Digital PCR to Determine the number of Transcripts from Single Neurons after Patch-Clamp Recording

Faragó, Nóra; Kocsis, Ágnes Katalin; Lovas, Sándor; Molnár, Gábor; Boldog, Eszter; Rózsa, Márton; Szemenyei, Viktor; Vámos, Enikő; Nagy, L; Tamás, Gábor; Puskás, László G.

doi:10.2144/000114029

Cited by 25 publications

(24 citation statements)

References 27 publications

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“…(p > 0.05, one-way ANOVA, n = 20 ROIs in 2 sections, see Materials and Methods). Since PV+INs are not the only IN types expressing δ-GABA A R, but other INs such as neurogliaform cells (NGCs) of the neocortex and the hippocampus also heavily express δ-GABA A Rs (Oláh et al, 2009; Faragó et al, 2013), we exmined δ-GABA A Rs distribution in areas where few PV+INs are present. Fig.…”

Section: Resultsmentioning

confidence: 99%

In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons

Ferando

Módy

2015

Neuropharmacology

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Perisynaptic and extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) mediate tonic conductances in many principal cells and interneurons (INs) of the central nervous system. δ-GABAARs comprise α4 subunits on principal cells of the neocortex and hippocampus, whereas they usually contain α1 on various INs. δ-GABAARs are highly plastic and exhibit a unique pharmacology, being insensitive to benzodiazepines, but sensitive to low concentrations of neurosteroids (NS). The neuroactive forms of several steroid hormones, NS act on δ-GABAARs as potent positive allosteric modulators leading to changes in neuronal excitability and network synchrony. In fact, δ-GABAARs on INs have been shown to control the dynamics of γ oscillations. During times of altered NS production, including stress, puberty, ovarian cycle and pregnancy, δ-GABAAR expression varies in different neurons regardless of the α subunits they contain. This plasticity has direct consequences for network functioning. In particular, plasticity of these receptors in pregnancy and the postpartum period affects CA3 γ oscillation frequencies (recorded in vitro), and during the ovarian cycle they underlie fluctuations in the amplitude of CA1 γ oscillations (recorded in vivo). Most δ-GABAAR-expressing INs in CA3 stratum pyramidale (SP) are parvalbumin (PV)+INs, whose fundamental role in γ oscillation generation and control has been extensively investigated. In this study we reduced or deleted δ-subunits in PV+INs, with the use of a PV/Cre-Gabrd/floxed genetic system. By means of immunohistochemistry we show that PV expression in PV-Gabrd−/− and PV-Gabrd+/− mice remains unaltered. Additionally, we confirm the absence of δ-GABAARs specifically from PV+INs. We find that in both PV-Gabrd−/− and PV-Gabrd+/− mice, CA3 γ oscillation frequencies measured in vitro were increased compared to Cre−/− controls. The increased frequencies could be lowered to control levels in PV-Gabrd+/− by the NS allopregnanolone (3α,5α-tetrahydroprogesterone, ALLO, 100 nM) but not by the synthetic δ-GABAAR positive allosteric modulator 4-Chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl] benzamide (DS-2, 10 µM). This is consistent with the idea that DS-2, in contrast to ALLO, selectively targets α4/δ-GABAARs but not the α1/δ-GABAARs found on INs. Therefore, development of drugs selective for IN-specific α1/δ-GABAARs may be useful in neurological and psychiatric conditions correlated with altered PV+IN function and aberrant γ oscillations.

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Section: Resultsmentioning

confidence: 99%

In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons

Ferando

Módy

2015

Neuropharmacology

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“…Animals used for this study also provided brain slices for other projects performed in parallel in the laboratory. Slice preparation and recordings were performed as previously described [13,21]. Recordings were obtained at approximately 36°C from cells visualised in layers I-III by infrared differential interference contrast videomicroscopy, at depths 60-130 μm from the surface of the slice.…”

Section: Methodsmentioning

confidence: 99%

Expression of GLP-1 receptors in insulin-containing interneurons of rat cerebral cortex

et al. 2019

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Aims/hypothesis Glucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons. Methods We harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5 mmol/l glucose) and hyperglycaemia (10 mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results. Results Single-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p < 0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development. Conclusions/interpretation Our results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells.

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“…After harvesting the cytoplasm of the recorded cells the samples were frozen in dry ice and of the OpenArray slide, cycling in the OpenArray NT cycler and data analysis was done as previously described (34) . For our dPCR protocol amplification, reactions with CT confidence values below 100 as well as reactions having CT values less than 23 or greater than 33 were considered primer dimers or background signals, respectively, and were excluded from the data set.…”

Section: Molecular Biological Analysismentioning

confidence: 99%

Automatic deep learning driven label-free image guided patch clamp system for human and rodent in vitro slice physiology

Koós

Oláh

Balassa

et al. 2020

Preprint

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Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. We have developed a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model was trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements were performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.

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Digital PCR to Determine the number of Transcripts from Single Neurons after Patch-Clamp Recording

Cited by 25 publications

References 27 publications

In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons

In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons

Expression of GLP-1 receptors in insulin-containing interneurons of rat cerebral cortex

Automatic deep learning driven label-free image guided patch clamp system for human and rodent in vitro slice physiology

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