Quantitative Electron Microscopic Methods Research Articles

Structural and Histochemical Changes in Rat Parietal Cortex Neurons during Biliary Drainage

The aim of the study reported here was to assess structural and metabolic changes in rat parietal cortex neurons during complete external bile drainage. Quantitative histochemical and electron microscopic methods were used. The findings showed that drainage of bile from the body led to gradual increases in the numbers of hyperchromic pyknotic neurons and ghost cells, death and reductions in the numbers of neurons, and increases in the number of glial cells in all layers of the parietal cortex. There was a gradual decrease in neuron size, with loss of sphericity (elongation). The cytoplasm showed decreases in succinate dehydrogenase, NADH, NADPH, and glucose-6-phosphate dehydrogenases, and decreases in RNA content, though these changes were accompanied by activation of lactate dehydrogenase and acid phosphatase. Nuclei and organelles (mitochondria, endoplasmic reticulum, Golgi complexes) underwent destructive changes, which were accompanied by increase in the numbers and sizes of lysosomes and phagosomes. Some neurons showed activation of the nuclear apparatus and increases in the relative content of free ribosomes. Thus, loss of bile from the body induced progressive increases in structural and metabolic changes in parietal cortex neurons, leading to the death of some and compensatory changes in others.

Systemic administration of lipopolysaccharide induces molecular and morphological alterations in the hippocampus

A systemic inflammatory reaction may have detrimental effects on the organism, including the central nervous system. Previous studies have indicated that lipopolysaccharide (LPS)-evoked systemic inflammation induces pathological alterations in the mouse midbrain, especially in the substantia nigra. The aim of the present study was to investigate whether the hippocampus is also affected after an intraperitoneal (i.p.) injection of LPS. We focussed on the dynamics of proinflammatory gene expression and the processes leading to neuronal cell death. A systemic inflammatory response in C57BL/6 mice was induced by an i.p. injection of LPS (1 mg/kg b.w.). The genetic, biochemical and morphological alterations were analysed up to 96 h after LPS administration using quantitative PCR, immunochemical, immunocytochemical and electron microscopic methods. Real-time PCR analysis indicated an altered expression of several genes, mainly responsible for arachidonic acid release and metabolism, in the hippocampus 96 h after the systemic administration of LPS. Three hours after LPS treatment, the level of mRNA for iNOS, COX-2 and TNFα was increased; then, after 6–24 h, it rose for TLR4 and cPLA2. The expression of 5-LOX and 12-LOX was increased at 12–24 and 24–48 h after LPS injection, respectively. Our data demonstrate for the first time the sequential activation of the expression of several pro-inflammatory genes responsible for the maintenance of the inflammatory response. Moreover, the electron microscopy studies presented the stimulation of apoptosis-inducing factor (AIF)-mediated death signalling and cathepsin B-related autophagy or necrosis. These biochemical and morphological alterations in the hippocampus, which were induced by systemic inflammation, may be responsible for the impairment of cognition function observed previously.

Structural and functional responses of extremity veins to long-term gravitational loading or unloading—lessons from animal systems

Long, transparent tubular tilt-cages were developed to maintain experimental rats either in 45 ∘ head-up (orthostasis model), or in 45 ∘ head-down body position (antiorthostasis model) for several weeks. In order to study the functional and structural changes in extremity blood vessels, also novel pressure angiograph systems, as well as special quantitative electron microscopic methods were applied. It was found that several adaptive mechanisms are activated in the lower limb superficial veins and microvessels of muscles when an organism is exposed to long-term (1–2 weeks) orthostatic-type gravitational load including a reversible amplification of the pressure-dependent myogenic response, tuning of the myogenic tone by Ca ++ - and voltage-sensitive K + channels in humans, augmentation of the intramural sympathetic innervation involving an increased nerve terminal density and synaptic vesicle count with functional remodeling, reorganization of vascular network properties (microvascular rarefaction in muscles, decreased branching angles in superficial veins), and responses of an endothelin and platelet-derived growth factor (PDGF) containing vesicle system in the endothelium. On the other hand, when applying long-term head-down tilting, the effects are dichotomous, e.g. it suppresses significantly the pressure-induced myogenic response, however does not diminish the adventitial sympathetic innervation density.

