endings into mature synaptic arrangements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in culture

The effect of the availability of synaptic targets on neuronal survival was tested by explanting the cochleovestibular ganglion from embryonic day 3-1/2 chick embryos and maintaining it in the presence or absence of appropriate synaptic target tissues for 14 days in culture. The targets were the inner ear, peripherally, and the myelencephalon, centrally. Light and electron microscopic observations showed that the ganglion cells in the explants with targets present had generally achieved a degree of differentiation comparable to that of their counterparts in embryonic day 14 embryos. The variety of cell types seen in the normal embryonic day 14 ganglia was also evident in vitro. In ganglia explanted without peripheral or central targets, few neurons survived. Ganglia explanted with either peripheral or central target intact showed considerably better survival than those explanted without any target. Ganglia explanted with only the peripheral target (the inner ear) survived equally as well as those with both central and peripheral targets. Ganglia cultured with the central target (myelencephalon) did not survive as well as those with peripheral targets. The effect of the peripheral target on the ganglion was less clear-cut when ganglia were first dissected from their targets and then recombined in culture. However, the results of such experiments in which nerve fascicles were traced in serial sections from ganglia to target areas, suggest that the actual innervation of target cells, as well as proximity of ganglia to target tissues, could influence neuronal survival. Establishment of innervation appeared to be selective, in that the closest available target area was not always the one contacted by the ganglionic fibers. The present findings are consistent with a role of neuron-target cell interactions in supporting neuronal survival in the cochleovestibular ganglion of the chick embryo. Both the central and the peripheral targets are implicated in trophic interactions with the sensory neurons.     

Identification under the electron microscope of climbing fibers and their synaptic contacts

Summary An attempt is made to identify, under the electron microscope, the climbing fibers of the cerebellum (in the cat) and their synaptic contacts with Purkinje cells and other cortical neurons. — Two kinds of axonal profiles, having synaptic contacts with primary and secondary dendrites of Purkinje neurons, can be recognized: One being terminal fibers densely packed with neurofilaments, having mainly contacts de passage with the dendrite surface, with small accumulations of synaptic vesicles at the presynaptic side of the contact. The others are rather knob-shaped contacts filled with synaptic vesicles and poor in neurofilaments. In chronically isolated folia, in which only local neurons and their processes have survived, all filamentous profiles have disappeared while vesicular ones are not appreciably reduced in number. It is inferred from this, that the neurofilamentous profiles correspond to climbing fibers, whereas the vesicular ones could be the endings of outer stellate axons, recurrent Purkinje axon collaterals, or ascending basket axon collaterals. — Similar two kinds of axon-terminal profiles are found in synaptic contact with Golgi and basket cell bodies. As in chronically isolated folia only the vesicular profiles survive, it is inferred that the climbing fiber has axo-somatic terminals on Golgi cells and basket cells as well. Previous information of this kind, gained with the light microscope and with degeneration studies, is thus substantiated with the aid of the electron microscope. The vesicular presynaptic profiles on Golgi and basket neurons are in the first case certainly and in the second with high probability endings of recurrent Purkinje axon collaterals. — The few axosomatic synapses found on outer stellate neurons may also be terminals of climbing fibers, but degeneration evidence for this is not conclusive. — The observations are summarized and evaluated from the functional point of view in a diagram, with consideration to recent physiological information on the function of climbing fibers.     

Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain

Macrophages and microglia in the developing and adult mouse brain have been identified by immunohistochemical localization of the macrophage-specific antigen F4/80 and monoclonal antibodies to the FcIgG1/2b (2.4G2) and type-three complement (Mac-1) receptors. In the adult mouse there are two classes of F4/80-positive cells; those associated with the choroid plexus, ventricles and leptomeninges and the microglia. The cells bearing Fc and complement receptors are indistinguishable, by their morphology and distribution, from those revealed by F4/80. During development macrophages invade the brain and can be followed through a series of transitional forms as they differentiate to become microglia. Macrophage invasion occurs when naturally dying cells are observed in large numbers and this is consistent with the idea that dying neurons and axons provide a stimulus for macrophage infiltration. Our results provide strong support for the hypothesis that the microglia are derived from monocytes and show that microglia possess receptors which would allow them to play a part in the immune defence of the nervous system.     

