Single-cell Reverse Transcriptase-polymerase Chain Reaction Research Articles

Insulin and leptin excite anorexigenic pro-opiomelanocortin neurones via activation of TRPC5 channels.

Pro-opiomelanocortin (POMC) neurones within the hypothalamic arcuate nucleus are vital anorexigenic neurones. Both the insulin receptor and leptin receptor are coupled to activation of phosphatidylinositide-3 kinase (PI3K) to regulate multiple functions that increase POMC neuronal excitability. Using whole-cell recording in several mammalian species, we have found that both insulin and leptin depolarised POMC neurones via activation of transient receptor potential (TRPC)5 channels. TRPC5 channels have been rigorously characterised as the downstream effector based on their biophysical properties, pharmacological profile, and localisation by immunocytochemistry and single-cell reverse transcriptase-polymerase chain reaction. By contrast, insulin and leptin hyperpolarise and inhibit neuropeptide Y/agouti-related peptide neurones via activation of KATP channels. As proof of principle, insulin given i.c.v. robustly inhibits food intake and increases O2 utilisation, CO2 production and metabolic heat production. Therefore, these findings indicate that the depolarisation/excitation of POMC neurones by insulin and leptin is preserved across mammalian species and the activation of TRPC5 channels is likely a major mechanism by which insulin and leptin regulate energy homeostasis in mammals.

Localisation of 11β-Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular Nuclei.

An accumulating body of evidence suggests that the activity of the mineralocorticoid, aldosterone, in the brain via the mineralocorticoid receptor (MR) plays an important role in the regulation of blood pressure. MR was recently found in vasopressin and oxytocin synthesising magnocellular neurosecretory cells (MNCs) in both the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus. Considering the physiological effects of these hormones, MR in these neurones may be an important site mediating the action of aldosterone in blood pressure regulation within the brain. However, aldosterone activation of MR in the hypothalamus remains controversial as a result of the high binding affinity of glucocorticoids to MR at substantially higher concentrations compared to aldosterone. In aldosterone‐sensitive epithelia, the enzyme 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) prevents glucocorticoids from binding to MR by converting glucocorticoids into inactive metabolites. The present study aimed to determine whether 11β‐HSD2, which increases aldosterone selectivity, is expressed in MNCs. Specific 11β‐HSD2 immunoreactivity was found in the cytoplasm of the MNCs in both the SON and PVN. In addition, double‐fluorescence confocal microscopy demonstrated that MR‐immunoreactivity and 11β‐HSD2‐in situ hybridised products are colocalised in MNCs. Lastly, single‐cell reverse transcriptase‐polymerase chain reaction detected MR and 11β‐HSD2 mRNAs from cDNA libraries derived from single identified MNCs. These findings strongly suggest that MNCs in the SON and PVN are aldosterone‐sensitive neurones.

Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops

The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ~50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.

Ca2+/CALMODULIN‐DEPENDENT PROTEIN KINASE II IN THE COCKROACH Periplaneta americana: IDENTIFICATION OF FIVE ISOFORMS AND THEIR TISSUES DISTRIBUTION

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²⁺ signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

The presence of HBV mRNA in the fertilized in vitro embryo of HBV patients confirms vertical transmission of HBV via the ovum.

This study aimed to confirm that vertical transmission of hepatitis B virus (HBV) can occur via the infected ovum. Specimens studied were obtained from discarded test-tube embryos from mothers with chronic HBV infection who had received in vitro fertilization treatment. Single-cell reverse transcriptase-polymerase chain reaction was used to detect HBV mRNA in the embryos. HBV mRNA was detected in the cleavage embryos of patients with chronic HBV infection, with a detection rate of 13.2% (5/38). The level of serum HBV DNA was not related to the HBV mRNA positivity rates in embryos. In this study, HBV mRNA was detected in test-tube embryos from HBV-infected mothers who had received in vitro fertilization treatment. This confirms the theory of vertical transmission of HBV via the ovum, thereby providing an important theoretical basis for further study on the mechanism of HBV vertical transmission, influencing factors and blocking measures.