Fine structural cytochemical analysis of homologous chromosome recognition, alignment, and pairing in Guinea pig spermatogonia and spermatocytes.

The nuclei of guinea pig spermatogonia and spermatocytes were studied by means of quantitative autoradiography and electron microscopic methods such as high-resolution cytochemistry, immunocytochemistry, and in situ hybridization. Our observations reveal, in the nucleus of spermatogonia type B, small lampbrush structures of extended chromatin not found in nonmeiotic cells. During meiotic interphase, pairs of parallel lampbrush structures become associated by numerous filaments. The formation of the synaptonemal complex is simultaneous with the extension of chromosomal axes in a continuous leptotene-zygotene stage. Some chromosomes do not recognize their homologs before the onset of the leptotene-zygotene stage and undergo classical leptotene and zygotene stages. The immunocytochemical localization of Dmc1 and Rad51 supports the idea that these proteins are not involved in homology search and final pairing. Immunolocalization of DNA, RNA polymerase II, heterogeneous nuclear ribonucleoproteins, small nuclear ribonucleoproteins, and the trimethyl-guanosin cap of small nuclear RNAs suggests that the chromatin of lampbrush structures transcribe hnRNA and that splicing is scarce. The results of quantitative autoradiography after [3H]uridine labeling show an intense transcription accompanied by a very slow export of RNA. In situ hybridization demonstrates the presence of RNA in the regions of homology recognition and pairing. These results lead us to propose that the RNA synthesized in the lampbrush structures is involved in the process of homology searching and recognition.

Hemidesmosomes Show Abnormal Association with the Keratin Filament Network in Junctional Forms of Epidermolysis Bullosa

Junctional epidermolysis bullosa is a group of hereditary bullous disorders resulting from defects in several hemidesmosome-anchoring filament components. Because hemidesmosomes are involved not only in keratinocyte-extracellular matrix adherence, but also in normal anchorage of keratin intermediate filaments to the basal keratinocyte membrane, we questioned whether this intracellular function of hemidesmosomes was also perturbed in junctional epidermolysis bullosa. We used quantitative electron microscopic methods to assess certain morphologic features of hemidesmosome-keratin intermediate filaments interactions in skin from normal subjects (n = 11) and from patients with different forms of junctional epidermolysis bullosa (n = 13). In addition, skin from patients with autosomal recessive epidermolysis bullosa simplex with plectin defects (n = 3) or with autosomal recessive dystrophic epidermolysis bullosa (n = 4) were included as controls. Values were expressed as a percentage of the total number of hemidesmosomes counted. In normal skin 83.3% +/- 3.3 (SEM) hemidesmosomes were associated with keratin intermediate filaments and 90.1% +/- 1.9 had inner plaques. In Herlitz junctional epidermolysis bullosa (laminin 5 abnormalities, n = 4) these values were reduced to 45.3% +/- 11.5 (p < 0.001; analysis of variance) and 50.3% +/- 12.8 (p < 0.001), respectively. In junctional epidermolysis bullosa with pyloric atresia (alpha6beta4 abnormalities, n = 3) the values were also reduced [41.8% +/- 7.0 (p < 0.001) and 44.5% +/- 5.7 (p < 0.001), respectively]. In the non-Herlitz group (laminin 5 mutations, n = 3) the counts were 66.7% +/- 7.1 (p > 0.05) and 70.5% +/- 8.5 (p < 0.05), and in skin from patients with bullous pemphigoid antigen 2 mutations (n = 3) the counts were 54.3% +/- 13.8 (p < 0.01) and 57.1% +/- 13.9 (p < 0.01). In epidermolysis bullosa simplex associated with plectin mutations the values were 31.9% +/- 8.9 (p < 0.001) for keratin intermediate filaments association and 39.9% +/- 7.1 (p < 0.001) for inner plaques. Findings in recessive dystrophic epidermolysis bullosa patients' skin were indistinguishable from normal control skin with inner plaques (90.5% +/- 2.5) and keratin intermediate filaments attachment (86.3% +/- 2.1). These findings suggest that the molecular abnormalities underlying different forms of junctional epidermolysis bullosa appear to affect certain critical intracellular functions of hemidesmosomes, such as the normal connections with keratin intermediate filaments. This may have important implications for the maintenance of basal keratinocyte integrity and resilience in junctional epidermolysis bullosa.