Binocular interaction in the fetal cat regulates the size of the ganglion cell population

During fetal development of the cat's visual system there is a marked overproliferation of optic nerve axons. In utero binocular interaction contributes to the severity of fiber loss since removal of an eye during gestation attenuates axon loss in the remaining optic nerve. The purpose of the present study was to determine whether this reduced loss of optic nerve fibers is due to a failure of retraction by supernumerary axon branches or to a reduction in ganglion cell death. To resolve this issue, we compared the number of ganglion cells and optic nerve fibers in adult cats which had one eye removed at known gestational ages. Retinal ganglion cells were backfilled with horseradish peroxidase and counts were made from retinal wholemounts. The axon complement was assessed with an electron microscopic assay. In the retinas of a normal cat we estimated 151,000 and 152,000 ganglion cells. The optic nerves of two other normal cats contained approximately 158,000 and 159,000 axons. In comparison, an animal enucleated on embryonic day 42 had 180,000 ganglion cells and 178,000 optic nerve fibers, while in an animal enucleated on embryonic day 51 the corresponding estimates were 182,000 and 190,000. The close agreement between cell and fiber counts indicates that axonal bifurcation does not contribute appreciably to the axon surplus in the optic nerve of prenatally enucleated cats. These results demonstrate that prenatal binocular interaction regulates the size of the mature retinal ganglion cell population.     

Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: A golgi study

Nucleus magnocellularis in the chicken consists predominantly of a population of mediu-msized cells which receive large, axosomatic endings from the auditory nerve. The morphological development of these cells and their auditory input were studied with the Golgi methods. At 7 1/2–9 days of incubation (embryonic days 7 1/2–9, staged according to the Hamburger-Hamilton series), cells in nucleus magnocellularis have several long, branched dendrites, which often end in bulbous swellings. By embryonic day 10, efferent axons have already grown out from the cells and characteristic terminal plexuses of these axons are seen in nucleus laminaris bilaterally. The dendrites of cells in nucleus magnocellularis have been replaced by a multitude of long somatic processes, giving the cell body a shaggy appearance. This arrangement is maintained up to embryonic day 15, when a remarkable second transformation occurs. The cells lose their somatic processes and present bald, round profiles. Around embryonic days 17–18 a primitive-looking process with a tip like a growth cone emerges from the cell body and somatic spines are evident. By days 19–20, one or two thin, frail dendritic processes can be seen.Correlated with this dramatic series of changes in the cells is a fixed sequence of transformations of the incoming axons. Around embryonic day 10, primary sensory axons in nucleus magnocellularis end in swellings resembling growth cones. Between days 11 and 13, following the explosive growth of somatic processes there is a corresponding expansion and ramification of the auditory nerve endings. On embryonic day 14, there is a condensation of the terminal axon branches, which now form a compact, highly branched plexus. Between days 16 and 17, the plexus coalesces into a calycine structure, now approaching its final form, the end-bulb of Held, which is achieved by embryonic days 19–20. The transformation of the plexus to the calycine form occurs around the same time that the cell loses its somatic processes.The parallel sequence in the morphogenetic stages of the assembly of the end-bulbs and their target cells evinces a correlation, if not a causal relationship between the sensory axons and the developing neurons. The arrangement of the somatic processes and axonal branches during the early, multipolar stage would provide an opportunity for optimum interactions between the synaptogenetic processes of the afferent axons and the target cells. The later morphological transformations could orchestrate the specific, cell-to-cell interactions which accompany, or even depend on the activity of the definitive end-bulb synapse.     