Pyramidal neurons in the superficial layers of rat retrosplenial cortex exhibit a late-spiking firing property

The rodent granular retrosplenial cortex (GRS) is reciprocally connected with the hippocampus. It is part of several networks implicated in spatial learning and memory, and is known to contain head-direction cells. There are, however, few specifics concerning the mechanisms and microcircuitry underlying its involvement in spatial and mnemonic functions. In this report, we set out to characterize intrinsic properties of a distinctive population of small pyramidal neurons in layer 2 of rat GRS. These neurons, as well as those in adjoining layer 3, were found to exhibit a late-spiking (LS) firing property. We established by multiple criteria that the LS property is a consequence of delayed rectifier and A-type potassium channels. These were identified as Kv1.1, Kv1.4 and Kv4.3 by Genechip analysis, in situ hybridization, single-cell reverse transcriptase-polymerase chain reaction, and pharmacological blockade. The LS property might facilitate comparison or integration of synaptic inputs during an interval delay, consistent with the proposed role of the GRS in memory-related processes.

Dopaminergic modulation of ganglion‐cell photoreceptors in rat

A novel class of photoreceptors, the intrinsically photosensitive retinal ganglion cells (ipRGCs), express the photopigment melanopsin and drive non-image-forming responses to light such as circadian photoentrainment, the pupillary light reflex and suppression of nocturnal melatonin production in the pineal. Because dendrites from one subclass of these cells - the M1-type ipRGCs - make presumptive synaptic contacts at sites of dopamine release from dopaminergic amacrine cells, they are prime targets for modulation by dopamine, a neuromodulator implicated in retinal circadian rhythms and light adaptation. In patch-clamp recordings from ipRGCs in intact rat retinas, dopamine attenuated the melanopsin-based photocurrent. We confirmed that this was the result of direct action on ipRGCs by replicating the effect in dissociated ipRGCs that were isolated from influences of other retinal neurons. In these recordings, the D1-family dopamine receptor agonist SKF38393 attenuated the photocurrent, caused a modest depolarization, and reduced the input resistance of ipRGCs. The D2-family agonist quinpirole had no effect on the photocurrent. Single-cell reverse-transcriptase polymerase chain reaction revealed that the majority of ipRGCs tested expressed drd1a, the gene coding for the D1a dopamine receptor. This finding was supported by immunohistochemical localization of D1a receptor protein in melanopsin-expressing ganglion cells. Finally, the adenylate cyclase activator forskolin, applied in combination with the phosphodiesterase inhibitor IBMX (isobutylmethylxanthine), mimicked the effects of SKF38393 on the ipRGC photocurrent, membrane potential and input resistance, consistent with a D1-receptor signaling pathway. These data suggest that dopamine, acting via D1-family receptors, alters the responses of ipRGCs and thus of non-image-forming vision.

Distinct Modes of AMPA Receptor Suppression at Developing Synapses by GluN2A and GluN2B: Single-Cell NMDA Receptor Subunit Deletion In Vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of thisswitch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence fora large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.

Pressor Effect of Apelin-13 in the Rostral Ventrolateral Medulla: Role of NAD(P)H Oxidase-Derived Superoxide

Microinjection of apelin-13 into the rostral ventrolateral medulla (RVLM) in the brainstem increases blood pressure in rats. In the present study, we tested the hypotheses that apelin-13 directly stimulates neuronal activity in neurons cultured from the brainstem and that NAD(P)H oxidase-derived reactive oxygen species are involved in this action of apelin-13. Microinjection of apelin-13 into the RVLM resulted in increases in arterial pressure and in renal sympathetic nerve activity in Sprague-Dawley rats. The pressor effect of apelin-13 was attenuated by the specific NAD(P)H-oxidase inhibitor gp91ds-tat. In neurons cultured from the ventral brainstem, spontaneous action potentials were recorded using current-clamp recording. Superfusion of neurons with apelin-13 (100 nM) increased the neuronal firing rate from 0.79 ± 0.14 to 1.45 ± 0.26 Hz (n = 7, P < 0.01) in angiotensin II receptor-like 1-positive neurons, identified with single-cell reverse transcriptase-polymerase chain reaction. Neither the angiotensin II type 1 receptor antagonist losartan nor the angiotensin II type 2 receptor antagonist 1-[[4-(dimethylamino)-3-methylphenyl[methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319) altered the positive chronotropic effect of apelin-13. Pretreatment of cells with either the reactive oxygen species scavenger superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD), 25 U/ml] or with gp91ds-tat significantly attenuated the chronotropic action of apelin-13. PEG-SOD and gp91ds-tat alone had no effect on basal neuronal firing. In addition, apelin-13 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in neuronal cultures. The superoxide generator xanthine-xanthine oxidase also increased neuronal activity in neurons, mimicking the neuronal response to apelin-13. These observations provide the first evidence that apelin-13 directly increases neuronal activity via stimulation of NAD(P)H oxidase-derived superoxide, a cellular signaling mechanism that may be involved in the pressor effect of apelin-13 in the RVLM.