Circadian rhythms in screening pigment and invaginating organelles in photoreceptor terminals of the housefly's first optic neuropile

Screening pigment granules occur in the synaptic terminals of photoreceptors in the fly's (Musca domestica, L.) compound eye. The granules resemble ommochrome granules in the overlying photoreceptor cell body. There are also two types of invagination into receptor terminals: capitate projections (from glial cells) and invaginations from neighboring receptor terminals. The number of profiles of these organelles in the first optic neuropile, the lamina, have been counted using single-section quantitative electron microscopic methods. Pigment granules are concentrated proximally in the terminal, toward the brain. The numbers change, increasing during the night (1 h after lights off) up to values more than twice the number 1 h after lights on, apparently by longitudinal migration of granules from the cell body into the terminal. Files entrained to day/night conditions and then held under constant darkness continue to exhibit changes in the numbers of profiles. Even though overall there were 80-90% fewer granule profiles than under day/night conditions, the numbers attained a peak many times higher at the end of the subjective day. Thus, the changes are endogenous, showing circadian rhythmicity. Although their significance is unknown, these changes parallel previously described circadian rhythms in the receptor terminals and their lamina monopolar-cell targets. The invaginations from receptor terminals were more numerous under day/night conditions than under constant darkness, and cycled in constant darkness, peaking at the end of subjective night. Capitate projections, by contrast, failed to change significantly under the experimental conditions analyzed, a lack of responsiveness they share with photoreceptor tetrad synapses.

Circadian rhythms in screening pigment and invaginating organelles in photoreceptor terminals of the housefly's first optic neuropile

Screening pigment granules occur in the synaptic terminals of photoreceptors in the fly's (Musca domestica, L.) compound eye. The granules resemble ommochrome granules in the overlying photoreceptor cell body. There are also two types of invagination into receptor terminals: capitate projections (from glial cells) and invaginations from neighboring receptor terminals. The number of profiles of these organelles in the first optic neuropile, the lamina, have been counted using single-section quantitative electron microscopic methods. Pigment granules are concentrated proximally in the terminal, toward the brain. The numbers change, increasing during the night (1 h after lights off) up to values more than twice the number 1 h after lights on, apparently by longitudinal migration of granules from the cell body into the terminal. Flies entrained to day/night conditions and then held under constant darkness continue to exhibit changes in the numbers of profiles. Even though overall there were 80–90% fewer granule profiles than under day/night conditions, the numbers attained a peak many times higher at the end of the subjective day. Thus, the changes are endogenous, showing circadian rhythmicity. Although their significance is unknown, these changes parallel previously described circadian rhythms in the receptor terminals and their lamina monopolar-cell targets. The invaginations from receptor terminals were more numerous under day/night conditions than under constant darkness, and cycled in constant darkness, peaking at the end of subjective night. Capitate projections, by contrast, failed to change significantly under the experimental conditions analyzed, a lack of responsiveness they share with photoreceptor tetrad synapses. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 517–529, 1997

Altered synaptic circuitry in the human temporal neocortex removed from epileptic patients.

Quantitative electron microscopic methods were used to study possible alterations in presumptive excitatory and inhibitory synaptic circuits in human neocortex removed from patients with intractable temporal lobe epilepsy. Synaptic density was compared between normal and abnormal regions as identified by Nissl staining and immunocytochemistry for the Ca2+-binding protein parvalbumin (PV). The normal regions showed a normal cytoarchitecture and normal pattern of staining for PV, whereas the abnormal regions displayed focal neuronal cell loss and a decrease in immunostaining for PV. In the abnormal regions the overall synaptic density (per 100 microm2 and per mm3) was approximately 30% higher than in normal regions, which corresponded to an increase of approximately 300 million synapses per mm3. The number of excitatory and inhibitory synapses was significantly higher and lower, respectively, than in normal regions. We suggest that these changes are a result of a focal sprouting of excitatory axon terminals and loss of inhibitory terminals which leads to hyperexcitatory synaptic circuits. These circuits may represent a neural substrate for the initiation or propagation of seizure activity in human epileptogenic neocortex.