The growth of synaptic endings in the mammalian brain: A study of the calyces of the trapezoid body

Summary The calyx of Held is a distinctive, axosomatic ending in the medial nucleus of the trapezoid body. An example of a growing synapse, its development, as visualized in rapid Golgi impregnations of age-graded littermates (cats, rats, rabbits, opossums), follows a morphological sequence: 1) The migratory, calycigenic axon ends in a sprouting growth cone with transient radiating processes; 2) On approaching the recipient neuron, the growth cone forms many thin, sinuous branches, spreading over the post-synaptic surface; 3) Replacing these are broad branches, which form the definitive calyciform synapse; 4) A network of calycine collaterals develops from collateral growth cones in a sequence similar to that of the parent calyx, but unaccompanied by sinuous branches. The sinuous branches seem to provide a developmental framework for the specific form of the calycine ending. The sprouting growth cones and radiating branches may relate to the establishment of the initial contacts by the presumptive synaptic members.Supported in part by U.S. Public Health Service Research Grant NB 06115 to Harvard University.     

Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.     

Nerve fibers in the sympathetic trunk of Rana pipiens: A quantitative electron microscope study

The numbers and diameters of nerve fibers were determined for the first time for all interganglionic rami of the sympathetic chain of the frog. The number of myelinated fibers ranged from 57 to 263 and was generally highest in the anterior portion (207) and lowest at the posterior end (57) of the chain. The exception was between the fourth and fifth ganglia where the middle splanchnic nerves arise — the number of myelinated fibers was highest here (263). Unmyelinated fiber counts were similar throughout the chain (417–814) except above and below the first sympathetic ganglion (7325 and 3026, respectively), and at the level where the middle splanchnic nerves arise (3780). Unmyelinated fibers constituted 75–97% of the fibers of the chain. The myelinated fibers ranged in diameter from 0.5 to 9.0 μ, 75% of them measuring between 1.5 and 3.0 μ. The diameter range for unmyelinated fibers was 0.1 to 5.0 μ with 78% being 0.1 to 1.0 μ.     

Immunohistochemical analysis of the early ontogeny of the neuropeptide Y system in rat brain

The distribution of neuropeptide Y in the developing rat brain was studied with immunocytochemistry, using the peroxidase-antiperoxidase method. Immunoreactive perikarya were first seen on embryonic day 13 and staining of fibres appeared from embryonic day 15 onwards: perikaryal staining was generally more intense prenatally than after birth. Areas rich in neuropeptide Y immunostaining included the monoaminergic regions of the brain stem from embryonic day 13 (especially the lateral reticular nucleus and the medullary reticular formation), the dorsal mesencephalon (with spots of immunoreactivity in the outer subventricular zone at embryonic days 13 or 14 and many cells and fibres in the inferior colliculus from embryonic days 16-20) and the olfactory tubercle/ventral striatum from embryonic day 15 until birth. The period of development of cortical neurones extended from embryonic day 19 until postnatal day 21. A hitherto unreported feature unique to neuropeptide Y was the presence in certain parts of the cerebral cortex of transient cells at the base of the cortical plate bearing radial processes which transverse its width. They were present from embryonic day 17 until postnatal day 4 and were maximally developed at embryonic days 20 or 21, contributing at this age a substantial fibre projection through the immature corpus callosum. The abundance of neuropeptide Y in the prenatal rat brain suggests it may play an important role in development.     

