Quantitative Electron Microscopic Analysis Research Articles

Effect of pinealectomy on the photoperiod-dependent changes of the specific secretory cells and à-subunit mRNA level in the chicken pars tuberalis

The effect of pinealectomy on the synthesis of the common alpha-subunit of glycoprotein hormones by the specific secretory cells of the pars tuberalis (PT-specific cells) was examined in male chicks. Expression of melatonin receptor (Mel(1c)) mRNA was demonstrated in the chick pars tuberalis by reverse transcription-polymerase chain reaction analysis. Northern blot analyses revealed that, after pinealectomy, common alpha-subunit mRNA levels were increased in the pars tuberalis of chicks kept under normal lighting (L:D=12 h:12 h), indicating that melatonin inhibits the synthesis of the alpha-subunit of PT-specific cells. Furthermore, alpha-subunit mRNA levels in the pars tuberalis were shown to display similar photoperiod-dependent changes in pinealectomized chicks as in intact animals. Levels of alpha-subunit mRNA in the pars tuberalis were decreased in both pinealectomized and control chicks kept under continuous light (L:D=24 h:0), whereas the levels were enhanced in pinealectomized chicks kept under extended darkness (L:D=1 h:23 h) and under normal lighting. Thus, pinealectomy did not affect the inhibition or stimulation of the alpha-subunit synthesis in the chicken pars tuberalis elicited by continuous light or extended darkness, respectively. Quantitative electron-microscopic analyses showed that, after exposure to continuous light for 30 days, many PT-specific cells were filled with enlarged secretory granules in both pinealectomized and control chicks. Exposure to extended darkness for 30 days caused an increase in the cytoplasmic and nuclear areas of the PT-specific cells. Secretory granules were however larger in pinealectomized than in intact control chicks. These results suggest that the activity of PT-specific cells is mainly regulated by photoperiod.

Abnormalities of Synapses and Neurons in the Hippocampus of Neuropsin-Deficient Mice

In the present study, we produced null-mutant mice of neuropsin, an extracellular matrix serine protease, to examine the neural functions of this protein particularly in the hippocampus. Golgi–Cox impregnation and Nissl-staining revealed morphological change of cell soma in the mutant mice compared to wild-type mice. However, Golgi–Cox impregnation revealed no apparent change in the dendritic arborization and spine density. Quantitative electronmicroscopic analysis revealed that number of asymmetrical synapses were significantly decreased in the stratum radiatum, the major terminal field of Schaffer-collaterals, whereas free boutons still holding synaptic vesicles but with no synaptic specialization were increased in number in the same microscopic fields. An increased number of parvalbumin-immunoreactive cells (known as fast spiking cells) in mutant was also observed. These results strongly suggest that neuropsin is involved in connectivity of a group of CA1 synapses and consequently in the hippocampal networking.

Experimental hypoxia of STZ-diabetic rat myocardium and protective effects of Ginkgo biloba extract: II. Ultrastructural investigation of microvascular endothelium

Four months after induction of diabetes by intraperitonal injection of 60 mg streptozotocin/kg body weight wistar rats were exposed to an acute respiratoric hypoxia of 20 min duration. One group of the rats received daily Ginkgo biloba extract EGb 761 (100 mg/kg body weight). By means of qualitative and quantitative electron microscopic analysis we compared the hypoxia-induced ultrastructural alterations of the myocardial microvascular endothelium in normal, diabetic, and EGb-protected rats. Aim of the study was to compare the hypoxia tolerance of myocardial microvessels of normal and diabetic rats and to test the possibility of antioxidative protection. The results revealed that only some ultrastructural microvascular parameters of diabetic rats were stronger altered after acute hypoxia than normal ones, e.g. capillary dilatation, number of lysosomes, frequency of vesicles and fused vesicles, endothelial swelling, and structural state of mitochondria. Other parameters exhibited less severe alterations than in healthy rats, as luminal blebbing and protrusions, endothelial vacuoles, mitochondrial swelling, and pericapillary debris. Protective effects of EGb could be demonstrated on endothelial swelling, blebbing, vesiculation and vesicular fusioning, partly on vacuolization, but not on mitochondrial parameters. The results were discussed on pathobiochemical background. EGb 761 was estimated to be protective against hypoxic damage on myocardial microvessels also in diabetic condition, but the study should be completed by inclusion of a reoxygenation interval.