Neuropeptide W has Cell Phenotype‐Specific Effects on the Excitability of Different Subpopulations of Paraventricular Nucleus Neurones

The administration of the neuropeptide W (NPW) and neuropeptide B (NPB) in rodents has been shown to influence the activity of a variety of autonomic and neuroendocrine systems. The paraventricular nucleus (PVN) is a major autonomic and neuroendocrine integration site in the hypothalamus, and neurones within this nucleus express the receptor for these ligands, NPB/W receptor 1 (NPBWR1). In the present study, we used whole cell patch clamp recordings coupled with single-cell reverse transcriptase-polymerase chain reaction to examine the effects of neuropeptide W-23 (NPW-23) on the excitability of identified PVN neurones. Oxytocin, vasopressin and thyrotrophin-releasing hormone neurones were all found to be responsive to 10 nm NPW-23, although both depolarising and hyperpolarising effects were observed in each of these cell groups. By contrast, corticotrophin-releasing hormone cells were unaffected. Further subdivision of chemically phenotyped cell groups into magnocellular, neuroendocrine or pre-autonomic neurones, using their electrophysiological fingerprints, revealed that neurones projecting to medullary and spinal targets were predominantly inhibited by NPW-23, whereas those that projected to median eminence or neural lobe showed almost equivalent numbers of depolarising and hyperpolarising cells. The demonstration of particular phenotypic populations of PVN neurones showing NPW-induced effects on excitability reinforces the importance of the NPB/NPW neuropeptide system as a regulator of autonomic function.

Serotoninergic Fine-Tuning of the Excitation–Inhibition Balance in Rat Visual Cortical Networks

Fundamental brain functions depend on a balance between excitation (E) and inhibition (I) that is highly adjusted to a 20-80% set point in layer 5 pyramidal neurons (L5PNs) of rat visual cortex. Dysregulations of both the E-I balance and the serotonergic system in neocortical networks lead to serious neuronal diseases including depression, schizophrenia, and epilepsy. However, no link between the activation of neuronal 5-hydroxytryptamine receptors (5-HTRs) and the cortical E-I balance has yet been reported. Here we used a combination of patch-clamp recordings of composite stimulus-locked responses in L5PN following local electrical stimulations in either layer 2/3 or 6, simultaneous measurement of excitatory and inhibitory conductance dynamics, together with selective pharmacological targeting and single-cell reverse transcriptase-polymerase chain reaction. We show that cortical serotonin shifts the E-I balance in favor of more E and we reveal fine and differential modulations of the E-I balance between 5-HTR subtypes, in relation to whether layer 2/3 or 6 was stimulated and in concordance with the specific expression pattern of these subtypes in pyramidal cells and deep interneurons. This first evidence for the functional segregation of 5-HTR subtypes sheds new light on their coherent functioning in polysynaptic sensory circuits.

Activation of δ-Opioid Receptors Reduces Excitatory Input to Putative Gustatory Cells Within the Nucleus of the Solitary Tract

