GABAA-receptor-mediated increase in intracellular Ca2+ concentration in the regenerating retina of adult newt

We used optical recording with the Ca(2+)-sensitive dye, fura-2, in living slice preparations from the newt retina at different stages of regeneration. gamma-Aminobutyric acid (GABA) induced pronounced [Ca(2+)](i) rise in progenitor cells and differentiating ganglion cells in the 'intermediate' stage of retinal regeneration. This [Ca(2+)](i) rise became less pronounced at the beginning of synapse formation in the late regenerating retina. At the late period of the late regenerating retina with the IPL thickness comparable to that of the control retina, GABA-induced [Ca(2+)](i) rise became undetectable or sometimes a small decrease in [Ca(2+)](i) was observed in regenerated ganglion cells. In contrast, N-methyl-d-aspartate (NMDA)-induced [Ca(2+)](i) rise appeared in premature ganglion cells and became prominent gradually as the regeneration proceeded. The [Ca(2+)](i) rise to GABA was mediated by GABA(A) receptors. This was shown by inhibition of GABA-induced Ca(2+) response with the preincubation of the GABA(A) receptor antagonist, bicuculline. The [Ca(2+)](i) rise due to GABA was suppressed in the absence of extracellular Ca(2+) or in the presence of the L-type voltage-gated Ca(2+) channel blocker, verapamil, suggesting that Ca(2+) may be entered through L-type Ca(2+) channels. Transient appearance of [Ca(2+)](i) rise to GABA during regeneration and origin of GABA-induced [Ca(2+)](i) rise were similar to those in the developing retina [J. Neurobiol. 24 (1993) 1600]. These similarities may suggest that common mechanisms may control neurogenesis and/or synaptogenesis during development and regeneration.     

GnRH receptor mRNA expression by in-situ hybridization in the primate pituitary and ovary

Gonadotrophin-releasing hormone (GnRH) receptors are presenton the ovary as well as in the anterior pituitary gland. GnRHanalogues may exert their actions in part via these ovarianreceptors. However, in the primate ovary, GnRH receptors areof low affinity and their significance is questionable. Theaim of the present study was to compare pituitary and ovarianexpression of the GnRH receptor mRNA by in-situ hybridizationto gain further information on the possible significance ofthe ovarian receptor. Pituitaries and ovaries were obtainedfrom two stump-tailed macaque monkeys and three marmoset monkeysat the mid-luteal phase of the ovulatory cycle. Human corporalutea were obtained during the early and mid-luteal phase andafter ‘rescue’ by human chorionic gonadotrophin(HCG) and a whole ovary obtained during the late luteal phase(n=1 per group). Frozen tissue sections were incubated witha 33P-labelled probe to the human GnRH receptor and exposedfor 4 weeks. All pituitary glands exhibited intense silver grainsin the anterior pituitary gland. In the ovaries, grains werepresent at low levels in the granulosa cells of antral follicles,just above tissue background in corpora lutea and indistinguishablefrom tissue background in the remaining ovarian compartments.These results demonstrate that the GnRH receptor mRNA in theprimate pituitary is present in sufficient quantities to beclearly detectable in the anterior pituitary gland by in-situhybridization. In contrast, in the human and monkey, ovary levelsof mRNA appear to be very low. corpus luteum/follicle/GnRH mRNA/in-situ hybridization     

Gene expression variation in the adult human retina

Despite evidence that differences in gene expression levels contribute significantly to phenotypic variation across individuals, there has been only limited effort to study gene expression variation in human tissue. To characterize expression variation in the normal human retina, we utilized a custom retinal microarray to analyze 33 normal retinas from 19 donors, aged 29-90 years. Statistical models were designed to separate and quantify biological and technical sources of variation, including age, gender, eye laterality, gene function and age-by-gender interaction. Although the majority of the 9406 genes analyzed showed relatively stable expression levels across different donors (for an average gene the expression level value of 95 out of a 100 individuals fell within a 1.23-fold range), 2.6% of genes showed significant donor-to-donor variation, with a false discovery rate of 10%. The mean expression ratio standard deviation was 0.15+/-0.8, log2, with a range of 0.09-0.99. Genes selectively expressed in photoreceptors showed higher expression variation than other gene classes. Gender, age and other donor-specific factors contributed significantly to the expression variation of multiple genes, and groups of genes with an age- and gender-associated expression pattern were identified. Our findings show that a significant fraction of gene expression variation in the normal human retina is attributable to identifiable biological factors. The greater expression variability of many genes central to retinal function (including photoreceptor-specific genes) may be partially explained by the dynamics of the vision process, and raises the possibility that photoreceptor gene expression levels may contribute to phenotypic diversity across normal adult retinas. In addition, as such diversity may result in different levels of disease susceptibility, exploring its sources may provide insights into the pathogenesis of retinal disease.     

Tyrosine hydroxylase mRNA in the dopaminergic neurons of young adult and aged mice by in situ hybridization

Tyrosine hydroxylase (TH) mRNA in the dopaminergic neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) in young adult and aged mice was detected and quantitated using in situ hybridization. Using 3H-labeled antisense RNA complementary to TH mRNA, these studies demonstrate the presence of TH mRNA in dopaminergic neurons of the SN and the VTA. Alternate sections stained immunocytochemically using TH-specific antiserum demonstrated that the neurons containing TH mRNA also contained TH protein. Quantitative analysis of the number of silver grains present over the dopaminergic neurons of the SN and VTA revealed no statistically significant difference between the two age groups. The results suggest that TH gene expression in dopaminergic neurons of the SN and VTA is not different in young adult and aged mice.     

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.     

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.     