The effects of light reversals on photoreceptor synaptogenesis in the fly Musca domestica.

The short-term influence of visual experience was studied in the first-order tetrad synapses of the first optic neuropil, or lamina, underlying the compound eye in the housefly (Musca domestica). We report the effects of single light reversals, in which flies reared in constant darkness were exposed to light or those in constant light were exposed to darkness, on this defined population of synapses. The synaptic population was characterized by quantitative electron microscopic methods using three parameters: (i) the number of synaptic contacts per photoreceptor terminal; (ii) the size of these synapses, given by the mean platform width of their presynaptic ribbons; and (iii) the proportion (up to approximately 20%) of small synapses-those having a ribbon platform less than 0.17 micron wide. The effects of light exposures in flies reared in constant darkness include decreased mean synaptic size and increased numbers of synapses. These effects are seen in flies of all ages (to 10 days). Long light exposures (2-6 h) are much less effective than short exposures (down to 20 s), with maximum effects obtained at 15 min (up to 45% more synapses). Small synapses are reasoned to be new junctions formed only recently; the decreased mean synaptic size and the increased number of synapses seen after short light exposures are both interpreted to result from a recent burst of synaptogenesis in the adult lamina. The effects of dark exposure in flies reared in constant light are the reciprocal of those seen in dark-reared flies exposed to light, but they are less pronounced. Although the function of such changes is not yet known, they may form part of the light adaptation mechanism of the photoreceptor, and occur along with a redistribution of other organelles involving membrane invaginations into its terminal. These changes occur against a background trend for control flies reared under constant conditions to have fewer, larger synapses with increasing age up to 10 days, an effect that is most pronounced in constant darkness, when synaptic number decreases by 21% and size increases by 13%.

Quantitative analysis of synaptogenesis in the inner plexiform layer of macaque monkey fovea.

Synaptogenesis has been tracked by using quantitative electron microscopic methods in the inner plexiform layer (IPL) of the developing Macaca monkey fovea from fetal day (Fd) 55 to Fd132. Vesicle-containing profiles were classified according to whether (1) they contained a ribbon indicating that they originated from a bipolar cell, or (2) the profile formed a junction. Group 2 was further subdivided by morphological characteristics into (2a) amacrine, (2b) bipolar, or (2c) unknown profiles. Ribbon-containing bipolar profiles are clearly identifiable at Fd55 when they occur at a density of 0.9/100 microns2. Bipolar synapses increase rapidly to 4.7/100 microns2 by Fd88, similar to their density at Fd132. Identifiable amacrine profiles forming a junction are rare at Fd55-68. By Fd88, amacrine synaptic density has jumped to 6.7/100 microns2 and continues to increase to 9.5/100 microns2 at Fd132. These quantitative data strongly suggest that, at the Macaca fovea, bipolar synaptogenesis both begins and ends before amacrine synaptogenesis. The large number of immature amacrine synaptic profiles and densities at Fd132 suggests that amacrine synapses continue to form after Fd132. This study confirms that cone-dominated monkey fovea has a different sequence of synaptogenesis than the rod-dominated peripheral retina (Nishimura and Rakic, [1985] J. Comp. Neurol 241:420-434). The data support the concept that synaptic developmental sequence is determined by the type of photoreceptor which dominates a particular retinal region or species. Bipolar ribbon synapses are observed in the outer half of the IPL at Fd55, are present in the inner IPL at Fd60, and then, with increasing age, are found throughout the IPL. This pattern strongly suggests that vertical OFF bipolar pathways form earlier than ON pathways in the IPL. In contrast, amacrine profiles are found throughout the IPL at the youngest ages, with an adult-like banding pattern present by Fd132.

Age-related changes in cartilage proteoglycans: quantitative electron microscopic studies.

Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules.

