scholarly journals A simplified electronic circuit for combined single-cell stimulation and recording using loose cell-attached electrodes

2017 ◽  
Author(s):  
Ben W. Strowbridge ◽  
R. Todd Pressler
Keyword(s):  
Patch Clamp ◽  
Integrated Circuits ◽  
Single Cell ◽  
Action Potentials ◽  
Present Report ◽  
Cell Activity ◽  
Synaptic Connections ◽  
Pulse Timing ◽  
Olfactory Bulb Slices

AbstractWhile tight-seal patch clamp recordings have found wide use in neuroscience and in other fields, the requirement to replace the glass pipette after every attempted recording represents an impediment to high throughput studies such as searching for monosynaptically connected pairs of neurons. Loose cell-attached recording was introduced in 2000 to circumvent this problem since it enabled combined recording and stimulation of visually-identified neurons without necessitating a tight (gigaohm) seal. Since the stimulus voltages required to evoke action potentials through low resistance seals are beyond the capacity of most commercial amplifiers, Barbour and Isope introduced a variation of classic patch clamp amplifier circuit that is able to deliver stimulus voltages that are effective in triggering action potentials under the loose cell-attached patch clamp configuration. The present report presents the design and operation of a simpler amplifier that contains only two integrated circuits and is able to effectively stimulate and record action potentials in mitral cells in rodent olfactory bulb slices. The addition of an accessory analog gating circuit enables manual control of the stimulus voltage with pulse timing controlled by a digital output from a computer. This system may be useful in studies that require surveying many potential pairs of neurons for synaptic connections and for sampling and manipulating single-cell activity in in vivo electrophysiology experiments.

scholarly journals CD200 promotes immunosuppression in the pancreatic tumor microenvironment

2020 ◽  
Vol 8 (1) ◽  
pp. e000189 ◽  
Author(s):  
Fouad Choueiry ◽  
Molly Torok ◽  
Reena Shakya ◽  
Kriti Agrawal ◽  
Anna Deems ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Pancreatic Tumor ◽  
Cell Activity ◽  
Immunofluorescent Staining ◽  
Cytokine Signaling ◽  
Pancreatic Cell ◽  
Tumor Bearing ◽  
Single Cell Rna Sequencing

BackgroundA significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC.MethodsImmunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein.ResultsWe found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/− MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein.ConclusionsThese results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.

Responses to acetylcholine of ganglion cells in an isolated mammalian retina

1976 ◽  
Vol 39 (6) ◽  
pp. 1220-1235 ◽  
Author(s):  
R. H. Masland ◽  
A. Ames
Keyword(s):  
Ganglion Cell ◽  
Action Potentials ◽  
Ganglion Cells ◽  
Cell Activity ◽  
Receptive Fields ◽  
Cholinergic Antagonists ◽  
Control Medium ◽  
High Concentrations ◽  
Evoked Activity

1. Rabbit retinas were isolated and superfused with a physiological medium. Ganglion cell activity was recorded during stimulation with focused light, and receptive fields were mapped. Receptive fields were identical to those found in vivo and did not change during a 6-h incubation. After the receptive field of a ganglion cell had been identified, acetylcholine or related agents were introduced singly or in combination into the medium, and their effect on the cell's spontaneous and light-evoked activity was observed. 2. Ganglion cells with on-center or directionally selective receptive fields were excited when ACh was added to the medium. The response to exogenous ACh was prevented by cholinergic antagonists. 3. These cells' spontaneous activity and response to light were enhanced by anticholinesterase and depressed by cholinergic antagonists. Antagonists varied in their ability to block the light-evoked response, with dihydro-beta-erythroidine the most effective. 4. Thresholds for ACh or the related agents were low, ranging from 1 to 40 muM; their effects were rapidly and completely reversed when the retina was returned to control medium. 5. In retinas incubated in medium containing 20 mM Mg2+ and 0.2 mM Ca2+, ganglion cells lost completely both their spontaneous and light-evoked activity, but retained their ability to generate action potentials in response to elevated K+. Ganglion cell activity rapidly returned to normal when the retina was returned to medium containing normal electrolytes. On-center and directionally selective cells were excited by ACh in retinas where synaptic transmission had been inhibited by 20 mM Mg2+ and 0.2 mM Ca2+. 6. The responses of on-center and directionally selective cells to ACh, to anticholinesterase, and to cholinergic antagonists in control medium indicate that the retina contains one or more synapses using ACh as a neurotransmitter. The response to ACh in retinas exposed to 20 mM Mg2+ and 0.2 mM Ca2+ suggests that at least one such synapse in on the ganglion cell itself. 7. Off-center cells were inhomogenous in their response to ACh. Although some responded just as the other classes of cell, the majority responded quite weakly and a subgroup was encountered which was entirely unaffected by even 1 mM ACh, by levels of physostigmine which inactivate virtually all retinal acetyl-cholinesterase, or by high concentrations of cholinergic antagonists. Only 2 of 20 off-cells tested in the presence of 20 mM Mg2+ and 0.2 mM Ca2+ were excited by ACh. Apparently ACh is not a primary transmitter for most off-cells.

scholarly journals Extracellular waveforms reveal an axonal origin of spikelets in pyramidal neurons

2018 ◽  
Vol 120 (4) ◽  
pp. 1484-1495 ◽  
Author(s):  
Martina Michalikova ◽  
Michiel W. H. Remme ◽  
Richard Kempter
Keyword(s):  
Experimental Data ◽  
Action Potential ◽  
Single Cell ◽  
Action Potentials ◽  
Pyramidal Neurons ◽  
Hippocampal Pyramidal Neurons ◽  
Coupled Cells ◽  
A Cell ◽  
Cell Firing

