scholarly journals A pool of postnatally-generated interneurons persists in an immature stage in the olfactory bulb

2018 ◽  
Author(s):  
Nuria Benito ◽  
Elodie Gaborieau ◽  
Alvaro Sanz Diez ◽  
Seher Kosar ◽  
Louis Foucault ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Olfactory Bulb ◽  
Immature Stage ◽  
Network Activity ◽  
Excitatory Synapses ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Synaptic Inputs ◽  
Glomerular Layer ◽  
Over Time

ABSTRACTCalretinin (CR)-expressing periglomerular (PG) cells are the most abundant interneurons in the glomerular layer of the olfactory bulb. They are predominately generated postnatally from the septal and dorsal sub-ventricular zones that continue producing them well into adulthood. Yet, little is known about their properties and functions. Using transgenic approaches and patch-clamp recording we show that CR(+) PG cells of both septal and dorsal origin have homogenous morphological and electrophysiological properties. They express a surprisingly poor repertoire of voltage-activated channels and fire, at most, one action potential. They also receive little synaptic inputs and NMDA receptors predominate at excitatory synapses. These properties, that resemble those of immature neurons, persist over time and limit the contribution of CR(+) PG cells in network activity. Thus, postnatally-generated CR(+) PG cells continuously supply a pool of latent neurons that unlikely participate in olfactory bulb computation but may provide a so far unsuspected reservoir of plasticity.

scholarly journals Electrophysiological Properties of Substantia Gelatinosa Neurons in the Preparation of a Slice of Middle-Aged Rat Spinal Cord

2021 ◽  
Vol 13 ◽  
Author(s):  
Yang Li ◽  
Shanchu Su ◽  
Jiaqi Yu ◽  
Minjing Peng ◽  
Shengjun Wan ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Patch Clamp ◽  
Molecular Mechanisms ◽  
Substantia Gelatinosa ◽  
Membrane Properties ◽  
Middle Aged ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Glutamatergic Neurons ◽  
Single Spike

A patch-clamp recording in slices generated from the brain or the spinal cord has facilitated the exploration of neuronal circuits and the molecular mechanisms underlying neurological disorders. However, the rodents that are used to generate the spinal cord slices in previous studies involving a patch-clamp recording have been limited to those in the juvenile or adolescent stage. Here, we applied an N-methyl-D-glucamine HCl (NMDG-HCl) solution that enabled the patch-clamp recordings to be performed on the superficial dorsal horn neurons in the slices derived from middle-aged rats. The success rate of stable recordings from substantia gelatinosa (SG) neurons was 34.6% (90/260). When stimulated with long current pulses, 43.3% (39/90) of the neurons presented a tonic-firing pattern, which was considered to represent γ-aminobutyric acid-ergic (GABAergic) signals. Presumptive glutamatergic neurons presented 38.9% (35/90) delayed and 8.3% (7/90) single-spike patterns. The intrinsic membrane properties of both the neuron types were similar but delayed (glutamatergic) neurons appeared to be more excitable as indicated by the decreased latency and rheobase values of the action potential compared with those of tonic (GABAergic) neurons. Furthermore, the glutamatergic neurons were integrated, which receive more excitatory synaptic transmission. We demonstrated that the NMDG-HCl cutting solution could be used to prepare the spinal cord slices of middle-aged rodents for the patch-clamp recording. In combination with other techniques, this preparation method might permit the further study of the functions of the spinal cord in the pathological processes that occur in aging-associated diseases.

scholarly journals Q&A: using Patch-seq to profile single cells

BMC Biology ◽  
2017 ◽  
Vol 15 (1) ◽  
Author(s):  
Cathryn R. Cadwell ◽  
Rickard Sandberg ◽  
Xiaolong Jiang ◽  
Andreas S. Tolias
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Patch Clamp ◽  
Single Cell ◽  
Single Cells ◽  
Cell Types ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Whole Cell Patch Clamp ◽  
Single Cell Rna Sequencing

Abstract Individual neurons vary widely in terms of their gene expression, morphology, and electrophysiological properties. While many techniques exist to study single-cell variability along one or two of these dimensions, very few techniques can assess all three features for a single cell. We recently developed Patch-seq, which combines whole-cell patch clamp recording with single-cell RNA-sequencing and immunohistochemistry to comprehensively profile the transcriptomic, morphologic, and physiologic features of individual neurons. Patch-seq can be broadly applied to characterize cell types in complex tissues such as the nervous system, and to study the transcriptional signatures underlying the multidimensional phenotypes of single cells.