Growth cone distribution patterns in the optic nerve of fetal monkeys: implications for mechanisms of axon guidance

The distribution of growth cones was studied in the optic nerve of monkeys during the first half of prenatal development using quantitative electron microscopic methods. Our aim was to test the hypothesis that ganglion cell growth cones extend predominantly along the surfaces of the nerve, just beneath the pia mater. A complete census of growth cones in cross sections of the nerve during the early phase of axon ingrowth, from embryonic day 39 (E39) to E41, demonstrates that growth cones are scattered within the majority of fascicles, even those located far from the surface of the nerve. By E45, growth cones are concentrated around the nasal, dorsal, and ventral edge of the optic nerve. They are less concentrated in the core and around the temporal edge. However, even as late as E49, virtually all fascicles in the nerve, whether deep or superficial, contain growth cones. Growth cones are dispersed within single fascicles and are often located far from glia. Thus, the newest fibers penetrate deep parts of the pathway and push through centers of densely packed bundles of older axons. This finding is consistent with the vagrant paths of growing axons reported in previous work on embryonic monkey optic nerve (Williams and Rakic, 1985). Our data challenge the hypotheses that growth cones extend selectively along the basal lamina, the pia mater, or glial end feet. Gradients found at later stages of development in the nerve are not due to a particular affinity of growth cones for non-neuronal substrata. The pattern we observed is much more likely to result from central-to-peripheral gradients in ganglion cell generation and possible associations between growth cones originating from the same regions of the retina.

Morphometric analysis of human epidermis treated with testosterone and dehydroepiandrosterone in organ culture

The influence of testosterone and dehydroepiandrosterone (DHA) on human epidermal differentiation in skin organ culture were studied with quantitative light and electron microscopic morphometric methods. The hormones had equal effects on the general tissue architecture at the concentrations of 10(-7) mol/l: they had a negligible effect on the thickness of the vital and cornified layers of epidermis, and on the type of keratinization, but caused a significant increase in the number of granular cells. The electron microscopic stereologic analyses showed that testosterone did not alter the size of either vital or cornified epidermal cells. Neither did it influence the quantities of major keratinocyte organelles (keratin filaments, desmosomes, ribosomes, mitochondria) in the different epidermal strata. However, the amounts of keratohyalin granules and keratinosomes were significantly higher in testosterone-treated granular cells than in controls. Qualitative electron-microscopic analysis of DHA-treated skin showed a similar change in the amount of keratohyalin granules and keratinosomes. The present findings suggest that although androgenic hormones exert little or no direct anabolic effect on epidermis, they may modulate keratinocyte maturation.

Neurogenic inflammation in the rat trachea. II. Identity and distribution of nerves mediating the increase in vascular permeability.

This study addresses the question of whether increased vascular permeability, which is a prominent feature of neurogenic inflammation in the respiratory tract, is mediated by sensory axons that end near venules in the airway mucosa. In these experiments, neurogenic inflammation was produced in the tracheal and bronchial mucosa of atropine-treated Long-Evans rats by electrical stimulation of the left or right superior laryngeal nerve and/or cervical vagus nerve. The particulate tracer Monastral blue was injected intravenously to localize the sites of increased vascular permeability, and microspectrophotometry was used to measure the amount of extravasated Monastral blue in the trachea and thereby quantify the increase in vascular permeability. In some rats, selective denervations were made to locate the cell bodies of neurons that mediate the increase in vascular permeability; in others, fluorescence immunohistochemistry and quantitative electron microscopic methods were used to determine which structures in the tracheal mucosa are innervated by these neurons. The study revealed that the vagally mediated increase in vascular permeability was sudden, transient (half-life = 2.4 min) and restricted to venules. Stimulation of the left or right superior laryngeal nerve increased the permeability of venules in the extrathoracic trachea, whereas stimulation of either vagus nerve increased vascular permeability in the intrathoracic trachea and bronchi. All nerves had bilateral effects in the trachea, but the vagus nerves had largely unilateral effects in the bronchi. Neurons that mediated the increase in venular permeability had their cells bodies in the jugular (superior sensory) ganglion of the vagus nerve or rostral portion of the nodose (inferior sensory) ganglion. Preganglionic autonomic vagal neurons in the brain stem were not essential for this increase in venular permeability. Few nerves identifiable by substance P-immunohistochemistry or electron microscopy were located near the affected venules, and no nerves were within 1 micron of the walls of venules. However, the epithelium and arterioles of the airway mucosa were densely innervated. All intraepithelial nerves were within 0.1 micron of epithelial cells, and at least two-thirds of nerves near arterioles were within 1 micron of the vessel walls. We conclude that the increase in venular permeability associated with neurogenic inflammation in the trachea and bronchi of rats is mediated by sensory axons that travel in the vagus nerves and superior laryngeal nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Energy Dissipation in Nuclear Ceramic Materials