Analysis of glomerular VEGF mRNA and protein expression in murine mesangioproliferative glomerulonephritis

Capillary repair is crucial in the healing of glomerulonephritis (GN). The vascular endothelial growth factor (VEGF) has pro-angiogenic properties and plays an important role in glomerular capillary regeneration. Habu Snake Venom (HSV) GN, a murine model for mesangioproliferative GN, was induced in uninephrectomized C57/BL6 mice. Glomerular damage and capillary repair were assessed using morphometry, stereology, and confocal laser scanning microscopy. Mesangiolytic glomeruli were microdissected (days 1,3,7,14) using laser capture microdissection technique. VEGF mRNA expression was analyzed by real-time polymerase chain reaction and compared to intact glomeruli of healthy controls. Spatiotemporal VEGF gene and protein expression was determined using nonradioactive in situ hybridization and immunohistochemistry. On day 1, diseased animals developed focal mesangiolysis paralleled by a significant decrease in length density of glomerular capillaries that gradually returned to baseline levels thereafter, indicating capillary growth in response to initial injury. Glomerular VEGF mRNA expression increased on day 3 and returned back to baseline and beyond at day 14 when the glomerular recovery process was completed. Similarly, glomerular VEGF protein expression tended to be higher on day 3. The present study documents temporarily increased glomerular VEGF gene and protein expression during the healing of HSV GN, suggesting a potential role of VEGF in the repair of mesangiolytic glomerular damage. Christian S. Haas and Valentina Campean contributed equally to this work.     

Differential expression of human telomerase catalytic subunit mRNA by In situ hybridization in pheochromocytomas

In pheochromocytomas, it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. We investigated the expression of human telomerase catalytic component (hTERT) mRNA, hTERT protein, Ki-67 antigen, and p27^kip1 in pheochromocytomas (27 benign, 7 suspected malignant, and 7 malignant), and evaluated the possibility of expressions of these proteins, and hTERT mRNA serve as diagnostic markers for predicting the biological behavior of these tumors. All tumors showed the classical histology and typical immunohistochemical pattern. By in situ hybridization, hTERT mRNA was expressed in 5/7 malignant tumors (defined as the presence of metastasis and/or extensive local invasion) as compared with 3/27 benign tumors. We examined the hTERT by immunohistochemistry to confirm the mRNA. hTERT mRNA expression was correlated with hTERT protein expression. All benign tumors exhibited no immunopositivity or <1% of cells stained for Ki-67 antigen. Six out of seven malignant tumors have shown either hTERT mRNA expression or Ki-67 immunoreactivity While no statistical difference in p27^kip1 expressions was observed among benign, malignant, and suspected malignant tumors, there was a statistical difference between the normal adrenal medulla samples and tumors (p<0.001). Thus, hTERT mRNA detection by in situ hybridization, hTERT expression, and Ki-67 antigen expression are all useful tools for differentiating malignant from benign pheochromocytomas.     

Fine structure of nerves in the regenerating limb of the newt Triturus

The structural integrity of some tissues and the regeneration of extremities in some vertebrates depends upon the nervous system. To investigate the structure of nerves exhibiting trophic function, nerves in regenerating forelimbs of adult newts, Triturus viridescens, were studied with the electron microscope. Nerve fibers sprout from the transected axons, 2–3 days after amputation of the limb, and invade all portions of the blastema and epidermis in large numbers. Regenerating nerve fibers contain a greatly increased amount of smooth-surfaced channels of endoplasmic reticulum containing moderately dense material, an increased number of microtubules, and large (1000 Å) membrane-bounded dense granules. The latter were not observed normally and could be distinguished from synaptic vesicles and dense-core vesicles thought to contain catechol amines. In larger nerves, the organelles are distributed in the peripheral axoplasm around a central zone containing neurofilaments. The relationship of the fine structural changes occurring during regeneration to the trophic action of nerves and the regeneration of nerve fibers is discussed. The tortuous membranous tubules of endoplasmic reticulum could serve as channels for the transport of substances, either trophic material or materials necessary for growth of the nerve, down the axon. Microtubules may play a role in the regulation of form of the growing axon and also be related to axoplasmic flow or migration of particles (e.g., granules) along their length. The large membrane-bounded dense granules appearing during regeneration resemble neurosecretory granules, which have been associated with regeneration in some invertebrates. These structures could, therefore, contain a trophic substance or hormone that is transported down the axon, released into the intercellular spaces, and controls subsequent regeneration of the limb.     

成年大鼠脑神经元aFGF基因mRNA表达

目的：为aFGF对成年大鼠脑内神经元的作用提供理论依据。方法：观察了成的大鼠脑内神经元mRNA表达分布情况。结果：成年大鼠脑皮质,海马,下丘离和中脑神经元核内均有不同程度的aFGF基因表达,结论：aFGF在脑多个部位神经元的表达可能与神经元的存活和功能活动的维持有关。     

用细胞原位杂交方法检测NPY mRNA在PC12细胞中的表达

目的：建立ＮＰＹ ｍＲＮＡ物细胞原位杂交检测方法,并以ＮＧＦ为诱导因子研究Ｄｅｘ对大鼠嗜铬细胞瘤ＰＣ１２细胞中ＮＰＹｍＲＮＡ表达的影响。方法：采用细胞原位杂交方法。方法：ＮＧＦ具有诱导ＮＰＹ基因表达的生物学效应且呈剂量效应关系;Ｄｅｘ对ＮＰｘＲＮＡ表达具有双相调节效应,即早期（〈８ｈ）可促进ＮＧＦ的诱导作用,而晚期（〉１６ｈ）则具有抑制作用（Ｐ〈０．０１）,但单独Ｄｅｘ对ＮＰＹｍＲＮＡ表达无显著影     

Autoradiographic localization of mas proto-oncogene mRNA in adult rat brain using in situ hybridization

The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medical part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.