Single-Cell RT-PCR Analysis of TRPC Channels Expressed in Rat Cholinergic, Dopaminergic, Noradrenergic, and Serotonergic Neurons in the Brain

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

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Combined single cell RT-PCR analysis with patch clamp recording in acute brain slices

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)47528-8 ◽

2000 ◽

Vol 82 ◽

pp. 12

Author(s):

Keisuke Tsuzuki ◽

Bertrand Lambolez ◽

Etienne Audinat ◽

James T. Porter ◽

Bruno Cauli ◽

...

Keyword(s):

Patch Clamp ◽

Single Cell ◽

Brain Slices ◽

Pcr Analysis ◽

Rt Pcr ◽

Patch Clamp Recording

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Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.5.f951 ◽

1998 ◽

Vol 274 (5) ◽

pp. F951-F957 ◽

Cited By ~ 6

Author(s):

Stephan Huber ◽

Bernd Schröppel ◽

Matthias Kretzler ◽

Detlef Schlöndorff ◽

Michael Horster

Keyword(s):

Patch Clamp ◽

Single Cell ◽

Collecting Duct ◽

Pcr Analysis ◽

Ureteric Bud ◽

Rt Pcr ◽

Outer Cortex ◽

Mrna Isoforms ◽

Pcr Products ◽

Pcr Techniques

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17( day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 ± 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

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Split single-cell RT-PCR analysis of Purkinje cells

Nature Protocols ◽

10.1038/nprot.2006.343 ◽

2006 ◽

Vol 1 (4) ◽

pp. 2143-2151 ◽

Cited By ~ 18

Author(s):

Shigeyuki Esumi ◽

Ryosuke Kaneko ◽

Yoshimi Kawamura ◽

Takeshi Yagi

Keyword(s):

Single Cell ◽

Purkinje Cells ◽

Pcr Analysis ◽

Rt Pcr

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Unicellular-Unilineage Erythropoietic Cultures: Molecular Analysis of Regulatory Gene Expression at Sibling Cell Level

Blood ◽

10.1182/blood.v93.10.3355.410k30_3355_3368 ◽

1999 ◽

Vol 93 (10) ◽

pp. 3355-3368 ◽

Cited By ~ 33

Author(s):

Benedikt L. Ziegler ◽

Robert Müller ◽

Mauro Valtieri ◽

Christa P. Lamping ◽

Christian A. Thomas ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Progenitor Cells ◽

Culture System ◽

Regulatory Gene ◽

Erythropoietin Receptor ◽

Pcr Analysis ◽

Control Mechanisms ◽

Rt Pcr ◽

Hematopoietic Progenitors

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34+ cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34+CD38− cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.

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Single-cell RT-PCR analysis of NK1 receptor mRNA in primary sensory neurons of the mouse

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)44941-x ◽

1997 ◽

Vol 73 ◽

pp. 109

Author(s):

Risa Tsurumi ◽

Tsugunobu Andoh ◽

Chihiro Tonda ◽

Yasushi Kuraishi

Keyword(s):

Single Cell ◽

Sensory Neurons ◽

Pcr Analysis ◽

Primary Sensory Neurons ◽

Nk1 Receptor ◽

Rt Pcr ◽

Receptor Mrna

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Membrane and Firing Properties of Glutamatergic and GABAergic Neurons in the Rat Medial Vestibular Nucleus

Journal of Neurophysiology ◽

10.1152/jn.00494.2004 ◽

2004 ◽

Vol 92 (5) ◽

pp. 3106-3120 ◽

Cited By ~ 49

Author(s):

Tomonori Takazawa ◽

Yasuhiko Saito ◽

Keisuke Tsuzuki ◽

Seiji Ozawa

Keyword(s):

