Single-cell Reverse transcriptase-PCR Research Articles

COX-2-Derived Prostaglandin E2 Produced by Pyramidal Neurons Contributes to Neurovascular Coupling in the Rodent Cerebral Cortex

Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE2) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE2 is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets. Brain function critically depends on a permanent spatiotemporal match between neuronal activity and blood supply, known as NVC. In the cerebral cortex, prostaglandins are major contributors to NVC. However, their biochemical identity remains elusive and their cellular origins are still under debate. Although astrocytes can induce vasodilations through the release of prostaglandins, the recruitment of this pathway during sensory stimulation is questioned. Using multidisciplinary approaches from single-cell reverse transcriptase-PCR, mass spectrometry, to ex vivo and in vivo pharmacology and optogenetics, we provide compelling evidence identifying PgE2 as the main prostaglandin in NVC, pyramidal neurons as their main cellular source and the vasodilatory EP2 and EP4 receptors as their main targets. These original findings will certainly change the current view of NVC.

Molecular characterization of individual D3 dopamine receptor-expressing cells isolated from multiple brain regions of a novel mouse model

Among dopamine receptors, the expression and function of the D3 receptor subtype is not well understood. The receptor has the highest affinity for dopamine and many drugs that target dopamine receptors.In this paper, we examined, at the single cell level, the characteristics of D3 receptor-expressing cells isolated from different brain regions of male and female mice that were either 35 or 70 days old. The brain regions included nucleus accumbens, Islands of Calleja, olfactory tubercle,retrosplenial cortex, dorsal subiculum, mammillary body,amygdala and septum. The expression analysis was done in the drd3-enhanced green fluorescent protein transgenic mice that report the endogenous expression of D3 receptor mRNA. Using single cell reverse transcriptase PCR, we determined if the D3 receptor-expressing fluorescent cells in these mice were neurons or glia and if they were glutamatergic, GABAergic or catecholaminergic. Next, we determined if the fluorescent cells co-expressed the four other dopamine receptor subtypes, adenylate cyclase V(ACV) isoform, and three different isoforms of G protein coupled inward rectifier potassium (GIRK) channels. The results suggest that D3 receptor is expressed in neurons,with region-specific expression in glutamatergic and GABAergic neurons. The D3 receptor primarily coexpressed with D1 and D2 dopamine receptors with regional, sex and age-dependent differences in the coexpression pattern. The percentage of cells co-expressing D3 receptor and ACV or GIRK channels varied significantly by brain region, sex and age. The molecular characterization of D3 receptor-expressing cells in mouse brain reported here will facilitate the characterization of D(3) receptor function in physiology and pathophysiology.

GABA, Its Receptors, and GABAergic Inhibition in Mouse Taste Buds

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

17Beta-estradiol regulation of T-type calcium channels in gonadotropin-releasing hormone neurons.

T-type calcium channels are responsible for generating low-threshold spikes that facilitate burst firing and neurotransmitter release in neurons. Gonadotropin-releasing hormone (GnRH) neurons exhibit burst firing, but the underlying conductances are not known. Previously, we found that 17beta-estradiol (E2) increases T-type channel expression and excitability of hypothalamic arcuate nucleus neurons. Therefore, we used ovariectomized oil- or E2-treated EGFP (enhanced green fluorescent protein)-GnRH mice to explore the expression and E2 regulation of T-type channels in GnRH neurons. Based on single-cell reverse transcriptase-PCR and real-time PCR quantification of the T-type channel alpha(1) subunits, we found that all three subunits were expressed in GnRH neurons, with expression levels as follows: Cav3.3 > or = Cav3.2 > Cav3.1. The mRNA expression of the three subunits was increased with surge-inducing levels of E2 during the morning. During the afternoon, Cav3.3 mRNA expression remained elevated, whereas Cav3.1 and Cav3.2 were decreased. The membrane estrogen receptor agonist STX increased the expression of Cav3.3 but not Cav3.2 in GnRH neurons. Whole-cell patch recordings in GnRH neurons revealed that E2 treatment significantly augmented T-type current density at both time points and increased the rebound excitation during the afternoon. Although E2 regulated the mRNA expression of all three subunits in GnRH neurons, the increased expression combined with the slower inactivation kinetics of the T-type current indicates that Cav3.3 may be the most important for bursting activity associated with the GnRH/LH (luteinizing hormone) surge. The E2-induced increase in mRNA expression, which depends in part on membrane-initiated signaling, leads to increased channel function and neuronal excitability and could be a mechanism by which E2 facilitates burst firing and cyclic GnRH neurosecretion.

Classification of NPY-Expressing Neocortical Interneurons

Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Because of the large electrophysiological, molecular, and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reverse transcriptase-PCR designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons, we used and compared different unsupervised clustering algorithms based on laminar location and electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively GABAergic and consistently disclosed three main types of NPY-expressing interneurons. (1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent and superficial, and substantially expressed the neuronal isoform of nitric oxide synthase. (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I coexpressed somatostatin and were the most excitable type. (3) Among fast-spiking and parvalbumin-positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles.