Neurogenesis of the nucleus accumbens septi and neighboring septal nuclei in the rhesus monkey: A combined [3H]thymidine and electron microscopic study

The time of origin and spatio-temporal pattern of neuron distribution in the nucleus accumbens septi and the lateral and medial septal nuclei were determined in the rhesus monkey. Autoradiographic analysis of 2 to 5 month old monkeys that had been exposed to a pulse of [³H]thymidine during embryonic or early postnatal days showed that neurons destined for the nucleus accumbens septi are generated over a period of approximately 50 days (between embryonic day 36 and 85 of the 165 day gestational period) whereas the neurons destined for the medial and lateral septal nuclei are produced for only 25 days (from embryonic day 36 to 62). The peak of proliferation of neurons destined for all three nuclei occurs synchronously around embryonic day 45. A number of small cells with round, densely stained nuclei were labeled in specimens injected after embryonic day 85; however, following combined light- and electron-microscopic analysis, these late generated cells were classified as glia.Neurons destined for the nucleus accumbens septi generated on different embryonic days did not exhibit spatio-temporal gradients in their distribution. In contrast, neurons destined to populate the lateral and medial septal nuclei displayed a prominent ‘outside-to-inside’ spatio-temporal gradient in which earlier generated neurons occupied positions closer to the pial surface and later forming neurons were positioned closer to the ventricular surface. Based on this evidence regarding the time span of neuronal production and the absence of spatio-temporal gradients, the mode of development of the nucleus accumbens septi more closely resembles the pattern of neurogenesis reported for the primate neostriatum than that in the adjacent septal nuclei.These findings indicate that the nucleus accumbens septi and neostriatum in primates may have a common embryological origin.     

A classification of Sharpey's fibers within the alveolar bone of the mouse: a high-voltage electron microscope study

In the remodeling interdental septum of the mouse, four types of Sharpey's fibers were observed. Classification of these fiber types was based on characteristics of their termination within the septum in relation to the resorption-related reversal line separating old and new bone. "Severed fibers" were located only within old bone and terminated at the reversal line. "Arborized fibers" were located only within new bone and terminated therein. "Adhesive fibers" were located within new bone and terminated within a heavy band of granular material at the reversal line. "Continuous fibers" had components within old and new bone. These components were connected across the reversal line by nonstriated fibrils. Adhesive fibers were the least numerous type; severed and arborized fibers were observed at nearly equal frequency. Continuous fibers were the most numerous type, their numbers being significantly greater than any of the other types (P less than .001). Mean numbers of continuous fibers were greater than the mean total of the three other fiber types (P less than .001). Mean numbers of severed, adhesive, and arborized fibers were not statistically different. The study suggested that continuous fibers could be transalveolar--that is, ones which pass through the septum without interruption joining fibers of the adjacent periodontal ligament. Maintenance of their spatial continuity appeared to require a connecting protein to orient new unit collagen fibrils to old ones in areas of reversal. Thus, transalveolar fiber bundles could be characterized as being composed of old and new segments joined by a connecting protein. As their unit collagen fibrils did not cross resorption-related reversal lines these fibers were spatially continuous but temporally discontinuous.     

Thrombus formation in a staged capillary membrane oxygenator associated with a microaggregate blood filter: a scanning electron microscope study

Blood contact at interfaces in extracorporeal devices is a source of traumas. Proteins are very rapidly adsorbed; then, depending on which proteins are left various degrees of platelet aggregation follow and thrombi develop. The scanning electron microscope reveals very instructive information on the morphology of the blood deposits which adhere to foreign surfaces. Oxygenators such as the Awad D, which is a staged one made of silicone rubber, become "thrombus invaded" after prolonged extracorporeal circulation in spite of adequate heparinization of the blood. In most cases, evaluation of membranes and devices is assessed with respect to transfer of blood gases. Careful examination of the morphology of deposits should be developed. This study shows the importance of blood flow rate, design, and materials.     

An electron microscope study of crystal calcium carbonate formation in the mouse otolith

The ultrathin sections of the developing otolith of mouse fetuses 15.5 days to 20 days after birth were observed with the aid of the electron microscope. The first step of otolith formation is an aggregation of organic clusters observed in the sacculus of the 15.5-day fetus. These organic structures are modified and assume a hexagonal shape in the 17.5-day fetus. The unmineralized stages of the otolith referred to as the preotolith, serve as the template for future development. One day after birth, at either end of the preotolith, minute tube-like structures develop in which needle-shaped crystallites are initiated. Crystallites continue to develop throughout the hexagonal template which give rise to a structure containing many regularly arranged needle-shaped crystallites. Based on the present observations, the mouse otolith is considered a multi-iso-oriented crystal.     

