Single-cell Reverse Transcription-polymerase Chain Research Articles

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Oral squamous cell carcinoma-released brain-derived neurotrophic factor contributes to oral cancer pain by peripheral tropomyosin receptor kinase B activation.

Oral cancer pain is debilitating and understanding mechanisms for it is critical to develop novel treatment strategies treatment strategies. Brain-derived neurotrophic factor (BDNF) signaling is elevated in oral tumor biopsies and is involved with tumor progression. Whether BDNF signaling in oral tumors contributes to cancer-induced pain is not known. The current study evaluates a novel peripheral role of BDNF-tropomyosin receptor kinase B (TrkB) signaling in oral cancer pain. Using human oral squamous cell carcinoma (OSCC) cells and an orthotopic mouse tongue cancer pain model, we found that BDNF levels were upregulated in superfusates and lysates of tumor tongues and that BDNF was expressed by OSCC cells themselves. Moreover, neutralization of BDNF or inhibition of TrkB activity by ANA12, within the tumor-bearing tongue reversed tumor-induced pain-like behaviors in a sex-dependent manner. Oral squamous cell carcinoma conditioned media also produced pain-like behaviors in naïve male mice that was reversed by local injection of ANA12. On a physiological level, using single-fiber tongue-nerve electrophysiology, we found that acutely blocking TrkB receptors reversed tumor-induced mechanical sensitivity of A-slow high threshold mechanoreceptors. Furthermore, single-cell reverse transcription polymerase chain reaction data of retrogradely labeled lingual neurons demonstrated expression of full-form TrkB and truncated TrkB in distinct neuronal subtypes. Last but not the least, intra-TG siRNA for TrkB also reversed tumor-induced orofacial pain behaviors. Our data suggest that TrkB activities on lingual sensory afferents are partly controlled by local release of OSCC-derived BDNF, thereby contributing to oral cancer pain. This is a novel finding and the first demonstration of a peripheral role for BDNF signaling in oral cancer pain.

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals

AbstractAntiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL. (Blood. 2003;102:2459-2465)

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.

TRPM8 Mediates Hyperosmotic Stimuli-Induced Nociception in Dental Afferents

Hyperosmolar sweet foods onto exposed tooth dentin evoke sudden and intense dental pain, called dentin hypersensitivity. However, it remains unclear how hyperosmolar stimuli excite dental primary afferent (DPA) neurons and thereby lead to dentin hypersensitivity. This study elucidated whether TRPM8, which is well known as a cold temperature– or menthol-activated receptor, additionally mediates nociception in response to hyperosmolar stimuli in adult mouse DPA neurons, which are identified by a fluorescent retrograde tracer: DiI. Single-cell reverse transcription polymerase chain reaction revealed that TRPM8 was expressed in subsets of DPA neurons and that TRPM8 was highly colocalized with TRPV1 and Piezo2. Immunohistochemical analysis also confirmed TRPM8 expression in DPA neurons. By using Fura-2-based calcium imaging, application of hyperosmolar sucrose solutions elicited calcium transients in subsets of the trigeminal ganglion neurons, which was significantly abolished by a selective TRPM8 antagonist: N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide (AMTB) hydrochloride. When we further examined changes of c-fos expression (a neuronal activation marker) in the spinal trigeminal nucleus after hyperosmolar stimulation onto exposed tooth dentin, c-fos mRNA and protein expression were increased and were also significantly reduced by AMTB, especially in the spinal trigeminal interpolaris-caudalis transition zone (Vi/Vc). Taken together, our results provide strong evidence that TRPM8 expressed in DPA neurons might mediate dental pain as a hyperosmosensor in adult mice.

Lineage marker expression on mouse hematopoietic stem cells

Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs.

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

Strategies to Obtain Diverse and Specific Human Monoclonal Antibodies From Transgenic Animals.

Techniques to obtain large quantities of antigen-specific monoclonal antibodies (mAbs) were first established in the 1970s when Georges Köhler and César Milstein immortalized antibody-producing mouse B-lymphocytes by fusion with myeloma cells (http://www.whatisbiotechnology.org/exhibitions/milstein). Combined with the expression of human antibodies in transgenic animals, this technique allowed upon immunization the generation of highly specific fully human mAbs for therapeutic applications. Apart from being extremely beneficial, mAbs are a huge success commercially. However, despite cell fusion generating many useful mAbs questions have been asked about which types of cells are prone to fuse and whether other methods may identify a wider range of binders. The discovery that expression libraries, using Escherichia coli or yeast, produced different specificities was intriguing and more recently Next-Generation Sequencing has identified wide-ranging usage with highly diverse and unique repertoires. Another strategy is the combination of flow cytometry sorting of antigen-binding B lymphocytes and single-cell reverse transcription polymerase chain reaction followed by reexpression, which has identified many high-affinity mAbs.

Extracellular ATP Induces Calcium Signaling in Odontoblasts

Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca2+ concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca2+-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X4 and P2X7 in odontoblasts. Overall, this study demonstrated heterogeneous expression of calcium-related ATP receptor subtypes in subsets of individual odontoblasts, suggesting extracellular ATP as a potential signal mediator for odontoblastic functions.

