The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Retrieval of four adaptive lineages in duiker antelope: evidence from mitochondrial DNA sequences and fluorescence in situ hybridization

Independent molecular markers (mitochondrial DNA sequences from two genes and fluorescence in situ hybridization with satellite DNA sequences as hybridization probes) were employed to investigate phylogenetic relationships among duiker antelope. When analyzed singly or taken together, the molecular and cytogenetic data allowed for the delimitation of four adaptive groups: the conservative dwarfs which are basal, a savanna specialist which groups apart from the forest duikers, the giant duikers, and the red duikers. Within the latter, a further subdivision comprising an east African and a west African red duiker clade is evident. The placement of the endangered zebra duiker and Aders' duiker remains problematic. Several of the nomenclatural divisions in current use are questioned by our results. These include the recognition of Philantomba as genus name for the blue and Maxwell's duiker and that Harvey's duiker be relegated to a subspecies of the Natal red duiker. We place our results in a biogeographic context and argue that duiker speciation has been driven predominantly by habitat fragmentation which probably led to the disruption of gene flow between geographic populations.     

Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Localization of liver fatty acid-binding protein and its mRNA in the liver and jejunum of rats: an immunohistochemical and in situ hybridization study

Summary The localization of liver fatty acid-binding protein (L-FABP) and its mRNA in the liver and jejunum was examined in normal and 3-day-fasted rats by means of immunohistochemistry using a specific antibody to L-FABP and in situ hybridization using a synthetic oligonucleotide complementary to L-FABP mRNA as probe. In the liver from normally fed rats, the signal for L-FABP mRNA in hepatocytes was distributed throughout the lobule, with higher intensity in the periportal than in the centrolobular region. After a 3-d fasting, the mRNA signal declined in intensity throughout the lobule, in accordance with the result of Northern blot analysis. Immunohistochemistry for L-FABP showed intralobular patterns of immunoreactivity similar to those of the mRNA signal in both fed and fasted animals. In the jejunum from fed rats, L-FABP-mRNA signal was abundant in the absorptive epithelial cells lining the lower two-thirds of villus and less abundant in the villus tip cells, while the intensity of L-FABP immunoreactivity remained high in the latter cells. Fasting brought about a downward shift of the mRNA signal to an area including the upper half of the crypt and the lower portions of villus, with decreased intensity in the rest of the villus. Immunohistochemistry also showed a downward extension of the immunoreactivity into the upper crypt area. The present results suggest that in situ hybridization is a useful tool to analyze regulations of the expression of L-FABP gene in the digestive organs in association with epithelial cell migration and dietary condition.