The application of image-analytical, indenting, and quantitative scanning electron microscopic methods on irradiated uranium and mixed-oxide fuel materials yields nonlinear thermodynamic operational data. Any oxide fuel material from a specific fabrication process undergoes its own means of transformation toward thermodynamic equilibrium through structural metamorphoses by irreversible processes far from equilibrium. The related dissipation energies vary with burn-up and temperature because of the variable capability of energy storage, due to specific structural reactions on nuclear impacts. This fact leads to a large variety of operational material properties. Preferable operational hyperelasticity and subsequent viscoelasticity can be predetermined by intentional selected kinetic processing during fuel fabrication.

Replacement of synaptic terminals in lamina II and Clarke's nucleus after unilateral lumbosacral dorsal rhizotomy in adult cats

Evidence from previous light-microscopic studies suggested that lumbosacral dorsal rhizotomy in cats elicits sprouting of converging undamaged systems into partially deafferented Clarke's nucleus and lamina II. We therefore applied quantitative electron-microscopic methods to determine whether this sprouting is associated with replacement of synaptic terminals (reactive reinnervation). We used stereological and morphometric methods to estimate terminal number per cross section in right and left lamina II and Clarke's nucleus in adult cats after acute and chronic unilateral (right-sided) lumbosacral deafferentation. Planimetric measurements of area indicated no significant shrinkage of either region as a result of the deafferentation; an increase in area occupied by glial cytoplasm (gliosis) equaled the decrease occupied by axonal components. The gliosis appears to persist indefinitely, although the degenerative debris stainable with conventional light-microscopic methods does not persist. Analysis of the synaptic population of lamina II reveals that the large central or "scalloped" terminals comprise a substantial fraction (greater than 40%) of the total area occupied by terminals in control material and that this population is largely lost upon deafferentation, leaving a large population of small terminals with spherical vesicles. Nevertheless, estimates of total terminal number indicate no difference between control and deafferented lamina II, suggesting a rapid and virtually complete replacement of lost dorsal root terminals by small terminals containing spherical vesicles. Terminal number in Clarke's nucleus also remains constant despite the loss of the dorsal root input. We conclude that there is also a virtually complete and rapid replacement of lost terminals in Clarke's nucleus by terminals containing spherical vesicles. These data provide an example of a case in which axonal sprouting demonstrated with light-microscopic methods is associated with electron-microscopic evidence of reactive reinnervation.

Evaluation of quantitative electron microscopic methods in the early diagnosis of cutaneous T-cell lymphomas

Evaluation of quantitative electron microscopic methods in the early diagnosis of cutaneous T-cell lymphomas

Polyribosomes under developing spine synapses: growth specializations of dendrites at sites of synaptogenesis.

