Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording

Reid R. Harrison; Ilya Kolb; Suhasa B. Kodandaramaiah; Alexander A. Chubykin; Aimei Yang; Mark F. Bear; Edward S. Boyden; Craig R. Forest

doi:10.1152/jn.00629.2014

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording

Journal of Neurophysiology ◽

10.1152/jn.00629.2014 ◽

2015 ◽

Vol 113 (4) ◽

pp. 1275-1282 ◽

Cited By ~ 9

Author(s):

Reid R. Harrison ◽

Ilya Kolb ◽

Suhasa B. Kodandaramaiah ◽

Alexander A. Chubykin ◽

Aimei Yang ◽

...

Keyword(s):

Patch Clamp ◽

Ion Channel ◽

Voltage Clamp ◽

Single Cells ◽

Patch Clamping ◽

Patch Clamp Recording ◽

Current Clamp ◽

Low Levels

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

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In vivo and in vitro Patch-clamp Recording Analysis of the Process of Sensory Transmission in the Spinal Cord and Sensory Cortex

Journal of PHYSIOLOGICAL ANTHROPOLOGY and Applied Human Science ◽

10.2114/jpa.24.93 ◽

2005 ◽

Vol 24 (1) ◽

pp. 93-97 ◽

Cited By ~ 3

Author(s):

Megumu Yoshimura ◽

Atsushi Doi ◽

Masaharu Mizuno ◽

Hidemasa Furue ◽

Toshihiko Katafuchi

Keyword(s):

Spinal Cord ◽

Patch Clamp ◽

Sensory Cortex ◽

Patch Clamp Recording ◽

Sensory Transmission ◽

Recording Analysis

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Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655971 ◽

1997 ◽

Vol 77 (02) ◽

pp. 376-382 ◽

Cited By ~ 15

Author(s):

Bruce Lages ◽

Harvey J Weiss

Keyword(s):

Platelet Rich Plasma ◽

Limited Range ◽

Dense Granule ◽

Receptor Desensitization ◽

Fura 2 ◽

Storage Pool ◽

Low Levels ◽

The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽

10.3390/molecules26061716 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1716

Author(s):

Kun Tong ◽

Ruotian Zhang ◽

Fengzhi Ren ◽

Tao Zhang ◽

Junlin He ◽

...

Keyword(s):

Ion Channels ◽

Patch Clamp ◽

Formalin Test ◽

Sodium Ion ◽

Inhibitory Potency ◽

Voltage Gated ◽

Patch Clamp Electrophysiology ◽

Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

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Synaptic background noise controls the input/output characteristics of single cells in an in vitro model of in vivo activity

Neuroscience ◽

10.1016/j.neuroscience.2003.08.027 ◽

2003 ◽

Vol 122 (3) ◽

pp. 811-829 ◽

Cited By ~ 162

Author(s):

J.-M Fellous ◽

M Rudolph ◽

A Destexhe ◽

T.J Sejnowski

Keyword(s):

Background Noise ◽

In Vitro Model ◽

Single Cells ◽

Input Output ◽

Vitro Model ◽

Output Characteristics

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DNA methylation specifies chromosomal localization of MeCP2.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.414 ◽

1996 ◽

Vol 16 (1) ◽

pp. 414-421 ◽

Cited By ~ 223

Author(s):

X Nan ◽

P Tate ◽

E Li ◽

A Bird

Keyword(s):

Dna Methylation ◽

Cultured Cells ◽

Centromeric Heterochromatin ◽

Intact Protein ◽

Chromosomal Protein ◽

Mouse Cells ◽

Low Levels ◽

Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

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Compensation of physiological motion enables high-yield whole-cell recording in vivo

10.1101/2020.06.09.143008 ◽

2020 ◽

Author(s):

William M. Stoy ◽

Bo Yang ◽

Ali Kight ◽

Nathaniel C. Wright ◽

Peter Y. Borden ◽

...

Keyword(s):

Single Cells ◽

Strong Dependence ◽

High Yield ◽

Patch Clamp Recording ◽

Gold Standard Method ◽

Whole Cell ◽

Whole Cell Recording ◽

Cell Recording ◽

Living Mouse

1.1.1AbstractWhole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode’s aperture and a cell’s membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane. We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette’s tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings in the deep in the brain of the living mouse, in the thalamus. This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26% (n=17/64) to 48% (n=32/66). Whole-cell recording yield improved from 10% (n = 9/88) to 24% (n=18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex. This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.

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Clonal analysis of basophil differentiation in bone marrow cultures from a Down's syndrome patient with megakaryoblastic leukemia

Blood ◽

10.1182/blood.v66.6.1278.bloodjournal6661278 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1278-1283

Author(s):

T Suda ◽

J Suda ◽

Y Miura ◽

Y Hayashi ◽

M Eguchi ◽

...

Keyword(s):

Single Cells ◽

Down's Syndrome ◽

Calcium Ionophore ◽

Down’S Syndrome ◽

Colony Growth ◽

Leukemic Cells ◽

Cytologic Examination ◽

Megakaryoblastic Leukemia

We present the in vitro differentiation of marrow cells from a patient with Down's syndrome accompanied by megakaryoblastic leukemia into basophils in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium, using a liquid culture and methylcellulose culture system. Identification of basophils was established by metachromatic staining with toluidine blue, transmission electron microscopy, and the presence of histamine. However, these basophils did not release histamine in response to calcium ionophore or chemotactic peptide. Samples from suspension cultures that contained 90% basophils showed chromosomal markers characteristic of leukemic cells (48, XY, +11, +21, t(1;15)) in all examined mitoses. The cellular composition of leukemic colonies grown in methylcellulose culture from single cells was studied using the micromanipulation technique. High plating efficiency and extreme predominance of basophil colonies were observed. In a total 137 cultures, 79 revealed colony growth. Of 59 colonies that were analyzed by cytologic examination, 46 were pure basophil colonies. These basophil colonies showed disperse morphology, similar to that of a normal basophil colony. The clonality of the basophil colonies and skewing of lineage expression were documented from leukemic single-cell cultures. These data showed that leukemic cells have the capacity for differentiation into some lineages that are not expressed in vivo.

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Self-organized cell migration across scales – from single cell movement to tissue formation

Development ◽

10.1242/dev.191767 ◽

2021 ◽

Vol 148 (7) ◽

pp. dev191767

Author(s):

Jessica Stock ◽

Andrea Pauli

Keyword(s):

Cell Migration ◽

Multiple Scales ◽

Single Cells ◽

Cell Movement ◽

Biological Response ◽

Collective Cell Migration ◽

Theoretical Concepts ◽

Self Organizing

ABSTRACTSelf-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on multiple scales. In this Review, we will explore self-organizing mechanisms that lay the foundation for both single and collective cell migration. Based on in vitro and in vivo examples, we will discuss theoretical concepts that underlie the persistent migration of single cells in the absence of directional guidance cues, and the formation of an autonomous cell collective that drives coordinated migration. Finally, we highlight the general implications of self-organizing principles guiding cell migration for biological and medical research.

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