Calcium-dependent regulation of cholecystokinin secretion and potassium currents in STC-1 cells

1993 ◽  
Vol 264 (6) ◽  
pp. G1031-G1036 ◽  
Author(s):  
A. W. Mangel ◽  
N. D. Snow ◽  
M. A. Misukonis ◽  
S. Basavappa ◽  
J. P. Middleton ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Reversal Potential ◽  
Barium Chloride ◽  
Cytosolic Calcium ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Electrophysiological Properties ◽  
Calcium Dependent ◽  
Control Value

Secretory and electrophysiological properties of STC-1 cells, a cholecystokinin-secreting cell line, were examined with a radioimmunoassay and patch-clamp recording techniques. Stimulation of cholecystokinin secretion was seen after exposure to agents anticipated to increase the level of intracellular calcium, including thapsigargin (8 microM), bombesin (50 nM), potassium-induced depolarization (50 mM), or after blockade of potassium channels with barium chloride (2 mM). The secretory effects of these agents were blocked by pretreatment with the calcium channel blocker diltiazem (1 microM). Whole cell patch-clamp recordings showed a hyperpolarizing shift in reversal potential after exposure to either thapsigargin (8 microM) or bombesin (50 nM) from a control value of -27 +/- 3 to -57 +/- 7 or -48 +/- 6 mV, respectively. This shift was in the direction of the reversal potential for potassium and was blocked by barium chloride (5 mM). Single-channel recordings from cell-attached membrane patches showed an inwardly rectifying potassium channel with channel open probability modulated by bombesin. These results indicate that in STC-1 cells a potassium current is increased by agents that stimulate CCK secretion, presumably by increasing the level of cytosolic calcium. STC-1 cells may serve as a model system to study the electrophysiological and secretory mechanisms involved in the release of cholecystokinin.

Patch-clamp recording from identified rat ciliary ganglion neurons in primary culture

1995 ◽  
Vol 73 (1) ◽  
pp. 65-71 ◽  
Author(s):  
M. E. M. Kelly ◽  
K. K. Johnson ◽  
P. C. Jackson
Keyword(s):  
Patch Clamp ◽  
Primary Culture ◽  
Outward Current ◽  
External Solution ◽  
Resting Membrane Potential ◽  
Transient Outward Current ◽  
Ciliary Ganglion ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Ganglion Neurons

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was −50 ± 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+ inward current and both sustained and transient outward K+ currents. Sustained outward K+ current was reduced (55–77%) by 5 mM tetraethylammonium and to a lesser extent (42–46%) by superfusion with nominally Ca2+ free external solution. Transient outward current was blocked by 100 μm 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to −50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+ and K+ currents giving rise to action potentials.Key words: fluorescent tracer, patch clamp, neuron, ciliary ganglion.

scholarly journals Nanocrown electrodes for robust and scalable intracellular recordings of cardiomyocytes for cardiotoxicity screening

2021 ◽  
Author(s):  
Zeinab Jahed ◽  
Yang Yang ◽  
Ching-Ting Tsai ◽  
Ethan P. Foster ◽  
Allister F. McGuire ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Technology Development ◽  
Device Fabrication ◽  
Great Promise ◽  
Success Rates ◽  
Patch Clamp Recording ◽  
Screening Assay ◽  
Drug Induced ◽  
Large Variability ◽  
Electrophysiological Properties

Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. However, the conventional patch clamp techniques for measuring iAPs are either laborious and low throughput or not suitable for measuring electrically connected cardiomyocytes. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. Nanoelectrodes show great promise, but the large variability in success rate, signal strength, and the low throughput of device fabrication have hindered them from being broadly adopted for proarrhythmia drug assessment. In this work, we developed vertically-aligned and semi-hollow nanocrown electrodes that are mechanically robust and made through a scalable fabrication process. Nanocrown electrodes achieve >99% success rates in obtaining intracellular access through electroporation, allowing reliable recordings of iAPs from human pluripotent stem-cell-derived cardiomyocytes (hPSC-CMs). The accuracy of nanocrown electrode recordings is validated by simultaneous patch clamp recording from the same cell. Nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four to five incremental drug doses. In this way, the dose-response data is self-referencing, which avoids the cell-to-cell variations inherent to hPSC-CMs. We are hopeful that this technology development is a step towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.

Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin

1996 ◽  
Vol 271 (2) ◽  
pp. C524-C532 ◽  
Author(s):  
Y. Tajima ◽  
K. Ono ◽  
N. Akaike
Keyword(s):  
Patch Clamp ◽  
Reversal Potential ◽  
Ionic Currents ◽  
Patch Clamp Recording ◽  
Ionic Selectivity ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Electrophysiological Properties ◽  
Perforated Patch ◽  
Ventricular Cells

Whole cell currents were recorded in single myocytes dissociated from guinea pig ventricles by the patch-clamp technique. The addition of 0.1 mg/ml gramicidin D, a cation-selective ionophore, into the pipette solution induced a gradual spontaneous perforation of the patch membrane under a conventional cell-attached configuration. The access resistance, measured at approximately 12 min after formation of a gigaohm seal, was 9.2 +/- 1.5 M omega (n = 12). The perforated patch membrane exhibited ionic selectivity for various monovalent cations, with a relative order of Cs+ (1.11) > K+ (1.0) > Na+ (0.65) >> tris(hydroxymethyl)aminomethane+ (approximately 0) but was not permeable for Cl-. Under the gramicidin-perforated patch recording configuration, the cells showed the typical electrophysiological properties for ventricular cells reported previously. The intracellular Cl- concentration, estimated from the reversal potential of the catecholamine-induced Cl- current, was 36.3 +/- 2.9 mM (n = 17). We thus conclude that the gramicidin-perforated patch recording mode provides a useful tool for recording the ionic currents while maintaining the intracellular Cl- concentration.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

scholarly journals A Temperature-Controlled Patch Clamp Platform Demonstrated on Jurkat T Lymphocytes and Human Induced Pluripotent Stem Cell-Derived Neurons

2020 ◽  
Vol 7 (2) ◽  
pp. 46 ◽  
Author(s):  
Jann Harberts ◽  
Max Kusch ◽  
John O’Sullivan ◽  
Robert Zierold ◽  
Robert H. Blick
Keyword(s):  
Stem Cell ◽  
T Lymphocytes ◽  
Patch Clamp ◽  
Pluripotent Stem Cell ◽  
Room Temperature ◽  
Standard Procedure ◽  
Induced Pluripotent Stem Cell ◽  
Electrophysiological Properties ◽  
Temperature Controlled ◽  
Induced Pluripotent

Though patch clamping at room temperature is a widely disseminated standard procedure in the electrophysiological community, it does not represent the biological system in mammals at around 37 °C. In order to better mimic the natural environment in electrophysiological studies, we present a custom-built, temperature-controlled patch clamp platform for upright microscopes, which can easily be adapted to any upright patch clamp setup independently, whether commercially available or home built. Our setup can both cool and heat the platform having only small temperature variations of less than 0.5 °C. We demonstrate our setup with patch clamp measurements at 36 °C on Jurkat T lymphocytes and human induced pluripotent stem cell-derived neurons. Passive membrane parameters and characteristic electrophysiological properties, such as the gating properties of voltage-gated ion channels and the firing of action potentials, are compared to measurements at room temperature. We observe that many processes that are not explicitly considered as temperature dependent show changes with temperature. Thus, we believe in the need of a temperature control in patch clamp measurements if improved physiological conditions are required. Furthermore, we advise researchers to only compare electrophysiological results directly that have been measured at similar temperatures since small variations in cellular properties might be caused by temperature alterations.

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