A scanning electron microscope study of aberrations in the prism pattern of rat incisor inner enamel

The prism pattern in the inner enamel of adult rat incisors was studied with the SEM in unfixed tissues that had been sectioned, ground, polished, and etched. Six different types of aberrations in the prism pattern were encountered: 1. Prism lamellae may be shorter than the mesio-lateral width of enamel. 2. Prism lamellae may deviate from a transverse orientation. 3. Prism lamellae may “fuse” or “bifurcate.” 4. Prisms of two adjacent lamellae may pursue a common course. 5. Prisms may change direction. 6. Variations exist in the outline of transversely cut prism profiles. Aberrations were observed at any distance from the dentino-enamel junction. These observations were used as a basis for an analysis of the movement of ameloblasts during rat incisor amelogenesis. It was concluded that it is physically possible for the ameloblasts to create the observed aberrations as they move along the path of the prisms. However, the aberrations seem to make it more difficult to understand the factors controlling ameloblast movement. Occasionally crystallite bridges connecting adjacent prisms were observed. A configuration resembling a bifurcating prism is presented.     

Evidence for the involvement of kainate receptors in synaptic transmission in the avian cochlear nucleus

Previous studies using various excitatory amino acid antagonists have shown that synaptic transmission between the auditory nerve and the cochlear nucleus of chickens (nuc. magnocellularis; NM) is mediated by non-N-methyl-D-aspartate (non-NMDA) receptors. In the present study we have attempted to define the subclass of non-NMDA receptor in the NM by examining the effects of various excitatory amino acid agonists on synaptically evoked field potentials in an in vitro preparation of the chicken brain stem. Both quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), whose actions operationally define the quisqualate receptor class, caused variable and weak depression of evoked responses in the NM, as did L-glutamate. Kainic acid, on the other hand, completely blocked postsynaptic responses at micromolar concentrations. We conclude that kainate-preferring non-NMDA receptors play a predominant role in mediating transmission in the NM.     

The formation and final structure of the oocyst wall ofEimeria acervulina: A transmission and scanning electron microscope study

Summary The formation and final structure of the oocyst wall ofEimeria acervulina is described, based on a detailed electron microscope study of the maturing oocysts. After fertilization of the macrogametocyte the wallforming bodies of type I progressively undergo disaggregation into smaller bodies and eventually move into spaces left by the pellicular membranes of the zygote, which simultaneously separate and elevate away from the zygote cytoplasm to form the outer layer of the oocyst wall. A newly formed membrane separates this layer from the cytoplasm. Following the formation of the outer layer, another membrane separates and elevates away from the cytoplasm, and the wall-forming bodies of type II, which by now have migrated to the periphery, move into the spaces and fuse together to form the inner layer of the oocyst wall. A newly formed membrane separates this layer from the cytoplasm. The wall of the young oocyst thus consists of two membrane-bound layers of approximately similar thickness; the outer layer being osmiophilic whilst the inner one is paler. An overlying membrane covers the oocyst. The surface of the oocyst wall was smooth in appearance when viewed with the scanning electron microscope.     

The area centralis of the retina in the cat and other mammals: focal point for function and development of the visual system

In many mammals, particularly species with frontalised eyes, a small region o retina is strongly specialised for high resolution, binocular vision. The region is typically located near the centre of the retina, a few millimetres temporal to the optic disc, and is termed the "area centralis" or, in some primates in which the specialisation is particularly well developed, the "fovea centralis". Where the specialisation is well developed, the area or fovea centralis dominates the organisation of the adult visual system. Studies of the histogenesis of the retina of the cat indicate that the process of retinal maturation is centred on the area centralis, which thus seems to be an organising focus in the ontogeny as well as the adult function of the visual system.