The rostral nucleus of the solitary tract (NST) is the first central relay in the gustatory pathway and plays a key role in processing and modulation of gustatory information. Here, we investigated the effects of opioid receptor agonists and antagonists on synaptic responses of the gustatory parabrachial nuclei (PbN)-projecting neurons in the rostral NST to electrical stimulation of the solitary tract (ST) using whole cell recordings in the hamster brain stem slices. ST-evoked excitatory postsynaptic currents (EPSCs) were significantly reduced by met-enkephalin (MetE) in a concentration-dependent fashion and this effect was eliminated by naltrexone hydrochloride, a nonselective opioid receptor antagonist. Bath application of naltrindole hydrochloride, a selective delta-opioid receptor antagonist, eliminated MetE-induced reduction of EPSCs, whereas CTOP, a selective mu-opioid receptor antagonist had no effect, indicating that delta-opioid receptors are involved in the reduction of ST-evoked EPSCs induced by MetE. SNC80, a selective delta-opioid receptor agonist, mimicked the effect of MetE. The SNC80-induced reduction of ST-evoked EPSCs was eliminated by 7-benzylidenenaltrexone, a selective delta1-opioid receptor antagonist but not by naltriben mesylate, a selective delta2-opioid receptor antagonist, indicating that delta1-opioid receptors mediate the reduction of ST-evoked EPSCs induced by SNC80. Single-cell reverse transcriptase-polymerase chain reaction analysis revealed the presence of delta1-opioid receptor mRNA in cells that responded to SNC80 with a reduction in ST-evoked EPSCs. Moreover, Western blot analysis demonstrated the presence of 40-kDa delta-opioid receptor proteins in the rostral NST tissue. These results suggest that postsynaptic delta1-opioid receptors are involved in opioid-induced reduction of ST-evoked EPSCs of PbN-projecting rostral NST cells.

Expression of Adenosine A2A Receptors in the Rat Lumbar Spinal Cord and Implications in the Modulation of N-Methyl-d-Aspartate Receptor Currents

The presence of A(2A) receptors in the dorsal horn of the spinal cord remains controversial. At this level, activation of N-methyl-d-aspartate (NMDA) receptors induces wind-up, which is clinically expressed as hyperalgesia. Inhibition of NMDA receptor currents after activation of A(2A) receptors has been shown in rat neostriatal neurons. In this study, we sought to establish the presence of adenosine A(2A) receptors in the lamina II of the rat lumbar dorsal horn neurons and investigated whether the activation of A(2A) receptors is able to modulate NMDA receptor currents. Experiments were conducted in the rat lumbar spinal cord. The presence of adenosine A(2A) receptor transcripts inside the lumbar spinal cord is assessed with the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Western blot experiments are performed at the same level. The RT-PCR technique is also performed specifically in the lamina II, and the presence of adenosine A(2A) receptor transcripts is assessed in neurons from the lamina II with the single-cell RT-PCR technique. The effect of adenosine A(2A) receptor activation on NMDA receptor currents is studied by the whole-cell configuration of the patch clamp technique. RT-PCR performed on the lumbar spinal cord revealed the presence of adenosine A(2A) receptor transcripts. Western blot experiments revealed the presence of A(2A) receptors in the lumbar spinal cord. RT-PCR performed on the substantia gelatinosa also revealed the presence of adenosine A(2A) receptor transcripts. Finally, single cell RT-PCR revealed the presence of adenosine A(2A) receptor transcripts in a sample of lamina II neurons. Patch clamp recordings showed an inhibition of NMDA currents during the application of a selective A(2A) agonist. These results demonstrate the presence of A(2A) receptor on neurons from the substantia gelatinosa of the rat lumbar dorsal horn and the inhibition of NMDA-induced currents by the application of a selective A(2A) receptor agonist. Therefore, A(2A) receptor ligands could modulate pain processing at the spinal cord level.

Growth Hormone‐Releasing Hormone and Glucocorticoids Determine the Balance Between Luteinising Hormone (LH) β‐ and LHβ/Follicle‐Stimulating Hormone β‐Positive Gonadotrophs and Somatotrophs in the 14‐Day‐Old Rat Pituitary Tissue in Aggregate Cell Culture

