Vesicular Glutamate Transporter 1 (VGLUT1)- and VGLUT2-containing Terminals on the Rat Jaw-closing γ-Motoneurons

Currently, compared to jaw-closing (JC) α-motoneurons, the information on the distribution and morphology of glutamatergic synapses on the jaw-closing (JC) γ-motoneurons, which may help elucidate the mechanism of isometric contraction of the JC muscle, is very limited. This study investigated the distribution and ultrastructural features of vesicular glutamate transporter 1 (VGLUT1)- and VGLUT2-immunopositive (+) axon terminals (boutons) on JC γ-motoneurons by retrograde tracing with horseradish peroxidase, electron microscopic immunocytochemistry, and quantitative analysis. About 35% of the boutons on identified JC γ-motoneurons were VGLUT+, and of those, 99% were VGLUT2+. The fraction of VGLUT1+ boutons of all boutons and the percentage of membrane of JC γ-motoneurons covered by these boutons were significantly lower than those for the JC α-motoneurons, revealed in our previous work. The bouton volume, mitochondrial volume, and active zone area of the VGLUT2+ boutons on the JC γ-motoneurons were uniformly small. These findings suggest that the JC γ-motoneurons, in contrast to the JC α-motoneurons, receive generally weak glutamatergic synaptic input almost exclusively from VGLUT2+ premotoneurons that form direct synapse with motoneurons.

Quantitative Ultrastructural Analysis of Endings Presynaptic to the Tooth Pulp Afferent Terminals in the Trigeminal Oral Nucleus

The ultrastructural parameters related to synaptic release of endings which are presynaptic to tooth pulp afferent terminals (p-endings) were analyzed to understand the underlying mechanism for presynaptic modulation of tooth pulp afferents. Tooth pulp afferents were labelled by applying wheat-germ agglutinin conjugated horseradish peroxidase to the rat right lower incisor, whereafter electron microscopic morphometric analysis with serial section and reconstruction of p-endings in the trigeminal oral nucleus was performed. The results obtained from 15 p-endings presynaptic to 11 labeled tooth pulp afferent terminals were as follows. P-endings contained pleomorphic vesicles and made symmetrical synaptic contacts with labeled terminals. The p-endings showed small synaptic release-related ultrastructural parameters: volume, 0.82 ± 0.45 µm³ (mean ± SD); surface area, 4.50 ± 1.76 µm²; mitochondrial volume, 0.15 ± 0.07 µm³; total apposed surface area, 0.69 ± 0.24 µm²; active zone area, 0.10 ± 0.04 µm²; total vesicle number, 1045 ± 668.86; and vesicle density, 1677 ± 684/µm². The volume of the p-endings showed strong positive correlation with the following parameters: surface area (r=0.97, P<0.01), mitochondrial volume (r=0.56, P<0.05), and total vesicle number (r=0.73, P<0.05). However, the volume of p-endings did not positively correlate or was very weakly correlated with the apposed surface area (r=-0.12, P=0.675) and active zone area (r=0.46, P=0.084). These results show that some synaptic release-related ultrastructural parameters of p-endings on the tooth pulp afferent terminals follow the "size principle" of Pierce and Mendell (1993) in the trigeminal nucleus oralis, but other parameters do not. Our findings may demonstrate a characteristic feature of synaptic release associated with p-endings.

Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice

OBJECTIVE : The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 μm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice. .

eNOS se correlaciona com a biogênese mitocondrial em corações com cardiopatia congênita e cianose / eNOS correlates with mitochondrial biogenesis in hearts of congenital heart disease with cyanosis

FUNDAMENTO: O programa de biogênese mitocondrial no coração parece apresentar remodelação adaptativa após estresse biomecânico e oxidativo. Os mecanismos adaptativos que protegem o metabolismo do miocárdio durante a hipóxia são coordenados, em parte, pelo óxido nítrico (NO). OBJETIVO: Observar a biogênese mitocondrial e expressão do óxido nítrico sintase (NOS) em corações de cardiopatia congênita com cianose; discutir a resposta mitocondrial à hipóxia crônica do miocárdio. MÉTODOS: Foram investigados 20 pacientes com defeitos cardíacos cianóticos (n = 10) ou acianóticos (n = 10). Foram estudadas amostras do miocárdio na via de saída ventricular direita, tomadas durante a operação. A análise morfométrica de mitocôndrias foi realizada por microscopia eletrônica de transmissão. A relação mtDNA/nDNA foi determinada com PCR em tempo real. Os níveis de transcrição da subunidade I da citocromo c oxidase (COXI), coativador-1α do receptor γ ativado por proliferador de peroxissoma (PGC-1α), o fator respiratório nuclear 1 (NRF1), e fator de transcrição mitocondrial A (Tfam) foram detectados por reação em cadeia da polimerase via transcriptase reversa (RT-PCR) ativado por fluorescência em tempo real. Os níveis proteicos de COXI e nNOS, iNOS e eNOS foram medidos por técnica de Western Blot. RESULTADOS: A densidade volumétrica mitocondrial (Vv) e a densidade numérica (Nv) foram significativamente elevadas em pacientes com cianose, em comparação com a cardiopatia congênita acianótica. MtDNA elevada e suprarregulação dos níveis de COXI, PGC-1 α, NRF1 e Tfam mRNA foram observadas em pacientes cianóticos. Os níveis de proteína de COXI e eNOS foram significativamente maiores no miocárdio de pacientes cianóticos que nos de acianóticos. Os níveis de transcrição do PGC-1α se correlacionam com os níveis de eNOS. CONCLUSÃO: A biogênese mitocondrial é ativada no miocárdio da via de saída ventricular na cardiopatia congênita com cianose, que ...

