Single-cell Reverse Transcription-polymerase Research Articles

The T-type calcium channel Ca V 3.2 regulates bladder afferent responses to mechanical stimuli.

The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

Thermo-Induced Coalescence of Dual Cores in Double Emulsions for Single-Cell RT-PCR

Single-cell reverse-transcription polymerase chain reaction (RT-PCR) has shown significant promise for transcriptional profiling of heterogeneous cells. However, currently developed microfluidic droplet-based methodologies for single-cell RT-PCR often require complex chip design to accommodate the associated multistep processes as well as customized detection platforms for high-throughput analysis. Herein, we proposed a dual-core double emulsion (DE)-based method to streamline the single-cell RT-PCR through thermo-induced coalescence of the dual cores. The dual-core DEs were produced by pairing two water-in-oil single emulsions containing a single-cell/lysis buffer and RT-PCR mix, respectively. After complete lysis of single cells in one of the cores, the dual-core DEs were merged by gentle heating, made possible by the optimized glycerol concentration present in the cores. Upon the coalescence of dual cores, the alkaline lysis buffer present in the core of the cell lysate was neutralized by the reaction buffer presented in the RT-PCR core, allowing TaqMan assay-based RT-PCR to occur effectively within the DEs. To demonstrate the potential of this streamlined dual-core platform, AKR1B10-positive A549 cells and AKR1B10-negative HEK293 cells were investigated via the TaqMan assay. Subsequently, specific transcript of AKR1B10 was readily available for quantitative profiling at the single-cell level using a commercially available flow cytometer in a high-throughput manner.

Attenuated regulatory function of the small-conductance Ca2+-activated K+ channel in detrusor smooth muscle cells excitability in an obese rat model.

Overactive bladder (OAB) is related to detrusor overactivity (DO), which is caused by the increased detrusor smooth muscle (DSM) cells excitability. Small-conductance Ca2+-activated K+ (SK) channels is a fundamental regulator of excitability and contractility in DSM cells. Obesity-related OAB is associated with the decreased expression and regulatory function of SK channels in DSM layer. However, the regulation role of SK channels in obesity-related OAB DSM cell excitability is still unknown. Here, we tested the hypothesis that obesity-related OAB is associated with reduced expression and activity of SK channels in DSM cells. Female Sprague-Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD) and weighed after 12weeks. We performed urodynamic study, single-cell quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and patch-clamp electrophysiology. Increased average body weights and urodynamically demonstrated OAB were observed in HFD rats. Single-cell qRT-PCR experiments discovered the decreased mRNA expression level of SK channel in DSM cell from HFD rats. Patch-clamp studies revealed that NS309, a SK channel activator, had an attenuated effect on membrane potential hyperpolarization in HFD DSM cells. In addition, the reduced whole cell SK channel currents were recorded in HFD DSM cells. Attenuated SK channels expression and function, which results in the increased DSM cells excitability and contributes to DO, is discovered in obesity-related OAB DSM cells, suggesting that SK channels might be potential therapeutic targets to control OAB.

Characterization and applications of nanobodies against Pseudomonas aeruginosa Exotoxin A selected from single alpaca B cells

We have established a highly efficient 96-well format based strategy to characterize nanobody (Nb) from an expressed alpaca Nb repertoire by combining heavy-chain antibody variable region gene cloning with Nb expression and reactivity profiling at the single cell level. Individual alpaca B lymphocytes were isolated based on B cell surface marker and membrane type antigen specific antibodies expression by fluorescence activated cell sorting (FACS). Single cell reverse transcription and polymerase chain reaction (PCR) amplification of CH1 and CH2 domain enable the identification and exclusion of B cell clones that express regular antibodies. The corresponding heavy-chain antibody variable region gene transcripts, which are CH1-CH2 domain negative, are then amplified from single B cell clones by nested PCR. Then Nbs were expressed in Escherichia coli cells and four Nbs showed high affinity against Pseudomonas aeruginosa Exotoxin A (PEA). Moreover, two PEA Nbs that recognize unique epitopes on PEA have been successfully applied to protect human cells from PEA induced apoptosis. In summary, our FACS and single cell reverse transcription polymerase chain reaction (RT-PCR) based strategy for identifying recombinant Nbs from single alpaca B cells allows highly efficient and unbiased characterization of the expressed Nb repertoires by sequence analysis, expression and parallel antibody reactivity testing.