Fourteen-day-old rat pituitary tissue represents an attractive model for studying cell population dynamics, particularly of gonadotrophs. Prolonged three-dimensional culture in serum- and hormone-free medium causes a striking decline in somatotroph abundance but a several-fold rise in monohormonal LH beta-positive cell number, whereas bihormonal gonadotrophs almost disappear. In the present study, we investigated whether these changes are inter-related by examining the effects of growth hormone-releasing hormone (GHRH) and glucocorticoids, two protagonist regulators of somatotrophs. Cells were identified by single cell reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence. Supplementation of the cultures for 2 weeks with GHRH (1 nm) did not augment the proportion of somatotrophs, but expanded the nonhormonal cell population. GHRH reduced the proportion of monohormonal luteinising hormone (LH)beta mRNA positive cells to approximately 50% of control, although the effect was not seen when these cells were visualised by immunostaining. Supplementation of the cultures with dexamethasone (4 nM) for 3 weeks partially rescued LH beta/follicle-stimulating hormone beta cells and fully rescued the GH mRNA cells in parallel with a decline in nonhormonal cell abundance, but strongly reduced bromodeoxyuridine labelling of GH-immunoreactive cells. As studied by patch-clamp single cell RT-PCR at the start of culture, GHRH caused an acute rise in intracellular [Ca(2+)] in some monohormonal GH cells, but at a higher incidence in cells expressing LH beta mRNA, alone or in combination with GH mRNA and/or pro-opiomelanocortin (POMC) mRNA. The present data suggest that, in the 14-day-old rat pituitary, the majority of GHRH target cells are cells expressing LH beta mRNA alone or in combination with GH and/or POMC mRNA. The data show co-regulation of gonadotroph and somatotroph population sizes by glucocorticoids and GHRH, with the former preserving bihormonal gonadotrophs and the latter repressing LH beta-only cell abundance. GHRH may not expand the somatotroph population unless glucocorticoid hormone is present to maintain terminal differentiation.

Terminal Nerve Gonadotrophin‐Releasing Hormone (GnRH) Neurones Express Multiple GnRH Receptors in a Teleost, the Dwarf Gourami (Colisa lalia)

Gonadotophin-releasing hormone (GnRH) peptide released from the terminal nerve (TN)-GnRH neurones of the dwarf gourami primarily modifies the electrical properties of various neurones, including the TN-GnRH neurones themselves. However, our knowledge on the expression of GnRH receptors (GnRHRs) in the TN-GnRH neurones is still limited. Here, we used the single-cell reverse transcriptase-polymerase chain reaction after whole-cell patch-clamp recording to study the distribution of various GnRHR types expressed in the individual TN-GnRH neurones. We found that TN-GnRH neurones express two of the three types of GnRHRs cloned in the dwarf gourami: GnRHR1-2 and -R2, but not -R1-1. Furthermore, in agreement with our previous findings, all TN-GnRH neurones contained mRNAs of salmon GnRH but not chicken GnRH-II.

Somatostatinergic modulation of firing pattern and calcium-activated potassium currents in medium spiny neostriatal neurons

Somatostatin is synthesized and released by aspiny GABAergic interneurons of the neostriatum, some of them identified as low threshold spike generating neurons (LTS-interneurons). These neurons make synaptic contacts with spiny neostriatal projection neurons. However, very few somatostatin actions on projection neurons have been described. The present work reports that somatostatin modulates the Ca2+ activated K+ currents (KCa currents) expressed by projection cells. These actions contribute in designing the firing pattern of the spiny projection neuron; which is the output of the neostriatum. Small conductance (SK) and large conductance (BK) KCa currents represent between 30% and 50% of the sustained outward current in spiny cells. Somatostatin reduces SK-type K+ currents and at the same time enhances BK-type K+ currents. This dual effect enhances the fast component of the afterhyperpolarizing potential while reducing the slow component. Somatostatin then modifies the firing pattern of spiny neurons which changed from a tonic regular pattern to an interrupted “stuttering”-like pattern. Semi-quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) tissue expression analysis of dorsal striatal somatostatinergic receptors (SSTR) mRNA revealed that all five SSTR mRNAs are present. However, single cell RT-PCR profiling suggests that the most probable receptor in charge of this modulation is the SSTR2 receptor. Interestingly, aspiny interneurons may exhibit a “stuttering”-like firing pattern. Therefore, somatostatin actions appear to be the entrainment of projection neurons to the rhythms generated by some interneurons. Somatostatin is then capable of modifying the processing and output of the neostriatum.

Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the Activation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for Extrapyramidal Side Effects of Serotonin Reuptake Inhibitors

The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.

