Single-cell Reverse Transcription-polymerase Chain Research Articles

Complex expression of natural killer receptor genes in single natural killer cells.

Human natural killer (NK) cells express several inhibitory and non-inhibitory NK receptors per cell. Understanding the expression patterns of these receptor genes in individual cells is important to understanding their function. Using a single-cell reverse transcription-polymerase chain reaction (RT-PCR) method, we analysed the expression of nine NK receptor genes in 38 resting CD56+ NK cells from peripheral blood of normal donors. We observed highly diverse patterns of receptor expression in these cells. No NK receptor is expressed universally in every CD56+ NK cell. The expressed receptor types per cell varied from two to eight. We specifically analysed the distribution of inhibitory (DL) and non-inhibitory (DS) killer immunoglobulin-like receptors (KIR). The frequency of individual receptor expression varied from 26% for 2DS2 to 68% for both 2DL1 and 2DL4. A comparison of the coexpression of DL and DS receptors showed a significant association in the expression of 2DL2 and 2DS2 (chi2=16.6; P<0.001) genes but no association between 2DL1 and 2DS1 or between 3DL1 and 3DS1 genes. Coexpression analysis of the 2DL1 and 2DL2 genes in 2DL4+ and 2DL4- cells showed a strong association in 2DL4+ but not in 2DL4- cells, suggesting a differential effect of the 2DL4 gene on the expression of 2DL1 and 2DL2 genes. Single-cell RT-PCR is a powerful tool to study multiple receptor gene expression ex vivo in individual NK cells and provides information about the expression pattern of KIR receptors that may suggest mechanisms of gene expression responsible for generation of the KIR repertoire.

A pTalpha-negative subpopulation of CD25+ TN thymocytes revealed by a transgenic marker.

We have recently generated 5'lambda5-huTAC mice, which express the human CD25 (huTAC) gene under the control of the 5'-flanking region of the mouse lambda5-gene. The huTAC-transgene was expressed in pre-B cells but neither in mature B cells nor in T cells of these mice. In this report we demonstrate that the transgene is also transiently expressed by adult CD25+ CD3-CD4-CD8- (triple negative, TN) thymocytes and in fetal thymocytes. The huTAC+, in contrast to the huTAC- subpopulation of the CD44+CD25+ TN cells, was unexpectedly found not to express the pTalpha-gene. Still the huTAC+CD44+CD25+ TN cells reconstituted the development of both alphabeta and gammadelta lineage cells equally efficiently as the pTalpha-expressing huTAC- fraction, demonstrating that this pTalpha-negative subpopulation contained precursors for both T-cell lineages. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that also in normal mice only a fraction of CD44+CD25+ and CD44-CD25+ TN cells expressed this gene. Taken together, these data indicate that huTAC transgene expression revealed a truly pTalpha-negative fraction of the CD44+CD25+ TN cells. The observation that not all precursors in the CD25+ TN population express the pTalpha-gene has important implications for the understanding of early T-cell development and T-cell lineage commitment.

First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H + ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all ‘patch–catch’ experiments and could be detected even in 2560-fold dilutions of the pollen contents. H + ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.

Correlation of the expression of kainate receptor subtypes to responses evoked in cultured cortical and spinal cord neurones

Responses of cultured rat cortical and spinal cord neurones to kainate (KA) have been related to the expression of KA receptor subunits revealed by single-cell reverse transcription polymerase chain reaction (RT-PCR). Semi-rapid application of KA evoked non-desensitizing responses with EC 50 values of 82 μM for cortical and 67 μM for spinal cord neurones. In the presence of concanavalin A, GYKI 53655 (100 μM) reduced responses of both types of neurone to KA by about 80% to leave a KA receptor-mediated response with an EC 50 of 4 μM on spinal cord neurones and 27 μM ( P<0.001) on cortical neurones. Ultra-fast application of KA to outside-out patches of cortical neurones evoked a non-decaying response which was reduced by about 30% by GYKI 53655 to reveal a transient response that desensitized by 92.5% with a time constant ( τ des) of 26.2 ms. Responses of spinal cord patches decayed by 47.8%. GYKI 53655 reduced the peak response by 8.3% and the residual response desensitized by 75.8%, with a τ des of 17.3 ms, all values being significantly smaller than for cortical neurones. Single-cell RT-PCR showed relative abundances of mRNAs for the KA receptors, GluR5, GluR6 and GluR7, of 12, 33 and 54% for cortical neurones and 38, 10 and 54% for spinal cord neurones, respectively. The relative abundances of KA1 and KA2 were 12 and 88% for cortical neurones, and 19 and 79% for spinal cord neurones, respectively. The most likely expression patterns of functional KA receptors is GluR6/KA2 for cortical neurones and GluR5/KA2 for spinal cord neurones.