Tetrodotoxin-sensitive voltage-gated sodium channels regulate bladder afferent responses to distension.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a prevalent, chronic bladder disorder that negatively impacts the quality of life for ∼5% of the western population. Hypersensitivity of mechanosensory afferents embedded within the bladder wall is considered a key component in mediating IC/BPS symptoms. Bladder infusion of voltage-gated sodium (Nav) channel blockers show clinical efficacy in treating IC/BPS symptoms; however, the current repertoire of Nav channels expressed by and contributing to bladder afferent function is unknown. We used single-cell reverse-transcription polymerase chain reaction of retrogradely traced bladder-innervating dorsal root ganglia (DRG) neurons to determine the expression profile of Nav channels, and patch-clamp recordings to characterise the contribution of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav channels to total sodium current and neuronal excitability. We determined the TTX-S and TTX-R contribution to mechanosensitive bladder afferent responses ex vivo and spinal dorsal horn activation in vivo. Single-cell reverse-transcription polymerase chain reaction of bladder-innervating DRG neurons revealed significant heterogeneity in Nav channel coexpression patterns. However, TTX-S Nav channels contribute the vast majority of the total sodium current density and regulate the neuronal excitability of bladder DRG neurons. Furthermore, TTX-S Nav channels mediate almost all bladder afferent responses to distension. In vivo intrabladder infusion of TTX significantly reduces activation of dorsal horn neurons within the spinal cord to bladder distension. These data provide the first comprehensive analysis of Nav channel expression within sensory afferents innervating the bladder. They also demonstrate an essential role for TTX-S Nav channel regulation of bladder-innervating DRG neuroexcitability, bladder afferent responses to distension, and nociceptive signalling to the spinal cord.

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

Toll-like receptor expression in pulmonary sensory neurons in the bleomycin-induced fibrosis model.

Airway sensory nerves are known to express several receptors and channels that are activated by exogenous and endogenous mediators that cause coughing. Toll-like receptor (TLR) s are expressed in nociceptive neurons and play an important role in neuroinflammation. However, there have been very few studies of TLR expression in lung-derived sensory neurons or their relevance to respiratory symptoms such as cough. We used the bleomycin-induced pulmonary fibrosis model to investigate the change in TLR expression in pulmonary neurons and the association of TLRs with transient receptor potential (TRP) channels in pulmonary neurons. After 2 weeks of bleomycin or saline administration, pulmonary fibrosis changes were confirmed using tissue staining and the SIRCOL collagen assay. TLRs (TLR 1–9) and TRP channel expression was analyzed using single cell reverse transcription polymerase chain reaction (RT-PCR) in isolated sensory neurons from the nodose/jugular ganglion and the dorsal root ganglion (DRG). Pulmonary sensory neurons expressed TLR2 and TLR5. In the bleomycin-induced pulmonary fibrosis model, TLR2 expression was detected in 29.5% (18/61) and 26.9% (21/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively. TLR5 was also detected in 55.7% (34/61) and 42.3% (33/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively, in the bleomycin-induced pulmonary fibrosis model. TLR5 was expressed in 63.4% of TRPV1 positive cells and 43.4% of TRPM8 positive cells. In conclusion, TLR2 and TLR5 expression is enhanced, especially in vagal neurons, in the bleomycin-induced fibrosis model group when compared to the saline treated control group. Co-expression of TLR5 and TRP channels in pulmonary sensory neurons was also observed. This work sheds new light on the role of TLRs in the control and manifestation of clinical symptoms, such as cough. To understand the role of TLRs in pulmonary sensory nerves, further study will be required.