Sensitization of Transient Receptor Potential Vanilloid 1 by the Prokineticin Receptor Agonist Bv8

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

Identification of the Cytolinker Protein Plectin in Neuronal Cellst-Expression of a Rodless Isoform in Neurons of the Rat Superior Cervical Ganglion

Plectin, a large (> 500 kDa) dumbbell-shaped cytolinker protein plays an important role in the organization of the cytoskeletal network and the maintenance of cell integrity in a wide variety of tissues and cell types. Earlier experiments revealed the presence of plectin in the central nervous system, whereas the expression in the peripheral nervous system remained unclear. Our results obtained with reverse transcriptase-PCR (RT-PCR) provide evidence that plectin is expressed in structures of the rat peripheral nervous system. In addition to well-characterized plectin transcripts we were able to reveal novel splicing variants affecting the region coding for the central rod domain. Previous studies report a high, but tissue-specific variability of the N-terminal domain of plectin due to alternatively spliced first coding exons and the optionally spliced small exons 2 alpha and 3 alpha. We demonstrate for the first time, using single-cell RT-PCR and immunocytochemistry, that plectin is expressed in neurons of the rat superior cervical ganglion (SCG). Plectin transcripts of single SCG neurons, starting with exon 1c as the first coding exon, contain the optionally spliced exon 2 alpha but lack exon 31. These data therefore suggest that plectin is expressed in rat SCG neurons as a rodless isoform with the molecular mass of 390 kDa.

Quantitative single-cell RT-PCR and Ca2+ imaging in brain slices

We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in individual cells of living brain slices. Quantification is achieved by using rapid-cycle, real-time PCR protocols and high-resolution external cDNA standard curves for the gene of interest. The method consists of several procedures, including cell soma harvest, reverse transcription, and an optimized cDNA purification step, which allowed us to quantify transcripts in small types of neurons, like cerebellar granule cells. Thus, we detected in single granule cells an average of 20 transcript copies of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon calcium imaging and quantitative RT-PCR in single Purkinje and granule cells, respectively, and identified distinct glutamate receptor-dependent Ca2+ responses in these two cell types. The approach was further tested by profiling the expression of the ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum. Our study revealed a developmental switch from an average of 15 NR2B copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell after P26. Taken together, our results demonstrate that the new method is rapid, highly sensitive, provides reliable results in neurons of various sizes, and can be used in combination with Ca2+ imaging.

Cholinergic Suppression of KCNQ Channel Currents Enhances Excitability of Striatal Medium Spiny Neurons

In response to glutamatergic synaptic drive, striatal medium spiny neurons in vivo transition to a depolarized "up state" near spike threshold. In the up state, medium spiny neurons either depolarize enough to spike or remain below spike threshold and are silent before returning to the hyperpolarized "down state." Previous work has suggested that subthreshold K+ channel currents were responsible for this dichotomous behavior, but the channels giving rise to the current and the factors determining its engagement have been a mystery. To move toward resolution of these questions, perforated-patch recordings from medium spiny neurons in tissue slices were performed. K+ channels with pharmacological and kinetic features of KCNQ channels potently regulated spiking at up-state potentials. Single-cell reverse transcriptase-PCR confirmed the expression of KCNQ2, KCNQ3, and KCNQ5 mRNAs in medium spiny neurons. KCNQ channel currents in these cells were potently reduced by M1 muscarinic receptors, because the effects of carbachol were blocked by M1 receptor antagonists and lost in neurons lacking M1 receptors. Reversal of the modulation was blocked by a phosphoinositol 4-kinase inhibitor, indicating a requirement for phosphotidylinositol 4,5-bisphosphate resynthesis for recovery. Inhibition of protein kinase C reduced the efficacy of the muscarinic modulation. Finally, acceleration of cholinergic interneuron spiking with 4-aminopyridine mimicked the effects of exogenous agonist application. Together, these results show that KCNQ channels are potent regulators of the excitability of medium spiny neurons at up-state potentials, and they are modulated by intrastriatal cholinergic interneurons, providing a mechanistic explanation for variability in spiking during up states seen in vivo.

Evidence for specific TRPM8 expression in human prostate secretory epithelial cells: functional androgen receptor requirement

TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

Voltage-Dependent Sodium Channels Are Expressed in Nonspiking Retinal Bipolar Neurons

Retinal bipolar neurons transmit visual information by means of graded synaptic potentials that spread to the synaptic terminal without sodium-dependent action potentials. Although action potentials are not involved, voltage-dependent sodium channels may enhance subthreshold depolarizing potentials in the dendrites and soma of bipolar cells, as they do in other CNS neurons. We report here that voltage-dependent sodium currents are observed in a subset of bipolar neurons from goldfish retina. Single-cell reverse transcriptase-PCR identified four different sodium channel alpha subunits in goldfish bipolar cells, putatively corresponding to the mammalian voltage-gated sodium channels Na(v)1.1, Na(v)1.2, Na(v)1.3, and Na(v)1.6. The amount of sodium current was largest in cells with smaller synaptic terminals, which probably represent cone bipolar cells. Localization of sodium channel immunoreactivity in goldfish retina confirmed the expression of voltage-gated sodium channels in cone bipolar cells of both ON and OFF types. Both immunocytochemical and physiological evidence suggests that the sodium channels are localized to the soma and dendrites where they may play a role in transmission of synaptic signals, particularly in the long, thin dendrites of cone bipolar cells.

Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.