Spikelets are small spike-like membrane depolarizations measured at the soma whose origin in pyramidal neurons is still unresolved. We investigated the mechanism of spikelet generation using detailed models of pyramidal neurons. We simulated extracellular waveforms associated with action potentials and spikelets and compared these with experimental data obtained by Chorev and Brecht ( J Neurophysiol 108: 1584–1593, 2012) from hippocampal pyramidal neurons in vivo. We considered spikelets originating in the axon of a single cell as well as spikelets generated in two cells coupled with gap junctions. We found that in both cases the experimental data can be explained by an axonal origin of spikelets: in the single-cell case, action potentials are generated in the axon but fail to activate the soma. Such spikelets can be evoked by dendritic input. Alternatively, spikelets resulting from axoaxonal gap junction coupling with a large (greater than several hundred μm) distance between the somata of the coupled cells are also consistent with the data. Our results demonstrate that a cell firing a somatic spikelet generates a detectable extracellular potential that is different from the action potential-correlated extracellular waveform generated by the same cell and recorded at the same location. This, together with the absence of a refractory period between action potentials and spikelets, implies that spikelets and action potentials generated in one cell may easily get misclassified in extracellular recordings as two different cells, albeit they both constitute the output of a single pyramidal neuron. NEW & NOTEWORTHY We addressed the origin of spikelets, using compartmental models of pyramidal neurons. Comparing our simulation results with published extracellular spikelet recordings revealed an axonal origin of spikelets. Our results imply that action potential- and spikelet-associated extracellular waveforms may easily get misclassified as two different cells, albeit they both constitute the output of a single pyramidal cell.

scholarly journals Functional In vivo Single-cell Transcriptome (FIST) Analysis Reveals Molecular Properties of Light-Sensitive Neurons in Mouse V1

2018 ◽  
Author(s):  
Jianwei Liu ◽  
Na Pan ◽  
Le Sun ◽  
Mengdi Wang ◽  
Junjing Zhang ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Three Dimensional ◽  
Whole Cell ◽  
Mrna Sequencing ◽  
Layer 2 ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Live Mouse

ABSTRACTVision formation is classically based on projections from the retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single neurons with specific functions in V1 still remain unknown. Some neurons in mouse V1 are tuned to light stimulus. To determine the molecular properties of light-stimulated neurons in layer 2/3 of V1, we developed a method of functional in vivo single-cell transcriptome (FIST) analysis that integrates sensory evoked calcium imaging, whole-cell electrophysiological patch-clamp recordings, single-cell mRNA sequencing and three-dimensional morphological characterization in a live mouse, based on a two-photon microscope system. In our study, 58 individual cells from layer 2/3 of V1 were identified as either light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of every single cell after individual functional tests were aspirated through the patch-clamp pipette for mRNA sequencing. Furthermore, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in the live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with high expression of genes related to transmission regulation, such as Rtn4r, Nr4a1, and genes involved in membrane transport, such as Na+/K+ ATPase, NMDA-type glutamatergic receptor, preferentially respond to light stimulation. Our findings demonstrate the ability of FIST analysis to characterize the functional, morphological and transcriptomic properties of a single cell in alive animal, thereby providing precise neuronal information and predicting its network contribution in the brain.

Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo

2007 ◽  
Vol 5 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Kazuo Kitamura ◽  
Benjamin Judkewitz ◽  
Masanobu Kano ◽  
Winfried Denk ◽  
Michael Häusser
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Cell Electroporation ◽  
Single Cell Electroporation

scholarly journals Single-cell profiling of Ebola virus infection in vivo reveals viral and host transcriptional dynamics

2020 ◽  
Author(s):  
Dylan Kotliar ◽  
Aaron E. Lin ◽  
James Logue ◽  
Travis K. Hughes ◽  
Nadine M. Khoury ◽  
...  
Keyword(s):  
Single Cell ◽  
Ebola Virus ◽  
Immune Cell ◽  
Temporal Dynamics ◽  
Cell Activity ◽  
Case Fatality ◽  
Protein Quantification ◽  
Cell Protein ◽  
Ebov Infection

SummaryEbola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting.

scholarly journals What single-cell stimulation has told us about neural coding

2015 ◽  
Vol 370 (1677) ◽  
pp. 20140204 ◽  
Author(s):  
Guy Doron ◽  
Michael Brecht
Keyword(s):  
Single Cell ◽  
Neural Coding ◽  
Single Neuron ◽  
Cell Activity ◽  
Brain Structures ◽  
Cell Stimulation ◽  
Precise Control ◽  
Neuronal Coding ◽  
Spike Patterns

In recent years, single-cell stimulation experiments have resulted in substantial progress towards directly linking single-cell activity to movement and sensation. Recent advances in electrical recording and stimulation techniques have enabled control of single neuron spiking in vivo and have contributed to our understanding of neuronal coding schemes in the brain. Here, we review single neuron stimulation effects in different brain structures and how they vary with artificially inserted spike patterns. We briefly compare single neuron stimulation with other brain stimulation techniques. A key advantage of single neuron stimulation is the precise control of the evoked spiking patterns. Systematically varying spike patterns and measuring evoked movements and sensations enables ‘decoding’ of the single-cell spike patterns and provides insights into the readout mechanisms of sensory and motor cortical spikes.

Sign in / Sign up

Export Citation Format

Share Document