C-Jun decreases voltage-gated K(+) channel activity in pulmonary artery smooth muscle cells.

Activity of voltage-gated K(+) (K(v)) channels controls membrane potential (E(m)) that regulates cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) by regulating voltage-dependent Ca(2+) channel function. A rise in [Ca(2+)](cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers vasoconstriction and stimulates PASMC proliferation. Whether c-Jun, a transcription factor that stimulates cell proliferation, affects K(v) channel activity in PASMCs was investigated. Infection of primary cultured PASMCs with an adenoviral vector expressing c-jun increased the protein level of c-Jun and reduced K(v) currents (I(K(V))) compared with control cells (infected with an empty adenovirus). Using single-cell reverse transcription-polymerase chain reaction, we observed that the mRNA level of Kv1.5 and the current density of I(K(V)) were both attenuated in c-jun-infected PASMCs compared with control cells and cells infected with antisense c-jun. Overexpression of c-Jun also upregulated protein expression of Kvbeta(2) and accelerated I(K(V)) inactivation. Furthermore, E(m) was more depolarized and [(3)H]thymidine incorporation was greater in PASMCs infected with c-jun than in control cells and cells infected with antisense c-jun. These results suggest that c-Jun-mediated PASMC proliferation is associated with a decrease in I(K(V)). The resultant membrane depolarization increases [Ca(2+)](cyt) and enhances PASMC growth.

Identification of purinergic receptors in retinal ganglion cells

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC–RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X 3–5, P2X 7). Immunohistochemistry with P2X 3,4 receptor subunit-specific antibodies showed a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

Impaired hyperpolarization in regenerated endothelium after balloon catheter injury.

Ca(2+)-activated K(+) (K(Ca)) channels control endothelial Ca(2+) homeostasis and the formation of vasodilators. After angioplasty, dysfunction of the regenerated endothelium leads to abnormal vasoregulation. In this study, we tested the expression and function of K(Ca) channels in regenerated endothelium at 6 weeks after balloon catheter injury of rat carotid arteries (CAs) by using single-cell reverse transcription-polymerase chain reaction, patch-clamp techniques, and analysis of vasoreactivity. In single regenerated endothelial cells (ECs), the percentage of ECs expressing the K(Ca) genes, rSK3 (12+/-8%) and rIK1 (22+/-9%), was significantly lower compared with the percentage of native ECs expressing these genes (rSK3 58+/-8%, rIK1 64+/-10%). In patch-clamp experiments, K(Ca) currents and acetylcholine-induced hyperpolarization were markedly reduced in regenerated ECs (shift of membrane potential -6+/-3 mV) compared with those in native ECs (shift of membrane potential -21+/-5 mV). In pressure myograph experiments, acetylcholine-induced dilation was impaired in reendothelialized CAs compared with normal CAs. Intraluminal application of the K(Ca) blocker apamin and charybdotoxin inhibited dilation by 30% in normal CAs but was without effect in reendothelialized CAs. Intraluminal application of 1-ethyl-2-benzimidazolinone (100 micromol/L), an opener of K(Ca) channels, evoked dilation by 29% in normal CAs but had no effect in reendothelialized CAs. In conclusion, the impaired expression of K(Ca) channels in regenerated endothelium results in defective hyperpolarization and impaired dilation. Thus, the impaired K(Ca) channel function contributes to functional alterations of regenerated endothelium after angioplasty.