Involvement of HCN Channel in Muscarinic Inhibitory Action on Tonic Firing of Dorsolateral Striatal Cholinergic Interneurons.

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum.

Tissue Plasminogen Activator Expression Is Restricted to Subsets of Excitatory Pyramidal Glutamatergic Neurons.

Although the extracellular serine protease tissue plasminogen activator (tPA) is involved in pathophysiological processes such as learning and memory, anxiety, epilepsy, stroke, and Alzheimer's disease, information about its regional, cellular, and subcellular distribution in vivo is lacking. In the present study, we observed, in healthy mice and rats, the presence of tPA in endothelial cells, oligodendrocytes, mastocytes, and ependymocytes, but not in pericytes, microglial cells, and astrocytes. Moreover, blockage of the axo-dendritic transport unmasked tPA expression in neurons of cortical and hippocampal areas. Interestingly, combined electrophysiological recordings, single-cell reverse transcription polymerase chain reaction (RT-PCR), and immunohistological analyses revealed that the presence of tPA is restricted to subsets of excitatory pyramidal glutamatergic neurons. We further evidenced that tPA is stored in synaptobrevin-2-positive glutamatergic synaptic vesicles. Based on all these data, we propose the existence of tPA-ergic neurons in the mature brain.

Asymmetrical allocation of JAK1 mRNA during spermatogonial stem cell division in Xenopus laevis.

During Xenopus spermatogenesis, each primary spermatogonium (PG), the largest single cell in the testis, undergoes mitotic divisions with a concomitant decrease in size to produce smaller differentiating spermatogonia. The spermatogonial stem cells (SSCs) occur in this PG population. Taking advantage of identifiable and isolatable properties of Xenopus SSCs, we examined JAK1 gene expression during the spermatogenesis because there have been reports on the important role of JAK/STAT pathway in regulating the status of SSCs in Drosophila and mouse. Surprisingly, insitu hybridization revealed the presence of JAK1 mRNA in the differentiating spermatogonia and primary spermatocytes as well as some PGs. Inhibition of JAK1 activity in the testis caused a decrease in percentage of BrdU-incorporating spermatogonia, suggesting that JAK1 was at least involved in regulation of spermatogonial proliferation. Interestingly, single cell reverse transcription-polymerase chain reaction (RT-PCR) clearly showed two different types of SSCs: SSCs with JAK1 mRNA (JAK1+ ) or without JAK1 mRNA (JAK1- ). Since JAK1- SSC level was increased by induction of testis regeneration, self-renewing SSCs were thought to be JAK1- . In addition, we found barrel-shaped PGs, in which JAK1 mRNA was localized asymmetrically to one half of the cell. The stainability with propidium iodide and morphology of two nuclei in the barrel-shaped PG were similar to those of PG nucleus. Based on the above observations, we propose the hypothesis that JAK1+ SSC is preparing for production of PGs destined to differentiate (destined PGs) and the accumulated JAK1 mRNA in the SSC is distributed exclusively into the destined PGs through mitotic division.

Diversity of GABAergic Interneurons in Layer VIa and VIb of Mouse Barrel Cortex

Neocortical layer VI modulates the thalamocortical transfer of information and has a significant impact on sensory processing. This function implicates local γ-aminobutyric acidergic (GABAergic) interneurons that have only been partly described at the present time. Here, we characterized 85 layer VI GABAergic interneurons in acute slices of mouse somatosensory barrel cortex, using whole-cell current-clamp recordings, single-cell reverse transcription-polymerase chain reaction, and biocytin labeling followed by Neurolucida reconstructions. Unsupervised clustering based on electrophysiological molecular and morphological properties disclosed 4 types of interneurons. The 2 major classes were fast-spiking cells transcribing parvalbumin (PV) (51%) and adapting interneurons transcribing somatostatin (SOM) (26%). The third population (18%) transcribed neuropeptide Y (NPY) and appeared very similar to neurogliaform cells. The last class (5%) was constituted by well-segregated GABAergic interneurons transcribing vasoactive intestinal peptide (VIP). Using transgenic mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we investigated the densities of GABAergic cells immunolabeled against PV, SOM, VIP, and NPY through the depth of layer VI. This analysis revealed that PV and NPY translating interneurons concentrate in the upper and lower parts of layer VI, respectively. This study provides an extensive characterization of the properties of layer VI interneurons.