Quantitative immunogold evidence that glutamate is a neurotransmitter in afferent synaptic terminals within the isthmo-optic nucleus of the pigeon centrifugal visual system

A quantitative electron microscopic analysis of glutamate (GLU) immunoreactivity using the post-embedding immunogold technique was carried out within the isthmo-optic nucleus (ION) of the pigeon centrifugal visual system (CVS). Measurements were performed in each of eight different categories of axon terminals, including those that were GABA-immunoreactive (-ir), considered representing control profiles and identified using a single or double-label immunocytochemical procedure. The results demonstrated that the glutamate immunogold particle densities for both mitochondrial and vesicular pools and for total surface area of bouton profiles were significantly higher in P1a, P1b and P2b terminals and not significantly different in P4 and P5 terminals compared to those recorded in control GABA-ir terminals (P2a, P2c, P3). Moreover, the values measured in GLU-ir positive profiles were all significantly higher than in either P4 or P5 terminals. The results suggest that tectal neurons, which provide the main input to the ION cells, are either inhibitory GABA-ir possibly associated with P2c and/or P3 terminals or excitatory GLU-ir via P1a, P1b and P2b terminals. Such differential effects of tectal afferents may be the basis for the modulation of centrifugal activity and consequently of end target retinal ganglion cell responses. The data are relevant to hypotheses implicating the avian CVS in mechanisms of selective enhancement of visual attention to either novel or meaningful stimuli within the visual field.

Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor.

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.

Interfacial adaptation of a Class II polyacid-modified resin composite/resin composite laminate restoration in vivo

The aim of this in vivo study was to evaluate the interfacial adaptation of Class II resin composite open sandwich restorations with a polyacid-modified resin composite as a stress-absorbing layer (PMRC/RC). Twenty Class II box-shaped, enamel-bordered cavities were prepared in 10 premolars scheduled to be extracted for orthodontic reasons. An open PMRC/RC sandwich restoration was placed in 1 of the cavities of each tooth. The first layer, PMRC, in the proximal box extended to the periphery in the cervical part of the cavity. The following RC layers were placed with a horizontally incremental technique. The PMRC was excluded from the control cavity. The teeth were extracted after 1 month and the interfacial adaptation of the restorations was studied with quantitative scanning electron microscope analysis using a replicate technique. Gap-free interfacial adaptation was observed for the PMRC/RC and RC restorations in cervical enamel in 97% and 73%, respectively (P = 0.006). The gap-free scores for dentin were 87% and 64%, respectively (P = 0.022). Excellent interfacial adaptation was observed in both groups for the occlusal enamel 99% and 100%, respectively. The adaptation to occlusal enamel for the direct resin composite restorations was significantly better than to dentin or cervical enamel. A higher frequency of enamel fractures was observed parallel to the cervical margins compared to the occlusal. No dentin fractures were observed in the experimental groups. The PMRC/RC sandwich technique showed a statistically significant improved interfacial adaptation to dentin and cervical enamel in Class II enamel-bordered cavities. The clinical significance of the differences has to be evaluated.

The organization of piriform cortex and the lateral olfactory tract following the loss of mitral cells in PCD mice.