Constitutive Expression of Functionally Active Cyclin-Dependent Kinases and Their Binding Partners Suggests Noncanonical Functions of Cell Cycle Regulators in Differentiated Neurons

Neurodegeneration in Alzheimer's disease and various experimental lesion paradigms are associated with an unscheduled upregulation of cell cycle-related proteins, indicating a link between cell cycle reactivation and neuronal death. Recent evidence, however, suggests that at least some of the canonical cell cycle regulators are constitutively expressed in differentiated neurons of the adult brain. Systematic investigations on the constitutive expression of cell cycle regulators in differentiated neurons in vivo, providing the basis for further insights into their potential role under pathological conditions, however, have not been carried out. Here, we demonstrate a constitutive neuronal expression of Cdks 1, 2, and 4; their activators cyclins D, A, B, and E; and their inhibitors p15(Ink4b), p16(Ink4a), p18(Ink4c), p19(Ink4d), p21(Waf1/Cip1), p27(Kip1), and p57(Kip2) within the neocortex of adult mice by western blot and immunocytochemistry. Expression was verified by single-cell reverse transcriptase-polymerase chain reaction applied to individual microscopically identified neurons captured with laser dissection. Immunoprecipitation and in vitro kinase assays revealed that Cdks 1, 2, and 4 are properly complexed to cyclins and exhibit kinase activity. This physiological expression of positive cell cycle regulators in adult neurons is clearly not related to neuronal proliferation. Taken together, our findings demonstrate a constitutive expression of functionally active cyclin-dependent kinases and their regulators in differentiated neurons suggesting a noncanonical role of cell cycle regulators potentially linked to neuronal plasticity and/or stability.

Molecular Basis for the GABAA Receptor-Mediated Tonic Inhibition in Rat Somatosensory Cortex

Fast inhibitory synaptic transmission is primarily mediated by synaptically released gamma-aminobutyric acid (GABA) acting on postsynaptic GABA(A) receptors. GABA acting on GABA(A) receptors produces not only phasic but also tonic inhibitions by persistent activation of extrasynaptic receptors. However, the mechanistic characteristics of tonic inhibition in the neocortex are not well-understood. To address this, we studied pharmacologically isolated GABA(A) receptor-mediated currents in neocortical pyramidal neurons in rat brain slices. Bath application of bicuculline blocked miniature inhibitory postsynaptic currents (mIPSCs) and produced an outward shift in baseline holding current (I(hold)). Low concentrations of SR95531, a competitive GABA(A) receptor antagonist, abolished mIPSCs but had no significant effect on I(hold). The benzodiazepine midazolam produced an inward shift in I(hold) by augmenting tonic GABA(A) receptor-mediated currents, which were significantly greater in layer V neurons than in layer II/III. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a relatively higher expressions of alpha1 and alpha5 subunit mRNA in layer V neurons. L-655708, an alpha5 subunit-specific inverse agonist, reduced tonic currents in layer V but not in layer II/III neurons, whereas zolpidem, an alpha1-subunit agonist, exerted equivalent effects in both layers. These data suggest that the alpha1 GABA(A) receptor subunit is generally involved in tonic inhibition in pyramidal neurons of the neocortex, whereas the alpha5 subunit is specifically involved in layer V neurons.

P2Y receptor‐mediated excitation in the posterior hypothalamus

Histaminergic neurons located in the posterior hypothalamus (tuberomamillary nucleus, TMN) project widely through the whole brain controlling arousal and attention. They are tonically active during wakefulness but cease firing during sleep. As a homeostatic theory of sleep involves ATP depletion and adenosine accumulation in the brain, we investigated the role of ATP and its analogues as well as adenosine on neuronal activity in the TMN. We show increased firing of rat TMN neurons by ATP, ADP, UTP and 2meSATP, indicating activation of receptors belonging to the P2Y family. Adenosine affected neither membrane potential nor firing of these cells. Single-cell reverse transcriptase-polymerase chain reaction revealed that P2Y1 and P2Y4 are prevailing receptors in TMN neurons. P2Y1 receptor mRNA was detected with a higher frequency in 2-week-old than in 4-week-old rats; in accordance, 2meSATP was more potent than ATP. Semi-quantitative real-time polymerase chain reaction revealed a developmental downregulation of mRNA levels for P2Y1 and P2Y4 receptors. Immunocytochemistry demonstrated neuronal and glial localization of the P2Y1 receptor protein. Network activity measured with multielectrode arrays in primary cultures made from the posterior hypothalamus was enhanced by UTP and 2meSATP (P2Y4 and P2Y1 agonists, respectively). ATP caused an inhibition of firing, which was reversed in the presence of suramin or gabazine [gamma-aminobutyric acid (GABA)A receptor antagonist], indicating that GABAergic neurons are preferentially activated by ATP in this network. Excitation of the wake-active TMN neurons by nucleotides and the lack of adenosine action may be important factors in sleep-wake regulation.