Growth and Differentiation Factors Derived from the N‐Terminal Domain Of Pro‐Opiomelanocortin

1. We describe a novel paracrine control system in the pituitary gland, consisting of peptides derived from the N-terminal fragment of pro-opiomelanocortin (N-POMC), for example POMC(1-74) and gamma3-melanocyte-stimulating hormone (MSH). 2. By searching the target cells of these N-POMC fragments, using the rise of intracellular free calcium as a response system and single cell reverse transcription-polymerase chain reaction of hormone mRNA as a cell type identification method, we found that a considerable number of cells in normal rat pituitary display combinatorial expression of different pituitary hormone genes (further referred to as 'multihormone mRNA cells'), without indication that all these cells also produce or store the respective hormones translatable from these mRNA. The N-POMC fragments POMC(1-74) and gamma3-MSH preferentially target particular subsets of these multihormone mRNA cells. 3. We discovered a potentially novel receptor for gamma3-MSH on these cells; more precisely, on cells coexpressing growth hormone and prolactin. The putative novel receptor displays properties highly divergent from those of the known gamma3-MSH receptor (i.e. the melanocortin-3 receptor) and even of all other melanocortin receptors cloned today.

Mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos

Objective: To compare the level of mitochondrial ATPase 6 gene expression in unfertilized oocytes and cleavage-stage embryos. Design: Reverse transcription polymerase chain reaction was performed in unfertilized oocytes and cleavage-stage embryos derived from tripronucleate embryos to determine ATPase 6 gene expression. Setting: Department of Obstetrics and Gynecology, Human Genetics Laboratory, Infertility Medical Center of CHA General Hospital, College of Medicine, Pochon CHA University, Seoul, Korea. Patient(s): Oocytes were obtained from infertile couples undergoing in vitro fertilization. Intervention(s): Unfertilized oocytes collected at 48 hours after retrieval and cleavage-stage embryos derived from tripronucleate embryos were prepared for evaluation of mitochondrial gene expression. Main Outcome Measure(s): Comparison of ATPase 6 gene expression by using single-cell reverse transcription polymerase chain reaction. Result(s): Expression of unfertilized oocytes decreased compared with early cleavage-stage embryos. Conclusion(s): Our findings of decreased ATPase 6 expression in unfertilized oocytes suggest that there may be a decrease in the mitochondrial functional capacity of oxidative phosphorylation.

Monitoring gene expression of TNFR family members by beta-cells during development of autoimmune diabetes.

Autoimmune diabetes results from destruction of pancreatic beta-cells by islet-infiltrating leukocytes. Different molecular mechanisms seem to be involved in this destruction but the results from many studies have not provided a clear picture so far. Therefore, we have developed a multiplex single-cell reverse transcription polymerase chain reaction to analyze the expression of genes of the tumor necrosis factor receptor (TNFR) family in pancreatic beta-cells during the development of autoimmune diabetes in a TCR-HA x INS-HA double transgenic as well as a non-obese diabetic (NOD) animal model. To this end we have followed the expression of cell surface receptors of the TNFR family in NOD mice as well as in double transgenic mice that express in their T cells class II MHC-restricted TCR specific for peptide 111 - 119 from influenza hemagglutinin (TCR-HA) as well as hemagglutinin under the control of the rat insulin promoter (INS-HA). Both types of mice develop insulitis and diabetes spontaneously. The data show a significant increase in the expression of Fas and TNFR2 (p75) during the development of insulitis, whereas TNFR1 (p55) is already expressed in beta-cells before the onset of insulitis. As ligands for these receptors are already expressed at high levels during the phase of insulitis, it is possible that beta-cell death is regulated by intracellular inhibitors of apoptosis pathways.

Adenosine receptor expression and modulation of Ca(2+) channels in rat striatal cholinergic interneurons.

Adenosine is a potent regulator of acetylcholine release in the striatum, yet the mechanisms mediating this regulation are largely undefined. To begin to fill this gap, adenosine receptor expression and coupling to voltage-dependent Ca(2+) channels were studied in cholinergic interneurons by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reaction. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly all of these interneurons (90%, n = 28) expressed detectable levels of A(1) adenosine receptor mRNA. A(2a) and A(2b) receptor mRNAs were less frequently detected. A(3) receptor mRNA was undetectable. Adenosine rapidly and reversibly reduced N-type Ca(2+) currents in cholinergic interneurons. The A(1) receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine completely blocked the effect of adenosine. The IC(50) of the A(1) receptor selective agonist 2-chloro-N6-cyclopentyladenosine was 45 nM, whereas it was near 30 microM for the A(2a) receptor agonist CGS-21680. Dialysis with GDPbetaS or brief exposure to the G protein (G(i/o)) alkylating agent N-ethylmaleimide also blocked the adenosine modulation. The reduction in N-type currents was partially reversed by depolarizing prepulses. A membrane-delimited pathway mediated the modulation, because it was not seen in cell-attached patches when agonist was applied to the bath. Activation of protein kinase C attenuated the adenosine modulation. Taken together, our results argue that activation of A(1) adenosine receptors in cholinergic interneurons reduces N-type Ca(2+) currents via a membrane-delimited, G(i/o) class G-protein pathway that is regulated by protein kinase C. These observations establish a cellular mechanism by which adenosine may serve to reduce acetylcholine release.

Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.

Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide's messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage-activated calcium channel subunit genes, alpha1A-D, alpha2, and beta1-4 are expressed in virtually all MNCs. However, the alpha1E subunit gene is not expressed at detectable levels in these cells. The expression of all of the beta-subunit genes in each MNC may account for the variations in physiological and pharmacological properties of the high voltage-activated channels found in these neurons. (Endocrinology 140: 5391-5401, 1999)

Human endothelium: endovascular biopsy and molecular analysis.

To develop a safe and reproducible method for harvesting viable vascular endothelium to analyze gene expression at sites of vascular lesions. Coaxial curved stainless-steel guide wires were used to obtain samples of endothelial cells from large arteries and veins in 29 patients undergoing routine endovascular procedures. Three immunocytochemical markers were used to identify cells as endothelial. Cellular viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of acetylated low-density lipoprotein, and cellular response to lipopolysaccharide. Single-cell reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study endothelial gene expression. Cells with endothelial morphology and immunoreactivity for von Willebrand factor, thrombomodulin, and angiotensin-converting enzyme were consistently obtained from iliac and carotid arteries and large veins (average yield [+/- standard error] from 26 iliac arteries, 262 endothelial cells +/- 45, 20%-30% of which were viable). These cells displayed induction of E-selection messenger RNA at PCR after exposure to lipopolysaccharide. Expression of vascular cell adhesion molecule 1 transcripts in endothelial cells increased with patient age (P < .01), whereas expression of intercellular adhesion molecule 1 did not. Viable endothelium can be obtained during routine angiography. Immunocytochemical and reverse transcription PCR analyses of these cells allowed determination of transcripts and proteins expressed by endothelium at sites of vascular lesions. Such information could aid in understanding mechanisms of vascular diseases and in clinical decision making.

Single-cell RT-PCR demonstrates expression of voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) in outer hair cells of rat cochlea

We investigated whether voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) are expressed in outer hair cells (OHCs) of rat cochlea using a single-cell reverse transcription–polymerase chain reaction (RT-PCR) technique. The OHCs were isolated from rat cochlea and the cytoplasm of each OHC was suctioned into a glass pipette containing RT-PCR reaction buffer with RNase inhibitor. RT-PCR revealed the presence of transcripts of ClC-1, ClC-2 and ClC-3, which were verified by DNA sequencing. The possible roles of these chloride channels in OHCs are discussed.

Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones

Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na +/Cl −-dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population.

Evidence for the preferential localization of Glutamate Receptor-1 subunits of AMPA receptors to the dendritic spines of medium spiny neurons in rat striatum

Although immunohistochemical studies have typically found the perikarya of striatal projection neurons to be devoid of immunohistochemical labelling for the GluR1 AMPA type glutamate receptor subunit, the striatal neuropil is rich in GluR1 immunolabelling and in situ hybridization histochemistry has indicated the presence of GluR1 message in many striatal neurons. To explore the possibility that GluR1 subunits may be synthesized by many striatal projection neurons, but selectively localized to their dendrites, we have used light-microscopic and electron-microscopic immunohistochemistry in combination with single-cell reverse transcription–polymerase chain reaction. Light-microscopic immunohistochemical studies confirmed the presence of abundant GluR1 immunoreactivity in the striatal neuropil in rats. Perikaryal labelling was restricted to neurons previously identified as parvalbuminergic neurons. Single-cell reverse transcription–polymerase chain reaction for individual striatal neurons in rats confirmed that most striatal projection neurons (i.e. containing either or both substance P message or enkephalin message) make GluR1 message. For example, 94% of enkephalin-containing neurons, 75% of substance P-containing neurons, and 87% of enkephalin and substance P co-containing neurons expressed GluR1 messenger RNA. Electron-microscopic immunohistochemistry revealed that GluR1 immunolabelling was prominent in 61% of dendritic spines and 53% of dendritic shafts. While prominent perikaryal GluR1 immunolabelling was observed only in a small population of interneurons, sparse perikaryal GluR1 immunolabelling was found associated with the rough endoplasmic reticulum, the Golgi apparatus, the outer membranes of the mitochondria, and the outer envelope of the nucleus of about 30% of striatal projection neurons (identified by their non-indented nuclei). These results indicate that striatal projection neurons selectively target GluR1 subunits to their spines and dendritic shafts. Our finding has implications for the functioning of striatal projection neurons and for the general issue of whether neurons can control the subcellular localization of glutamate receptors.