Homozygous Purkinje Cell Degeneration (PCD) mice exhibit a selective loss of olfactory bulb mitral cells (MCs) after 4 months of age. This selective degeneration leaves a subpopulation of denervated granule cells which establish new reciprocal dendro-dendritic synapses with unaffected tufted cells (TCs) (14). This suggests a capacity for plasticity in TCs and raises the question of whether a comparable degree of reorganization occurs in their axonal terminals in piriform cortex (PC) following the loss of MCs. Homozygous (experimental) and heterozygous (control) PCD mice were routinely perfused and processed for electron microscopy. A quantitative electron microscopic analysis was performed on radially oriented micrograph montages spanning from the pia into layer II of PC. After MC loss in the experimental animals there was a decrease in density of larger myelinated axons in the lateral olfactory tract (LOT). Myelinated axons in the LOT had a mean cross-sectional diameter of 1.26 +/- 0.04, and 0.81 +/- 0.025 microm in the control and experimental mice, respectively. In superficial layer I of PC, control mice had presynaptic axonal terminals from mitral and tufted cells with characteristic electron lucent (light) profiles establishing asymmetric synapses with pyramidal cell dendrites. In contrast, the experimental mice showed a decrease in electron lucent terminals and a robust increase in electron dense (dark) presynaptic associational terminals. Although the overall synaptic density did not differ between the control and experimental mice (16.40 +/- 0.94 and 18.10 +/- 0.96 synapses/100 microm2, respectively), an overall decrease in the thickness of Layer 1 suggests that the total number of synapses decreases following MC loss. In addition to the apparent increase of associational terminals, the diameter of terminal enlargements increased as well as the number of multiple synaptic contact per terminals in the experimental animal, suggesting further compensatory mechanisms for the loss of MC presynaptic terminals.

Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons.

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).

Clinical evaluation of different adhesive systems for restoring teeth with erosion lesions

This investigation evaluated the performance of a resin-modified glass ionomer, a compomer, and a bonding system/composite combination for the restoration of cervical erosion lesions without cavity preparation. Forty-eight lesions (11 patients) were restored with a bonding agent/composite combination [Prime & Bond 2.1/PrismaTPH (P & B/TPH); DeTrey/Dentsply], a compomer (Dyract; DeTrey/Dentsply), or a light-curing glass ionomer (Fuji II LC; Fuji). The materials were randomly assigned to the patients in triplets. No cavity preparation was performed. The procedures strictly followed the manufacturers' instructions. The restorations were evaluated clinically, using modified USPHS criteria, and by quantitative scanning electron microscope (SEM) analysis, at baseline and 12 months. The clinical data were statistically evaluated with the Pearson chi-square test, the SEM data (criterion gap formation) were analyzed with the Mann-Whitney U-test and error rates method. Clinically, two restorations could not be evaluated. One Dyract restoration failed. With respect to marginal discoloration, recurrent caries and contour, no significant differences could be found between the materials. The surface texture of P & B/TPH and Dyract was significantly better than that of Fuji II LC at baseline and 12 months. Compared to P & B/TPH and Fuji II LC, Dyract revealed a significant decrease in marginal integrity between baseline and 12 months. In SEM analysis, gap formation was determined as follows: baseline, enamel interface: 4% Dyract= 4% Fuji >2% P & B/TPH and dentin interface: 11% Dyract >9% P & B/TPH >2% Fuji; 12 months, enamel interface: 15% Dyract >4% Fuji >3% P & B/TPH and dentin interface: 11% P & B/TPH >6% Fuji >5% Dyract. The error rates method revealed no significant differences, in general, between the three materials with regard to gap formation. In conclusion, the restorations of erosion lesions with different classes of adhesive materials were well retained after 12 months. None of the materials studied revealed superiority over the other materials. All materials revealed shortcomings with respect to either surface texture, marginal integrity or color stability clinically and for all materials gap formation was recorded in the SEM evaluation.

Enhanced expression of AMPA receptor protein at perforated axospinous synapses.

We performed a quantitative electron microscopic analysis of the middle third of the molecular layer in the dentate gyrus of rat, using material processed with postembedding gold labeling for the glutamate receptor subunits GluR1, GluR2/3, or NMDAR1. Perforated axospinous synapses were at least twice as likely as non-perforated ones to express detectable levels of AMPA receptor subunits, whereas no significant differences in NMDA receptor expression were observed. These data imply that perforated synapses may be especially potent, and are consistent with the hypothesis that insertion of AMPA receptor protein into the postsynaptic membrane of previously silent synapses contributes to long-term potentiation.

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction.

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.

Repression of the A8L gene, encoding the early transcription factor 82-kilodalton subunit, inhibits morphogenesis of vaccinia virions.

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the Escherichia coli lac operator system. The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels. Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication. Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.