We have previously reported that there is a dramatic increase in polyribosomes associated with dendritic spines during periods of synapse growth induced by denervating lesions. We suggested that polyribosomes at the postsynaptic site may somehow be involved in the growth of synapses. To evaluate this hypothesis further, the present study determines whether synapses which are growing in the developmental period also have accumulations of polyribosomes. We examined the dentate gyrus of the developing rat electron microscopically at 7, 10, 15, 20, and 28 days of age, which spans the major period of synaptogenesis in this structure. Qualitative observations revealed dramatic accumulations of polyribosomes under spine synapses in the youngest animals (7 and 10 days of age). With synapse development, the accumulations of polyribosomes became less dramatic, so that by 28 days of age, the neuropil of the dentate gyrus appeared qualitatively mature. To determine the relationship between polyribosomes under spine synapses and synapse development, quantitative electron microscopic methods were use to evaluate synapse density (number of synapses/100 micron 2), and the incidence of polyribosome-containing spines (proportion of spine synapses with underlying polyribosomes) in the neuropil of the dorsal blade of the dentate gyrus at each age. An inverse relationship was found between synapse density and the proportion of spines with polyribosomes. Synapse density increased in an almost linear fashion between 7 and 28 days of age to levels which were actually somewhat higher than in mature rats, whereas the incidence of polyribosome-containing spines was highest at the youngest ages and decreased with development. Thus, polyribosomes were most prominent under spine synapses during the period of maximal synapse growth. These results, together with our previous observations of increased numbers of polyribosomes under spines during lesion-induced growth, suggest that the polyribosomes represent a structural specialization of dendrites at sites of synapse construction. We propose that they produce protein(s) that are involved in synapse growth.

Neuronal and synaptic structure of the dorsal lateral geniculate nucleus in normal and monocularly deprived Macaca monkeys.

The dorsal lateral geniculate nucleus (dLGN) of Macaca monkeys was studied by Golgi and quantitative electron microscopic (EM) methods to determine if differences in neuronal morphology exist which might correlate with the known physiological separation of X-type cells into the parvocellular and Y-type cells into the magnocellular laminae. Monocularly lid-sutured Macaca monkeys were also studied by quantitative EM methods to compare the synaptic organization within laminae innervated by the deprived and nondeprived retinae. We have divided our sample of Golgi-impregnated neurons into three groups: Types A, B, and C. Type A neurons comprise the majority of the projections cells and are quite heterogeneous in their overall morphology. Type B neurons have long dendrites with multiple appendages; some have a locally ramifying beaded axon Type C neurons are characterized by dendrites which are mainly restricted to the interlaminar zones. We found Type A and B neurons in both the parvocellular and magnocellular laminae. The cell bodies of Type C neurons lay within the interlaminar zones or the parvocellular laminae. All three types contributed dendrites to the interlaminar zones. No significant differences in Golgi morphology other than overall size were found in parvocellular or magnocellular laminae that would explain the previously demonstrated electrophysiological differences. Terminal profiles and synapses in the parvocellular, magnocellular, and interlaminar zones were classified and counted using quantitative EM methods. RSD and F terminals were most numerous in all three zones. RLP terminals were rare in the interlaminar zones. A new type of terminal, RMD was found in the magnocellular interlaminar zones. The laminar and interlaminar zones have the same overall synaptic density, but differed in types of synaptic terminals. The only quantitative difference between any of these regions was in the magnocellular laminae where the counts showed 70% more F terminals per unit area. The same quantitative methods were applied to the laminar and interlaminar zones of dLGNs from monocularly lid-sutured monkeys. We found no qualitative or quantitative difference between the synaptology of zones receiving input from the deprived retina compared to the open eye retina, nor between any regions of the dLGN in deprived monkeys compared to normal monkeys.

Circadian and cyclic LHRH variations in the organum vasculosum of the lamina terminalis of female and male rats.

The organum vasculosum of the lamina terminalis (OVLT) was investigated by immunofluorescence and quantitative electron microscopic methods, and its LHRH content was measured at intervals during the estrous cycle and in male rats. A well-defined circadian rhythm in the LHRH content was demonstrated in the OVLT. The highest value was observed on the day of proestrus with peaks at noon and in the late afternoon. The amount of LHRH was about the same in the male as in the female rats on the day of diestrus 1. The OVLT in both sexes contained LHRH-positive fibers and terminals, but less immunofluorescence was visible on the day of diestrus and in the male animals. Quantitative electron microscopic analysis shows that in the OVLT nerve terminals, abutting on the basement membrane, more granulated vesicles (80-90 nm in diameter) are present on the day of proestrus than on any other day of the cycle or in the male. Our results suggest that the OVLT may participate in some way in the control of the cyclic gonadotrophic function of the anterior pituitary and/or in the behavioral effect of sexual hormones.