Single Cell ◽

Firing Pattern ◽

Vestibular Nucleus ◽

Cholinergic Neurons ◽

Gabaergic Neurons ◽

Membrane Properties ◽

Medial Vestibular Nucleus ◽

Pcr Analysis ◽

Rt Pcr ◽

Electrophysiological Properties

In previous studies, neurons in the medial vestibular nucleus (MVN) were classified mainly into 2 types according to their intrinsic membrane properties in in vitro slice preparations. However, it has not been determined whether the classified neurons are excitatory or inhibitory ones. In the present study, to clarify the relationship between the chemical and electrophysiological properties of MVN neurons, we explored mRNAs of cellular markers for GABAergic (glutamic acid decarboxylase 65, 67, and neuronal GABA transporter), glutamatergic (vesicular glutamate transporter 1 and 2), glycinergic (glycine transporter 2), and cholinergic neurons (choline acetyltransferase and vesicular acetylcholine transporter) expressed in electrophysiologically characterized MVN neurons in rat brain stem slice preparations. For this purpose, we combined whole cell patch-clamp recording analysis with single-cell reverse transcription–polymerase chain reaction (RT-PCR) analysis. We examined the membrane properties such as afterhyperpolarization (AHP), firing pattern, and response to hyperpolarizing current pulse to classify MVN neurons. From the single-cell RT-PCR analysis, we found that GABAergic neurons consisted of heterogeneous populations with different membrane properties. Comparison of the membrane properties of GABAergic neurons with those of other neurons revealed that AHPs without slow components and a firing pattern with delayed spike generation (late spiking) were preferential properties of GABAergic neurons. On the other hand, most glutamatergic neurons formed a homogeneous subclass of neurons exhibiting AHPs with slow components, repetitive firings with constant interspike intervals (continuous spiking), and time-dependent inward rectification in response to hyperpolarizing current pulses. We also found a small number of cholinergic neurons with various membrane properties. These findings clarify the electrophysiological properties of excitatory and inhibitory neurons in the MVN, and the information about the preferential membrane properties may be useful for identifying GABAergic and glutamatergic MVN neurons electrophysiologically.

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Encephalomyelitis Associated with Akabane Virus Infection in Adult Cows

Veterinary Pathology ◽

10.1354/vp.39-2-269 ◽

2002 ◽

Vol 39 (2) ◽

pp. 269-273 ◽

Cited By ~ 37

Author(s):

J. K. Lee ◽

J. S. Park ◽

J. H. Choi ◽

B. K. Park ◽

B. C. Lee ◽

...

Keyword(s):

Spinal Cord ◽

Virus Infection ◽

Early Stage ◽

Neuronal Loss ◽

Ventral Horn ◽

Pcr Analysis ◽

Akabane Virus ◽

Rt Pcr ◽

Adult Cattle ◽

The Brain

Between August and September 2000, five 2–7-year-old cows in Korea exhibited neurologic signs and were diagnosed as infected with Akabane virus based on the results of histopathology, immunohistochemistry, serology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss was noted in the brain and the ventral horn gray matter of the spinal cord. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigen was detected in the brain and spinal cord, mainly in degenerating neurons and glial cells. RTPCR analysis revealed a target band of expected size in four cows. This is the first report on an outbreak of natural Akabane virus infection in adult cattle.

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Guinea Pig Kisspeptin Neurons Are Depolarized by Leptin via Activation of TRPC Channels

Endocrinology ◽

10.1210/en.2010-1285 ◽

2011 ◽

Vol 152 (4) ◽

pp. 1503-1514 ◽

Cited By ~ 87

Author(s):

Jian Qiu ◽

Yuan Fang ◽

Martha A. Bosch ◽

Oline K. Rønnekleiv ◽

Martin J. Kelly

Keyword(s):

Guinea Pig ◽

Single Cell ◽

Kinase Inhibitor ◽

Reproductive Function ◽

Janus Kinase ◽

Burst Firing ◽

Resting Membrane Potential ◽

Trpc Channels ◽

Rt Pcr ◽

Inward Current

Abstract Hypothalamic kisspeptin neurons are critical for driving reproductive function, but virtually nothing is known about their endogenous electrophysiological properties and the effects of leptin on their excitability. Therefore, we used the slice preparation from female guinea pigs to study the endogenous conductances and the effects of leptin on kisspeptin neurons. We targeted the arcuate kisspeptin neurons using visualized-patch whole-cell recording and identified kisspeptin neurons using immuocytochemical staining for kisspeptin or single cell RT-PCR. We also harvested dispersed arcuate neurons for analysis of expression of channel transcripts. Kisspeptin neurons exhibited a relatively negative resting membrane potential, and eighty percent of the neurons expressed a pacemaker current (h-current) and a T-type Ca2+ current. Furthermore, the glutamate receptor agonist N-methyl D-aspartic acid depolarized and induced burst firing in kisspeptin neurons. Leptin activated an inward current that depolarized kisspeptin neurons and increased (burst) firing, but leptin hyperpolarized NPY neurons. Lanthanum, a TRPC-4,-5 channel activator, potentiated the leptin-induced inward current by 170%. The leptin-activated current reversed near −15 mV and was abrogated by the relatively selective TRPC channel blocker 2-APB. The leptin effects were also blocked by a Janus kinase inhibitor, a phosphatidylinositol 3 kinase inhibitor, and a phospholipase Cγ inhibitor. In addition, the majority of these neurons expressed TRPC1 and -5 and phospholipase Cγ1 based on single cell RT-PCR. Therefore, guinea pig kisspeptin neurons express endogenous pacemaker currents, and leptin excites these neurons via activation of TRPC channels. The leptin excitatory effects on kisspeptin neurons may be critical for governing the excitatory drive to GnRH neurons during different nutritional states.

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