Synaptic plasticity of 5-hydroxytryptamine-immunoreactive terminals in the phrenic nucleus following spinal cord injury: A quantitative electron microscopic analysis

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)-immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT-immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT-labeled terminals formed axodendritic contacts, but some 5-HT-labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection.

Synaptic plasticity of 5‐hydroxytryptamine–immunoreactive terminals in the phrenic nucleus following spinal cord injury: A quantitative electron microscopic analysis

Synaptic plasticity of 5‐hydroxytryptamine–immunoreactive terminals in the phrenic nucleus following spinal cord injury: A quantitative electron microscopic analysis

The effects of tracheal occlusion and release on type II pneumocytes in fetal lambs

Fetal tracheal occlusion (TO) has been shown to lead to lung hyperplasia in various animal models, and this procedure has already been carried out in human fetuses with congenital diaphragmatic hernia (CDH). However, the authors previously showed that TO caused a decrease in type II pneumocytes. Purpose: The aim of this study is to examine the effects of TO and release on type II pneumocytes. Method: TO was carried out with a Swan Ganz or Fogarty catheter in fetal sheep at 116 to 118 days of gestation. TO was maintained for 2 weeks followed by deflation of the balloon for 1 week before delivery, in group 1; in group 2, TO was maintained for 19 days and released 2 days before delivery. Group 3 consisted of previously reported animals who had TO maintained until birth. Unoperated twins served as controls. All specimens were analyzed using the surfactant protein C (SP-C) mRNA as a specific marker for type II pneumocytes. We used Northern Blot and in situ hybridization techniques to quantify total SP-C and the density of type II cells. Electron microscopy (EM) was also used to evaluate and quantitate type II cells. Results: TO resulted in significant lung growth in all groups. In situ hybridization and Northern Blot analysis showed that there was a complete recovery of type II cells in group 1 versus controls. Quantitative EM analysis confirmed these findings. In group 2 the number of type II cells was decreased but there was an increase in SP-C content per type II cell versus group 3. Conclusion: Lung growth after TO appears to occur at the expense of type II cell differentiation. This effect is reversible with the release of TO before birth in this animal model.

Lack of gonadotropin-positive feedback in the male rat is associated with lack of estrogen-induced synaptic plasticity in the arcuate nucleus.

We have demonstrated that estrogen induces synaptic reorganization in the hypothalamic arcuate nucleus of female rats during the ovarian cycle and proposed that estrogen-induced synaptic retraction plays a role in the disinhibition of gonadotropin secretion that occurs during the afternoon of proestrus. This so-called positive feedback of gonadotropins is developmentally determined. It is present in female rats and absent in males. To confirm the role of the estrogen-induced synaptic retraction in positive feedback, we tested whether administration of estrogen to male rats also fails to induce synaptic remodeling of the arcuate nucleus. Male and female rats were gonadectomized and studied as follows. One month following gonadectomy, animals received either a single injection of estradiol (100 micrograms/animal in sesame oil; 12 males and 12 females) or vehicle (6 males and 6 females). Twenty-four hours following injections, all animals in the vehicle-injected group and 6 animals of each sex in the treatment groups were sacrificed, while the rest of the animals were killed 48 h following the hormone injections (6 per group). As expected, quantitative electron microscopic analysis of the female arcuate nuclei revealed that compared to oil-injected controls, estradiol induced drastic decreases in the overall synapse counts by 24 h (121 +/- 10 vs. 74 +/- 5 synapses/1,000 microns membrane; p < 0.05). Synaptic counts had recovered to control levels by 48 h. On the contrary, in males, estradiol treatment did not cause changes in the total synapse counts at either time. As a further control, the lack of an estrogen-induced gonadotropin surge in long-term castrate males was also confirmed. Our study confirmed that in males estradiol does not alter the net synaptology of the arcuate nucleus or cause gonadotropin positive feedback. This is in clear contradistinction to females which show both synaptic plasticity and gonadotropin-positive feedback upon receiving exogenous or endogenous estrogen. The lack of estrogen-induced synaptic plasticity may be an underlying mechanism in the abolishment of positive gonadotropin, feedback in developing males and the development of constant estrus in aging female rats.