BACKGROUND: Mitochondrial biogenesis program in heart appears to exhibit adaptive remodeling following biomechanical and oxidative stress. The adaptive mechanisms that protect myocardium metabolism during hypoxia are coordinated in part by nitric oxide (NO). OBJECTIVE: To observe mitochondrial biogenesis and nitric oxide synthase (NOS) expression in hearts of congenital heart disease with cyanosis, discuss mitochondrial response to chronic hypoxia in myocardium. METHODS: 20 patients with cyanotic (n=10) or acyanotic cardiac defects (n=10) were investigated. Samples from the right ventricular outflow tract myocardium taken during operation were studied. Morphometric analysis of mitochondria was performed with transmission electron microscope. Relative mtDNA/nDNA ratio was determined with real-time PCR. Cytochrome c oxidase subunit I (COXI), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (Tfam) transcript levels were detected by real-time fluorescent RT-PCR. COXI and nNOS, iNOS and eNOS protein levels were measured with western blot. RESULTS: Mitochondrial volume density (Vv) and numerical density (Nv) were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease. Elevated mtDNA and up-regulated COXI, PGC-1α, NRF1 and Tfam mRNA levels were observed in cyanotic patients. Protein levels of COXI and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic patients. PGC-1α transcript levels correlated with the levels of eNOS. Conclusion: Mitochondrial biogenesis is activated in right ventricular outflow tract myocardium in congenital heart disease with cyanosis, which could be the adaptive response to chronic hypoxia and possibly involves eNOS up-regulation. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0).

Ultrastructural analysis of low-threshold mechanoreceptive vibrissa afferent boutons in the cat trigeminal caudal nucleus / 대한해부학회지

Ultrastructural parameters related to synaptic release and their correlation with synaptic connectivity were analyzed in the low-threshold mechanoreceptive vibrissa afferent boutons in laminae III and IV of the trigeminal caudal nucleus (Vc). Rapidly adapting vibrissa afferents were intra-axonally labeled, and quantitative ultrastructural analyses with serial sections were performed on the labeled boutons and their presynaptic endings (p-endings). The volume of the labeled boutons was widely distributed from small to large ones (0.8~12.3 microm3), whereas the p-endings were small and uniform in size. The volume of the labeled boutons was positively correlated with the ultrastructural parameters such as mitochondrial volume (correlation coefficient, r=0.96), active zone area (r=0.82) and apposed surface area (r=0.79). Vesicle density (r=-0.18) showed little correlation to the volume of labeled boutons, suggesting that the total vesicle number of a bouton is proportional to its volume. In addition, the bouton volume was positively correlated with the number of p-endings (r=0.52) and with the number of dendrites postsynaptic to the labeled bouton (r=0.83). These findings suggest that low-threshold mechanoreception conveyed through vibrissa afferents is processed in a bouton size-dependent manner in the Vc, which may contribute to the sensory-motor function of laminae III/IV in Vc.

Effects of AG60 on Gastric Chief Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric epithelial cells and the gastric chief cells of the mouse inoculated with Ehrlich carcinoma cells in the inguinal area following administration of acriflavine-guanosine composition (AG60). Healthy adult ICR mice were divided into normal and experimental groups. In the experimental groups, each mouse was inoculated with 1x10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. The day following the 7th injection of saline or AG60, each mouse was injected with methyl-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, gastric tissues were taken and fixed in 10% buffered neutral formalin. Deparaffinized sections were coated with autoradiographic emulsion EM-1 and dried, and then placed in a light-tight box. The number of labeled epithelial cells in the gastric mucosae were observed and calculated. And for electron microscopic observation, gastric tissues were prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granules and mitochondria in the gastric chief cells were observed and calculated. On the autoradiographic study, number of labeled cells in the area of 3.5 mm width (6 micrometer thickness) of mouse gastric mucosae of normal control, tumor control and AG60-treated groups were 319.7+/-66.46, 343.7+/-47.72 and 102.3+/-54.99 respectively. On the electron microscopic study, the size of zymogen granule in the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.74+/-0.208 micrometer, 1.18+/-0.291 micrometer and 0.97+/-0.259 micrometer, respectively. And the mitochondrial size of the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.86+/-0.364 micrometer, 1.02+/-0.466 micrometer and 0.92+/-0.390 micrometer, respectively. And in the AG60 treated group, most chief cells did not show any difference in ultrastructure, except that myelin figures were more frequently observed, in comparison with that of nornmal control group. From the above results, AG60 may suppress the DNA synthesis of the gastric epithelial cells, but does not results severe fine structural defect on the gastric chief cells. These results suggest that AG60 is expected as one of the most effective anticancer drugs.