D2 dopamine receptors reduce N-type Ca2+ currents in rat neostriatal cholinergic interneurons through a membrane-delimited, protein-kinase-C-insensitive pathway.

Dopamine has long been known to regulate the activity of striatal cholinergic interneurons and the release of acetylcholine. Yet, the cellular mechanisms by which this regulation occurs have not been elucidated. One way in which dopamine might act is by modulating voltage-dependent Ca2+ channels. To test this hypothesis, the impact of dopaminergic agonists on Ca2+ channels in neostriatal cholinergic interneurons was studied by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reactions. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly, all interneurons tested (90%, n = 17) coexpressed D2 (short and long isoforms) and D1b (D5) dopamine receptor mRNAs. D1a receptor mRNA was found in only a small subset (20%) of the sample and D3 and D4 receptor mRNAs were undetectable. D2 receptor agonists rapidly and reversibly reduced N-type Ca2+ currents. D1b/D1a receptor activation had little or no effect on Ca2+ currents. The D2 receptor antagonist sulpiride blocked the effect of D2 agonists. Dialysis with guanosine-5'-O-(2-thiodiphosphate) or brief exposure to the G protein (Gi/o) alkylating agent N-ethylmaleimide also blocked the D2 modulation. The reduction in N-type currents was neither accompanied by kinetic slowing nor significantly reversed by depolarizing prepulses. The D2 receptor effects were mediated by a membrane-delimited pathway, because the modulation was not seen in cell-attached patches when agonist was applied to the bath and was not disrupted by perturbations in cytosolic signaling pathways known to be linked to D2 receptors. Activation of M2 muscarinic receptors occluded the D2 modulation, suggesting a shared signaling element. However, activation of protein kinase C attenuated the M2 modulation without significantly affecting the D2 modulation. Taken together, our results suggest that activation of D2 dopamine receptors in cholinergic interneurons reduces N-type Ca2+ currents via a membrane-delimited, Gi/o class G protein pathway that is not regulated by protein kinase C. This signaling pathway may underlie the ability of D2 receptors to reduce striatal acetylcholine release.

Lobster shal: comparison with Drosophila shal and native potassium currents in identified neurons

The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyte expression system.

Single cell reverse transcription-polymerase chain reaction (RT-PCR) and the GABA-A receptor

Single cell reverse transcription-polymerase chain reaction (RT-PCR) and the GABA-A receptor

Cell-specific expression of theα9 n-ACh receptor subunit in auditory hair cells revealed by single-cell RT-PCR

Single-cell reverse transcription polymerase chain reaction was carried out in three different cell types from the organ of Corti of the four-day old rat. For this purpose, pieces of the organ of Corti were mounted under a differential-interference contrast video microscope. Two different mounting configurations were used to allow imaging of cells from two almost orthogonal angles. This method afforded unequivocal recognition of various cell types in the vital tissue, and extraction of nucleus and cytoplasm of specified individual cells with a patch pipette. Messenger RNA encoding the alpha 9 acetylcholine (ACh) receptor subunit was detected and sequenced from individual outer hair cells and inner hair cells, but was not found in Deiters' cells. The identical Deiters' cells were positive for a P2x receptor subunit. This indicates cell-specific expression of the alpha 9 subunit in inner hair cells and outer hair cells and supports the hypothesis that this subunit contributes to calcium (Ca2+) permeable ionotropic ACh receptors (ACh-R). ACh-dependent Ca2+ concentration increase has been observed in both outer hair cells and inner hair cells.