Metabolic and neurochemical plasticity of gamma-aminobutyric acid-immunoreactive neurons in the adult macaque striate cortex following monocular impulse blockade: quantitative electron microscopic analysis.

The purpose of the present study was to examine the effects of retinal impulse blockade on gamma-aminobutyric acid (GABA)-immunoreactive (GABA-IR) neurons in cytochrome oxidase (CO)-rich puffs of the adult monkey striate cortex. Specifically, we wished to know if changes occurred in their CO activity, GABA immunoreactivity, and synaptic organization. A double-labeling technique, which combined CO histochemistry and postembedding GABA immunocytochemistry on the same ultrathin sections, was used to reveal simultaneously the distribution of the two markers. We quantitatively compared changes in GABA-IR neurons of deprived puffs (DPs) with respect to non-deprived puffs (NPs) 2 weeks after monocular tetrodotoxin treatment. We found that the proportion of darkly CO reactive mitochondria in GABA-IR neurons of DPs drastically decreased to about half of those in NPs. There was a greater reduction of CO levels in GABA-IR axon terminals than in their cell bodies and dendrites. In contrast, most non-GABA-IR neurons displayed no significant change in their CO levels. Morphologically, GABA-IR neurons and axon terminals in DPs showed a significant shrinkage in their mean size. GABA immunoreactivity, as indicated by the density of immunogold particles in GABA-IR neurons, declined in DPs, and a greater decrease was also found in axon terminals than in cell bodies or dendrites. Moreover, the numerical density of GABA-IR axon terminals and synapses in DPs was significantly reduced without changes in that of asymmetric and symmetric synapses. Thus, the present results support the following conclusions: 1) Oxidative metabolism and neurotransmitter expression in GABA-IR neurons are tightly regulated by neuronal activity in adult monkey striate cortex; 2) GABA-IR neurons are much more vulnerable to functional deprivation than non-GABA-IR ones, suggesting that these inhibitory neurons have stringent requirement for sustained excitatory input to maintain their heightened oxidative capacity; and 3) intracortical inhibition mediated by GABA transmission following afferent deprivation may be decreased in deprived puffs, because the oxidative capacity and transmitter level in GABAergic neurons, especially in their axon terminals, are dramatically reduced.

White matter of the cerebellum of the chicken (Gallus domesticus): a quantitative light and electron microscopic analysis of myelinated fibers and fiber compartments.

Low magnification light microscopic examination of the white matter in appropriately stained avian and mammalian cerebellum reveals a mediolateral succession in which areas of large, heavily myelinated fibers alternate with areas containing nearly exclusively small fibers. A large fiber accumulation (LFA) and its medially adjoining small fiber area (SFA) form a fiber compartment, which, with related parts of cortex and central nuclei, constitutes a so-called cerebellar module. The composition and the apparent mediolateral heterogeneity of cerebellar fiber compartments was quantified in the chicken by morphometrical analysis of myelinated fiber profiles in light (LM) and electron (EM) microscopic micrographs. In LM versus EM, approximately 37% of the myelinated fiber population is neglected. This deficit concerns profiles that are smaller than 1.2 micron2 (diameter < 1.2 microns). EM analysis is therefore considered a prerequisite and forms the main part of this study. The myelinated fiber population has a left-skewed log normal size distribution. Ninety-nine percent of the myelinated fibers fall within the range of 0.1 to 20 microns2 (diameter = 0.4-5.0 microns) and 90% are even smaller than 7 micron2 (diameter < 3.0 microns). Small fibers are abundant in both parts of the compartment. Statistical comparisons provide quantitative confirmation of the LM distinction of LFAs and SFAs. It appears, moreover, that, apart from typical LFAs and SFAs, transitional zones rather than sharp borders can be distinguished between the two. The medial border of the LFA appears to be more sharply defined than its lateral border. Distinct mediolateral fluctuations were found with respect to fiber density (166-243 fibers/1,000 microns2), mean profile area (2.4-4.0 microns2), and interspace (31-47%). These differences reflect the contrast between LFA (lower density, larger mean profile area) and SFA (higher density, smaller mean profile area). The interspace discriminates less well between LFA and SFA but is often smaller in the LFA and larger in the SFA. The presented quantitative characteristics of mediolateral heterogeneity in the cerebellar fiber layer can be used as reference for morphometric studies on the different fiber systems of the cerebellar white matter and the functional organization of the compartments.