Translational Traits of Non-Small-Cell Lung Carcinoma with Drug Resistances by Dual mRNA and Protein Quantification at the Single Cell Level

Translation in eukaryotic cells is a fundamental biological process that has been investigated widely for understanding gene expression. To comprehensively understand gene expression, single cell level observation of both transcript and protein is desirable for translational analysis. The simultaneous observation of both transcript and protein, however, remains challenging due to the requirements of a highly sensitive and multi-modal platform. Here, we report a robust microdevice enabling both transcript and protein quantification from a single cell with a large number of cells per assay. Using 25600 microwells, we quarantined single cells for observing protein expression through immunostaining and sequentially mRNA expression via single cell reverse transcription polymerase chain reaction. Based on this correlation between transcript and protein, 2-dimensional cytometric representation from single cells is possible. The relationship between transcript and proteome of three NSCLC cell lines (HCC827, H1650 and H1975) with different levels of drug resistances to epidermal growth factor tyrosine kinase inhibitors clearly indicates unique translational traits of each cell line. Our data demonstrated that the most drug resistant cell line, H1975, showed least translational efficiency in cMET whereas the least drug resistant cell line, HCC827, showed most translational efficiency. Cytometric platforms, like the one introduced here, may ultimately allow researchers to quantify the evolution of cancerous tissues enabling detailed monitoring of tumor progression.

Distribution and functional expression of Kv4 family α subunits and associated KChIP β subunits in the bed nucleus of the stria terminalis

Regulation of BNSTALG neuronal firing activity is tightly regulated by the opposing actions of the fast outward potassium current, IA , mediated by α subunits of the Kv4 family of ion channels, and the transient inward calcium current, IT . Together, these channels play a critical role in regulating the latency to action potential onset, duration, and frequency, as well as dendritic back-propagation and synaptic plasticity. Previously we have shown that Type I-III BNSTALG neurons express mRNA transcripts for each of the Kv4 α subunits. However, the biophysical properties of native IA channels are critically dependent on the formation of macromolecular complexes of Kv4 channels with a family of chaperone proteins, the potassium channel-interacting proteins (KChIP1-4). Here we used a multidisciplinary approach to investigate the expression and function of Kv4 channels and KChIPs in neurons of the rat BNSTALG . Using immunofluorescence we demonstrated the pattern of localization of Kv4.2, Kv4.3, and KChIP1-4 proteins in the BNSTALG . Moreover, our single-cell reverse-transcription polymerase chain reaction (scRT-PCR) studies revealed that mRNA transcripts for Kv4.2, Kv4.3, and all four KChIPs were differentially expressed in Type I-III BNSTALG neurons. Furthermore, immunoelectron microscopy revealed that Kv4.2 and Kv4.3 channels were primarily localized to the dendrites and spines of BNSTALG neurons, and are thus ideally situated to modulate synaptic transmission. Consistent with this observation, in vitro patch clamp recordings showed that reducing postsynaptic IA in these neurons lowered the threshold for long-term potentiation (LTP) induction. These results are discussed in relation to potential modulation of IA channels by chronic stress.

Substance P Sensitizes P2X3 in Nociceptive Trigeminal Neurons

Peripheral inflammation produces pain hypersensitivity by sensitizing nociceptors. Potentiation of P2X3 receptor activity in nociceptors may play an important role in this peripheral sensitization. However, we do not fully understand how P2X3 activity is elevated in inflammation. Thus, we investigated whether P2X3 activity in trigeminal nociceptive neurons is regulated by the neurokinin-1 (NK-1) receptor that is activated by an inflammatory mediator, substance P. Single-cell RT-PCR and immunohistochemistry revealed that NK-1 in nociceptive neurons was mainly co-expressed with P2X3. Ca2+ imaging and whole-cell patch-clamp recordings indicated that both substance P and Sar-substance P, a selective NK-1 agonist, significantly potentiated α,β-meATP-induced currents and [Ca2+]i responses in nociceptive neurons. These potentiating effects were completely blocked by GR82334, a specific NK-1 antagonist. Our results demonstrate that substance P sensitizes P2X3 receptor through the activation of NK-1, thus warranting these receptors as possible targets for pain therapy in the orofacial region. Abbreviations: α,β-methylene adenosine 5′-triphosphate (ATP), α,β-meATP; neurokinin-1, NK-1; single-cell reverse-transcription polymerase chain-reaction, single-cell RT-PCR; [Sar9,Met(O2)11]-substance P, Sar-substance P.