Quantitative Ultrastructural Analysis of Periodontal Afferent Terminals in the Trigeminal Motor Nucleus / 대한해부학회지

Little is known about processing mechanism of sensory input from the periodontal ligaments to the trigeminal motor nucleus for the control of chewing force and modulation of chewing pattern. Low threshold mechanoreceptive periodontal afferent was labeled with horseradish peroxidase by use of intra-axonal injection technique and investigated with electron microscopy. Quantitative ultrastructural analysis was performed on the 39 serially reconstructed labeled boutons in the trigeminal motor nucleus in cat. Labeled bouton contained clear spherical vesicles and one or two large dense cored vesicles. Most of labeled boutons were dome or round shape. All the analysed labeled boutons were presynaptic to dendritic shaft or distal dendrite and those presynaptic to soma or proximal dendrite were not observed. A large number of labeled boutons (46.2%) were postsynaptic to one or two presynaptic pleomorphic vesicle containing endings. Synaptic triad, in that a presynaptic ending which is presynaptic to the labeled bouton, in turn, is presynaptic to dendrite that is postsynaptic to the labeled bouton, was observed in 10.3% of the labeled boutons. Most of the labeled boutons showed simple synaptic organization, in that 64.1% of the labeled boutons made synaptic contacts with one or two neuronal profiles. One (2.6%) of the 39 analyzed labeled boutons showed synaptic contacts with 5 or more neuronal profiles. Labeled bouton volume, mitochondrial volume, apposed surface area and active zone area showed wide variation. These ultrastructural parameters were positively correlated with bouton volume. The values for apposed surface area and active zone area with presynaptic p-endings, in contrast to those with postsynaptic dendrites, showed narrow range and had little correlation with bouton volume. The present study revealed characteristic features on ultrastructural parameters of labeled boutons from periodontal afferent which is involved in periodontal masseteric reflex, and that influence on the postsynaptic trigeminal motoneurons showed wide variability.

Processing Mechanism of Sensory Information Originated from the Oral Cavity in the Trigeminal Nucleus Oralis / 대한해부학회지

To analyze the synaptic characteristics of axon terminals originated from the tooth pulp in the trigeminal nucleus oralis, labeling of tooth pulp afferents with wheat-germ agglutinin conjugated horseradish peroxidase and morphometric analysis with electron microscopic photographs were performed. The results obtained from 23 labeled endings were as follows. All of the labeled boutons contained clear and round synaptic vesicles (dia. 45~55 nm). 3 (13.64%) out of 23 labeled endings have 20~105 dense cored vesicles and do not make synaptic contacts with p-endings. But remaining 20 labeled endings (86.36%) almost do not have dense cored vesicles and 12 of them make synaptic contacts with p-endings. The mean number of synaptic contacts was 2.61+/-2.06 and the postsynaptic profiles were usually middle or distal dendrite and dendritic spine (1.74+/-1.36) rather than soma or proximal dendrite. The mean number of synaptic contacts with pendings was 0.87+/-1.01. And the frequency of the synaptic triads were 0.39+/-0.58. The vesicle density was 993.23+/-267.41/mum(2). The volume of labeled bouton was 3.54+/-2.20 mum(3) and highly correlated (P < 0.01) with surface area (11.78+/-4.92 mum(2), r = 0.95), total apposed surface area (2.90+/-1.56 mum(2), r=0.72), total active zone area (0.61+/-0.37 mum(2), r = 0.82), mitochondrial volume (0.75+/-0.53 mum(3), r = 0.94), the number of synaptic vesicles (2621.30+/-1473.61, r= 0.91) and the number of synaptic contacts (r = 0.76). These results suggest that there are two groups of tooth pulp afferent terminals according to the presence of dense cored vesicles in the trigeminal nucleus oralis. And the sensory processing mechanism of each groups may be different. And the "size principle" of Pierce & Mendell (1993) is also applicable to these terminals.