Diphtheria Toxin-mediated Ablation of Parietal Cells in the Stomach of Transgenic Mice

The self-renewing epithelial populations present in the gastric units of the mouse stomach are descended from a multipotent stem cell and undergo an orderly migration-associated differentiation followed by apoptosis. The steady state census of the three principal cell types (acid-producing parietal cells, mucus-producing pit cells, and pepsinogen and intrinsic factor-producing zymogenic cells) is accurately controlled, despite marked differences in the rates of migration of each lineage. A transgenic mouse model has been created to define functional interrelationships between the proliferation, differentiation, and death programs of these lineages. Nucleotides -1035 to +24 of the noncatalytic beta subunit gene of mouse H+/K+-ATPase were used to direct expression of an attenuated diphtheria toxin A subunit in the parietal cell lineage. These transcriptional regulatory elements are not active in members of the pit and zymogenic lineages. Stomachs, prepared from postnatal day 28-80 transgenic mice and their normal littermates, were subjected to single- and multilabel immunohistochemical studies as well as qualitative and quantitative light and electron microscopic morphologic analyses. The toxin produced complete ablation of differentiated parietal cells. Loss of parietal cells was accompanied by a 5-fold increase in the number of undifferentiated granule-free cells located in the proliferative compartment of gastric units. This amplified population of granule-free cells included the multipotent stem cell as well as committed precursors of the pit and zymogenic lineages. Loss of mature parietal cells was also associated with (i) a block in the differentiation program of the zymogenic lineage with an accumulation of pre-neck cells and a depletion of their neck and mature zymogenic cell descendants, and (ii) an approximately 2-fold amplification of pit cells. These findings are consistent with the notion that epithelial homeostasis within gastric units is maintained by instructive interactions between their different cell lineages. Unlike pit and zymogenic cells, parietal cells complete their differentiation in the gastric unit's proliferative compartment before undergoing a bipolar migration along the unit. Thus, the mature parietal cell is in a strategic position to influence decision-making among gastric epithelial cell precursors and to modulate the migration-associated terminal differentiation programs of the pit and zymogenic lineages.

Spine loss in experimental epilepsy: quantitative light and electron microscopic analysis of intracellularly stained CA3 pyramidal cells in hippocampal slice cultures.

The sequence of neuronal alterations resulting from epileptic activity is poorly understood. In the hippocampus of some epileptic patients, there is a loss of certain neuronal types in the hilar region and in CA3. The neuronal alterations preceding this degeneration probably affect synaptic structures. Here we have estimated the number of dendritic spines, major postsynaptic elements of hippocampal neurons, in defined dendritic segments of identified (intracellularly stained) CA3 pyramidal neurons in "epileptic" slice cultures of hippocampus and in control cultures. Slice cultures were prepared from five- or six-day-old rat pups and maintained in vivo for 23 days before epileptic activity was induced by application of the convulsants bicuculline and picrotoxin for three days. Individual CA3 pyramidal neurons were then intracellularly injected with horseradish peroxidase, and the number of dendritic spines was counted in proximodistal dendritic segments by applying the Sholl method. In addition, the total dendritic length was measured and the branching index evaluated. The number of spines on CA3 pyramidal cell dendrites in the "epileptic" cultures was found to be decreased by 40%. This spine loss affected proximal and peripheral dendritic segments of the CA3 pyramidal neurons to a similar extent. No significant differences were observed between control and "epileptic" cultures in dendritic length or in the branching index. Quantitative electron microscopic analysis did not reveal differences between "epileptic" cultures and control cultures in the spine area of the labelled CA3 pyramidal cells, indicating that there was a real spine loss, not just a reduction in the size of the spines. We conclude that epileptic activity causes morphological alterations in defined postsynaptic compartments of hippocampal pyramidal cells surviving under these conditions.