Characterization of the expression pattern of adrenergic receptors in rat taste buds

Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of α1A, α1B, α1D, α2A, α2B, α2C, β1, and the β2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine β-hydroxylase (DβH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell reverse transcription polymerase chain reaction (RT-PCR). These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play modulatory roles in processing of gustatory information similar to other peripheral sensory systems such as the retina, cochlea, and olfactory bulb.

Chemical identification of nestin-immunoreactive neurons in the rat basal forebrain: A re-examination

This study explores recently identified neurons that express the protein nestin in the medial septum–diagonal band of Broca (MS–DBB) of adult rats and humans. These nestin positive neurons from MS–DBB are known to project to the hippocampus and frontal cortex of the brain. However, their chemical identification has not been fully elucidated. In this study, we further investigated the chemical identity of the nestin-immunoreactive (ir) neurons in rats using double immunofluorescence labeling and single cell reverse transcription polymerase chain reaction (RT-PCR) techniques. The results of double labeling showed that all nestin-ir neurons exhibited choline acetyltransferase (ChAT) immunoreactivity in the MS–DBB of the basal forebrain. Conversely, only about 43% of the ChAT-ir neurons showed nestin immunoreactivity. In addition, a vast majority of the nestin-ir neurons (95%) were nerve growth factor receptor (NGFR) positive. The nestin-ir neurons were highly distributed in the rostral and intermediated regions of the MS–DBB. Single cell RT-PCR results showed that 90% of the nestin mRNA expressed neurons displayed ChAT mRNA expression as well, but neither the mRNA of the proteins glutamic acid decarboxylases 67 (GAD67) nor vesicular glutamate transporters (VGLUTs). These results provide the first evidence that nestin-ir neurons in the basal forebrain are a subpopulation of the classic cholinergic neurons. With high NGFR proteins activated in the nestin-ir neurons, we propose that the presence of nestin in the cholinergic neurons might be related to the survival and plasticity of the cholinergic neurons.

Functional Analysis of Embryonic Stem Cell–Derived Glial Cells after Integration into Hippocampal Slice Cultures

Embryonic stem (ES) cell-derived neural progenitor cells (ESNPs) generated in vitro are multipotent progenitors which can differentiate into oligodendrocytes, astrocytes, and neurons. Given the exciting prospects for ES cell-based treatments of neurological disorders, several studies investigated the migration, integration, and differentiation of grafted ESNPs into neurons and glial cells. However, little is known about the functional properties of transplanted ESNPs on the single cell level. In this study, we combined electrophysiology, single cell reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry to determine the developmental time course of molecular and functional properties of ES cell-derived glial precursors (ESGPs) after deposition onto hippocampal slice cultures. Based on functional criteria, donor cells possessed three different phenotypes. During an observation period of 3 weeks after engraftment, the proportion of donor cells with a passive current pattern (type 3) continuously increased. The majority of these cells expressed astroglial markers. Type 3 host cells underwent similar developmental changes. In contrast, donor and host cells expressing time- and voltage-dependent currents (types 1, 2) displayed different developmental profiles. Importantly, type 2 donor and host cells also differed in the expression of inwardly rectifying K(+) channels. This suggests that despite several similarities in overall current phenotypes and timing of maturation, many donor cells integrated into host tissue but did not acquire the full set of ion channels present in their native counterparts. These findings emphasize the need to carefully characterize ES cell-derived progeny aimed for neural repair and cell-mediated gene transfer strategies.