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Somatostatin Inhibits Gonadotropin Releasing Hormone Neuronal Activities in Juvenile Mice

Background: The gonadotropin releasing hormone (GnRH) neurons perform a pivotal function in the central regulation of fertility. Somatostatin (SST) is an important neuromodulatory peptide in the central nervous system and alters neuronal activities via G protein-coupled SST receptors. A number of studies have shown that SST modulates the reproductive axis at the hypothalamic level. However, the precise action mechanisms of SST and related receptor subtypes have yet to be fully understood. In this study, we evaluated the direct effects of SST on GnRH neurons in juvenile mice. Methods: Juvenile (postnatal days, < PND 30) GnRH-GFP transgenic mice expressing green fluorescent protein were used in this study. Acute coronal brain slices containing the preoptic area were prepared and all identified GnRH neurons were recorded using the gramicidin perforated-patch clamp technique; type II SST receptor (SSTR2) mRNA expression was evaluated via single cell reverse transcription-polymerase chain reaction (RT-PCR). Results: SST caused membrane hyperpolarization, depolarization, no response, or membrane hyperpolarization with a reduction of action potential. Most (57.7%, 30/52) of the GnRH neurons tested were hyperpolarized by SST and this SST-induced hyperpolarization was found to be concentration-dependent. The percentage of responses, membrane potential changes (MPC), and resting membrane potential (RMP) by SST were not significantly different in juvenile male and female GnRH neurons. The SST-induced hyperpolarization was maintained in the presence of tetrodotoxin (TTX), a sodium channel blocker, and an amino acid blocking cocktail (AABC) containing AP-5 (NMDA receptor antagonist), CNQX (non-NMDA glutamate receptor antagonist), picrotoxin (GABAA receptor antagonist), and strychnine (glycine receptor antagonist). SSTR2 mRNA was expressed on 10 (38%) among 26 GnRH neurons. Seglitide, an SSTR2 agonist, mimicked this SST-induced hyperpolarization (11/23 47.8%) and this response was maintained in the presence of TTX and AABC. Conclusion: Our data show that SST can exert potent inhibitory action against GnRH neuronal excitability via SSTR2 activation in juvenile mice. (Endocrinol Metab 26:210-217, 2011)

Facilitatory Actions of Serotonin Type 3 Receptors on GABAergic Inhibitory Synaptic Transmission in the Spinal Superficial Dorsal Horn

Analgesic effects of serotonin (5-hydroxytryptamine [5-HT]) type 3 (5-HT3) receptors may involve the release of gamma-aminobutyric acid (GABA) in the spinal dorsal horn. However, the precise synaptic mechanisms for 5-HT3 receptor-mediated spinal analgesia are not clear. In this study, we investigated whether GABAergic neurons in the superficial dorsal horn (SDH) express functional 5-HT3 receptors and how these 5-HT3 receptors affect GABAergic inhibitory synaptic transmission in the SDH, by using slice preparations from adult glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice. Tight-seal whole cell recordings from GFP-positive and -negative neurons showed that 5-HT3 receptor-specific agonist 2-methyl-serotonin (2-Me-5-HT) induced inward currents in a substantial population of both GFP-positive and -negative neurons. Additionally, we confirmed expression of 5-HT3 receptors in both types of neurons by single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis. Further, GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs)-both those evoked by electrical stimulation and those occurring spontaneously in tetrodotoxin (i.e., miniature IPSCs [mIPSCs])-were recorded from GFP-negative neurons. 2-Me-5-HT increased the amplitude of the evoked IPSCs and the frequency of mIPSCs. The amplitude of mIPSCs was not affected by 2-Me-5-HT, suggesting that 5-HT augments GABAergic synaptic transmission via presynaptic mechanisms. The present observations indicate that 5-HT3 receptors are expressed on both somadendritic regions and presynaptic terminals of GABAergic neurons and regulate GABAA receptor-mediated inhibitory synaptic transmission in the SDH. Taken together, these results provide clues for the underlying mechanisms of the antinociceptive actions of 5-HT3 receptors in the spinal dorsal horn.

Modulation of firing activity by ATP in dopamine neurons of the rat substantia nigra pars compacta

ATP acts as a neurotransmitter or co-neurotransmitter in many areas of the CNS and peripheral nervous systems; however, little is known about the expression and functional role of purinoceptors (P2) in midbrain dopaminergic neurons. Therefore, we investigated P2X receptor expression and regulation of spontaneous firing activity in dopaminergic neurons of the substantia nigra pars compacta (SNc) in rats using patch-clamp and Ca2+-imaging techniques. In most neurons, application of ATP (1 μM–1 mM) increased firing rate dose-dependently (EC50=1.26±0.26 μM, n=45). When the P2-receptor agonists such as 2-methylthio-adenosine 5′-triphosphate (2-MeSATP) or ATPγS were applied or pressure-applied to the neuron, the firing activity increased together with a rise in cytosolic Ca2+ concentration ([Ca2+]c), but application of beta,gamma-methylene ATP (P2X1, 3 agonist) or methylthio-adenosine 5′-diphosphate (P2Y1 agonist) had no effect. In many neurons, the effect of ATP was abolished by the application of the P2-receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). When ATP was applied in a Ca2+-free solution, there was no detectable change in [Ca2+]c, suggesting that ATP does not release Ca2+ from intracellular stores. In the single-cell reverse transcription polymerase chain reaction (RT-PCR), we found that 65% of dopaminergic neurons expressed mRNAs for P2X receptors; positive amplifications of P2X6 (57.1%), P2X2/6 (25.0%), and P2X4 mRNA (17.9%), respectively. From the above results, we could conclude that ATP modulates firing activities in the rat SNc dopaminergic neurons, possibly via P2X2, P2X2/6, and/or P2X4 receptors.

NaCl responsive taste cells in the mouse fungiform taste buds

Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells (∼44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells (∼56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.