Quantitative and Ultrastructural Analysis of Afferent Terminals from Tooth Pulp in the Trigeminal Principal Sensory Nucleus / 대한해부학회지

To analyze the synaptic characteristics of axon terminals originated from tooth pulp in the trigeminal principal sensory nucleus, and find morphological evidence that p-endings on these terminals use GABA as neurotransmitter, labeling of tooth pulp afferents with wheat -germ agglutinin conjugated horseradish peroxidase, postembedding immunocytochemistry and morphometric analysis were performed. The results obtained were as follows. The labeled boutons contained clear and round synaptic vesicles, and the mean number of synaptic contacts was 4.21 +/- 2.58. The postsynaptic profiles were usually middle or distal dendrites and dendritic spines rather than soma or proximal dendrites. The mean number of synaptic contacts with p -endings was 2.62 +/- 2.01. The volume of labeled bouton was 4.39 +/- 3.08 micro meter 3 and highly correlated (P<.01) with surface area (r = 0.96), total apposed surface area (r = 0.84), total active zone area (r = 0.84), mitochondrial volume (r = 0.95), the number of synaptic vesicles (r = 0.92), the number of synaptic profiles (r = 0.76) and the number of synaptic p -endings (r = 0.67). The p -endings contained pleomorphic synaptic vesicles. The volume of p -ending was 0.91 +/- 0.47 micro meter 3 and highly correlated (P<.01) with surface area (r = 0.94), total apposed surface area (r = 0.76), total active zone area (r = 0.62), mitochondrial volume (r = 0.70) and the number of synaptic vesicles (r = 0.94). P -endings were GABA immunoreactive. These results suggest that tooth pulp afferent terminals in trigeminal principal sensory nucleus may be related to complex processing of sensory information and presynaptic p -endings using GABA as neurotransmitter, are important in this sensory processing. The "size principle" of Pierce and Mendel (1993) is also applicable to tooth pulp afferent terminals and presynaptic p -endings.

Morphological Change with the Induction of Cisplatin Resistance from the Bladder Cell Lines / 대한비뇨기과학회지

PURPOSE: When Managing metastatic bladder tumors, to overcome the resistance mechanism of cisplatin is a main problem to be solved. The objective is to confirm the changes of general and ultrastructural morph ology with the induction of cisplatin resistance from the bladder cell line. MATERIALS AND METHODS: The samples of this investigation are 2ng/ml-cisplatin resistant human bladder cell lines T24R2 established by SNUH Urology and the drug resistant bladder cell lines T24 was obtained from ATCC, as a control group. We cultured the resistant cell line on the slide and observed it using light microscopy to see the general morphology. For the ultrastructural morphology, we fixed cultured cells, made an epon block, sliced an ultrathin section and observed it using H-71000 EM. RESULTS: Under light microscopy, the cytoplasm of the resistant cell line shows a plumper pattern than that of the parent cell. Under electronmicroscopy, the chromatin of the resistant cell line has a relatively finely dispersed chromatin pattern when compared to the parent cell line, which shows a coarse and aggregated chromatin pattern. Within the cytoplasm, the mitochondrial volume, dilated rough endoplasmic reticulum, polyribosomes and ribosomes are moderately increased in the resistant cell line when compared to the parent cell line. In particular, we found a great amount of double membrane vesicle near the cell surface and pinocytic vesicles on the surface, which are seldom observed within the parent cells. CONCLUSIONS: We concluded that the cisplatin resistant human bladder cell lines (T24R2) underwent a morphological change with the induction of cisplatin resistance, and we hypothesize that the resistant cell's ultrastructure, which shows morphological change, will be involved in the drug resistance mechanism. Regarding this matter, further research will be needed.

Electron Microscopic Study of Cervical Carcinoma Tumor Cells after Chemotherapy / 대한부인종양콜포스코피학회잡지

Our aim was to identify histologic changes in cervical carcinoma tumor cells due to chemotherapy,by electron microscopic examination. Cisplatin and 5-fluorouracil were used between March 1, 1994 and February 28, 1995 on a total of 16 patients. The treatment schedule consisted of 75 mg/m2 cisplatin via iv with hyperhydration on Day 1, in combination with 1000 mg/m2 5-fluorouracil via continuous iv on Day 1 to 5. The treatment was repeated every 4 weeks with a maxinum of 3 courses. Ultrastructurally, chemotherapy induced apoptosis,indicated by condensation of nucleus and cytoplasm,fragmentation of nuclei, and apoptotic bodies. Due to repeat chemotherapy, increased levels of desmosomes were noted and parts of tumor nests were replaced by mature squamous cells. Tumor nests were reduced in size, mitochondria exhibited swelling, the endoplasmic reticulum was dilated, and infiltration of inflammatory cells increased after chemotherapy. These results suggest that chemotherapy induces apoptosis and injury to the cytoplasm and nuclei of tumor cells. However, the exact selection mechanism of the biochemical pathway of cell death is not fuUy understood. Further study of the molecular biologic actions of cell death after chemotherapy is warranted.