Molecular characterization of T-type Ca 2+ channels responsible for low threshold spikes in hypothalamic paraventricular nucleus neurons

The hypothalamic paraventricular nucleus (PVN) is composed of functionally heterogeneous cell groups, possessing distinct electrophysiological properties depending on their functional roles. Previously, T-type Ca 2+ dependent low-threshold spikes (LTS) have been demonstrated in various PVN neuronal types, including preautonomic cells. However, the molecular composition and functional properties of the underlying T-type Ca 2+ channels have not been characterized. In the present study, we combined single cell reverse transcription–polymerase chain reaction (RT-PCR), immunohistochemistry and patch-clamp recordings to identify subtypes of T-type Ca 2+ channels expressed in PVN cells displaying LTS (PVN-LTS), including identified preautonomic neurons. LTS appeared at the end of hyperpolarizing pulses either as long-lasting plateaus or as short-lasting depolarizing humps. LTS were mediated by rapidly activating and inactivating T-type Ca 2+ currents and were blocked by Ni 2+. Single cell RT-PCR and immunohistochemical studies revealed Cav3.1 (voltage-gated Ca 2+ channel) as the main channel subunit detected in PVN-LTS neurons. In conclusion, these data indicate that Cav3.1 is the major subtype of T-type Ca 2+ channel subunit that mediates T-type Ca 2+ dependent LTS in PVN neurons.

Nicotinic acetylcholine receptor expression by directionally selective ganglion cells

Acetylcholine (ACh) enhances the preferred direction responses of directionally selective ganglion cells (DS GCs; Ariel & Daw, 1982; Ariel & Adolph, 1985) through the activation of nicotinic acetylcholine receptors (nAChRs; Ariel & Daw, 1982; Massey et al., 1997; Kittila & Massey, 1997). DS GCs appear to express at least two types of nAChRs, those that are sensitive to the partially subtype-specific antagonist methyllycaconitine (MLA), and those that are MLA-insensitive (Reed et al., 2002). Our purpose was to confirm the expression of alpha7 nAChRs by DS GCs and to assess the contributions of other nAChR subtypes to DS GC responses. Using choline as a nAChR partially subtype-specific agonist, we found that the majority of DS GCs demonstrated responses to choline while under synaptic blockade. The blockade or reduction of choline-induced responses by bath application of nanomolar (nM) concentrations of MLA provided direct evidence that the choline responses were mediated by alpha7 nAChRs. Because choline is a partial agonist for alpha3beta4 nAChRs (Alkondon et al., 1997), the residual choline responses are consistent with mediation by alpha3beta4 nAChRs. Additionally, a subset of DS GCs responded to nicotine but not to choline, indicating the expression of a third nAChR subtype. The pharmacological results were supported by single cell reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry experiments. The expression of alpha7 and specific non-alpha7 nAChR subtypes was correlated with the preferred direction. This indicates the possibility of differential responses to ACh depending on the direction of movement. This is the first description of differential expression of multiple nAChR subtypes by DS GCs.

Involvement of transient receptor potential vanilloid-1 in calcium current inhibition by capsaicin

Capsaicin has been used as a topical analgesic to treat diverse pain conditions. We investigated the molecular mechanisms that mediate the inhibition of high-voltage-activated calcium channel currents (ICa) using trigeminal ganglion neurons and a heterologous expression system. Capsaicin inhibited ICa in capsaicin-sensitive trigeminal ganglion neurons, but not in capsaicin-insensitive neurons. Single-cell reverse-transcription polymerase chain reaction revealed the expression of TRPV1 only in capsaicin-sensitive neurons. Capsaicin inhibited ICa in transient receptor potential vanilloid-1-expressing C2D7 cells stably expressing human N-type calcium channels, whereas capsaicin failed to inhibit ICa in naïve C2D7 cells with no endogenous transient receptor potential vanilloid-1 expression. Calcium influx via transient receptor potential vanilloid-1 is not likely to play a critical role in capsaicin-induced ICa inhibition in trigeminal ganglion neurons. ICa inhibition might be one of the mechanisms for the analgesic effect of capsaicin.

Human immunoglobulin variable region gene analysis by single cell RT-PCR

This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cμ, Cγ, Cκ and/or Cλ). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.

Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons.

The molecular and functional properties of neuronal nicotinic acetylcholine receptors (nAChRs) were characterized from CA1 neurons in rat hippocampal slices using single-cell reverse-transcription polymerase chain reaction (RT-PCR) in conjunction with whole-cell patch-clamp recordings. We analysed the presence of the neuronal nAChR subunit mRNAs alpha2-7 and beta2-4, along with the mRNA for the GABAergic markers GAD (glutamic acid decarboxylase) 65 and 67 isoforms, and VGAT (vesicular GABA transporter) in interneurons from the stratum radiatum and stratum oriens, and in CA1 pyramidal neurons. Functional nAChR-mediated currents were detected in both interneuron populations, but not in pyramidal neurons. The neuronal nAChR subunit mRNAs detected in > 20 % of the populations examined were alpha4, alpha5, alpha7 and beta2-4 in stratum radiatum interneurons; alpha2, alpha3, alpha4, alpha7, beta2 and beta3 subunits in stratum oriens interneurons; and beta2-4 in pyramidal neurons. High levels of GABAergic marker mRNAs were detected in both interneuron populations, but not in pyramidal neurons. Significant co-expression of nAChR subunits within individual neurons was detected for alpha3 + alpha5, alpha4 + beta2, alpha4 + beta3, alpha7 + beta2, beta2 + beta3 and beta3 + beta4. The kinetics of the nAChR-mediated currents in response to the application of 100 microM ACh were significantly correlated with the expression of particular nAChR subunits. The alpha3, alpha7 and beta2 nAChR subunits were individually correlated with a fast rise time, the alpha2 nAChR subunit was correlated with a medium rise time, and the alpha4 nAChR subunit was correlated with a slow rise time. The alpha2 and alpha4 nAChR subunits were also significantly correlated with slow desensitization kinetics.

Effects of cyanide and hypoxia on membrane currents in neurones acutely dissociated from the rostral ventrolateral medulla of the rat

Previous reports suggested that some neurones located in the rostral ventrolateral medulla (RVL) can act as fast oxygen sensors which enhance the sympathetic activity and blood pressure independent of peripheral chemoreceptors. The aim of this study was to compare hypoxic responses of different subpopulations of RVL neurones to ascertain whether the hypoxic sensitivity is restricted to one group of these neurones. Whole-cell patch-clamp recordings were made from acutely dissociated neurones obtained from RVL of P13–P19 rats. Short-lasting hypoxia (1–2 min) was evoked by pressure injection of NaCN or lowering pO 2. Cells projecting to the upper thoracic segments were retrogradely labelled with fluorescent beads. Catecholaminergic (CA) or non-catecholaminergic (non-CA) neurones were identified using single-cell reverse-transcription polymerase chain reaction (RT-PCR) or immunocytochemistry. Recordings were made from 38 neurones (26 spinally-projecting, 12 non-spinal) using Cs +/TEA or K +-containing pipettes. In most of the cells tested with slow depolarising ramp commands (78%; including spinally-projecting and non-spinal neurones, as well as CA and non-CA neurones), NaCN or hypoxia evoked a reversible increase of the sustained inward current. Extracellular application of 1 mM Co 2+ or 25 nM TTX revealed three components of the hypoxia-sensitive inward current which resembled the persistent sodium ( I NaP), low threshold calcium (LVA Ca 2+) and high threshold calcium (HVA Ca 2+) currents. The NaCN or hypoxia induced increase of the current could also be observed during step commands. Recordings with K +-containing pipettes during similar depolarising ramps revealed, in addition, a reversible increase of I K in 78% of tested cells (in all four types of examined neurones). These results are consistent with the concepts that RVL neurones can act as a central oxygen sensor. However, in contrast to the previously published data demonstrating that in pentobarbital anaesthetised rats only the barosensitive and spinally projecting cells were affected by a short-lasting hypoxia, our findings obtained with dissociated RVL neurones indicate that sensitivity to hypoxia is widely distributed within this part of the medulla oblongata.