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Neuron May 2023The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in...
The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
Topics: Mice; Animals; Action Potentials; Rhodopsin; Angiotensin-Converting Enzyme 2; Neurons; Mutation
PubMed: 37015225
DOI: 10.1016/j.neuron.2023.03.009 -
Journal of Voice : Official Journal of... Mar 2023The vibration of the vocal folds produces the primary sound for the human speech. The vibration depends mainly on the pressure, airflow of the lungs, and the material...
OBJECTIVES
The vibration of the vocal folds produces the primary sound for the human speech. The vibration depends mainly on the pressure, airflow of the lungs, and the material properties of the vocal folds. In order to change them, muscles in the larynx stretch the vocal folds. This interplay is rarely investigated, but can give insight in the complex process of speech production. Most material properties studies are damaging the tissue; therefore, a nondestructive one is desired.
METHODS
An ex vivo phonation experiment combined with the dynamic Pipette Aspiration Technique is used to investigate 10 porcine larynges, under manipulations of different adduction and elongation levels. For each manipulation, the near surface material properties of the vocal folds are measured as well as different phonation parameters like the subglottal pressure, glottal resistance, frequency, and stiffness. Thereby, a high-speed camera was used to record the vocal fold movement.
RESULTS
On most of the measured parameters, the manipulations do show an effect. Both manipulations lead to a higher phonation frequency and an increase of the stiffness of the tissue. Comparing both manipulations, the elongation results in higher elasticity values than the adduction. Different measurement parameters have been compared with each other and correlations could be found. Where the strongest correlation are found among the elasticity values of different frequencies. But it can also be seen that the elasticity values correlate with phonation parameters.
CONCLUSION
It was possible to produce a data set of 560 measurements in total. To our knowledge, this is the first time Pipette Aspiration Technique was combined with ex vivo phonation measurements for combined measurements. The amount of measurement data made it possible to carry out statistic investigations. The effect of the manipulations on material properties as well as on phonation parameters could be measured and different correlations could be found. The results lead to the hypothesis that the stretch does not have a huge effect on the material properties of the lamina propria, but more on the underlying muscle.
PubMed: 37005126
DOI: 10.1016/j.jvoice.2023.03.001 -
Pharmaceutical Research May 2023Intratracheal delivery and consistent dosing of dry powder vaccines is especially challenging in mice. To address this issue, device design of positive pressure dosators...
PURPOSE
Intratracheal delivery and consistent dosing of dry powder vaccines is especially challenging in mice. To address this issue, device design of positive pressure dosators and actuation parameters were assessed for their impacts on powder flowability and in vivo dry powder delivery.
METHODS
A chamber-loading dosator assembled with stainless-steel, polypropylene or polytetrafluoroethylene needle-tips was used to determine optimal actuation parameters. Powder loading methods including tamp-loading, chamber-loading and pipette tip-loading were compared to assess performance of the dosator delivery device in mice.
RESULTS
Available dose was highest (45%) with a stainless-steel tip loaded with an optimal mass and syringe air volume, primarily due to the ability of this configuration to dissipate static charge. However, this tip encouraged more agglomeration along its flow path in the presence of humidity and was too rigid for intubation of mice compared to a more flexible polypropylene tip. Using optimized actuation parameters, the polypropylene pipette tip-loading dosator achieved an acceptable in vivo emitted dose of 50% in mice. After administering two doses of a spray dried adenovirus encapsulated in mannitol-dextran, high bioactivity was observed in excised mouse lung tissue three days post-infection.
CONCLUSIONS
This proof-of-concept study demonstrates for the first time that intratracheal delivery of a thermally stable, viral-vectored dry powder can achieve equivalent bioactivity to the same powder, reconstituted and delivered intratracheally. This work may guide the design and device selection process for murine intratracheal delivery of dry powder vaccines to help progress this promising area of inhalable therapeutics.
Topics: Animals; Mice; Administration, Inhalation; Powders; Biological Products; Polypropylenes; Steel; Dry Powder Inhalers; Particle Size; Aerosols
PubMed: 36991226
DOI: 10.1007/s11095-023-03492-2 -
ENeuro Apr 2023Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input....
Midbrain dopamine (DA) neurons are among the best characterized pacemaker neurons, having intrinsic, rhythmic firing activity even in the absence of synaptic input. However, the mechanisms of DA neuron pacemaking have not been systematically related to how these cells respond to synaptic input. The input-output properties of pacemaking neurons can be characterized by the phase-resetting curve (PRC), which describes the sensitivity of interspike interval (ISI) length to inputs arriving at different phases of the firing cycle. Here we determined PRCs of putative DA neurons in the substantia nigra pars compacta in brain slices from male and female mice using gramicidin-perforated current-clamp recordings with electrical noise stimuli applied through the patch pipette. On average, and compared with nearby putative GABA neurons, DA neurons showed a low, nearly constant level of sensitivity across most of the ISI, but individual cells had PRCs showing relatively greater sensitivity at early or late phases. Pharmacological experiments showed that DA neuron PRCs are shaped by small-conductance calcium-activated potassium and Kv4 channels, which limit input sensitivity across early and late phases of the ISI. Our results establish the PRC as a tractable experimental measurement of individual DA neuron input-output relationships and identify two of the major ionic conductances that limit perturbations to rhythmic firing. These findings have applications in modeling and for identifying biophysical changes in response to disease or environmental manipulations.
Topics: Mice; Male; Female; Animals; Dopaminergic Neurons; Mesencephalon; Pars Compacta; Action Potentials
PubMed: 36973012
DOI: 10.1523/ENEURO.0445-22.2023 -
Journal of Virological Methods May 2023We present the point-of-care (POC) molecular diagnostic solution, evaluated during COVID-19 pandemic caused by SARS-CoV-2 (Dec 2021). The POC comprised of a complete...
We present the point-of-care (POC) molecular diagnostic solution, evaluated during COVID-19 pandemic caused by SARS-CoV-2 (Dec 2021). The POC comprised of a complete platform from self-sampling to RT-PCR testing of SARS-CoV-2 and its variants on portable Compact Q Real time polymerase chain reaction system. The multiplex assay was designed to target S, ORF1, and N genes of SARS-CoV-2 genome in a single tube with RNaseP as endogenous internal control. The present POC enables high accuracy (>95%) and high-throughput testing with a turnaround time of 45 min. It provides a unique platform from self-sample collection to report generation with rapid protocol, pipette and expert-free operation, long shelf-life stability and room temperature storage which enable to increase the efficiency of molecular testing. This novel test named "CoviSwift™ COVID-19 S PLUS RAPID PCR KIT" has been approved by CDSCO, Indian National Regulatory Authority, India, and is in use for clinical settings in India.
Topics: Humans; SARS-CoV-2; COVID-19; Point-of-Care Systems; Reverse Transcriptase Polymerase Chain Reaction; Pandemics; Sensitivity and Specificity; Real-Time Polymerase Chain Reaction; COVID-19 Testing
PubMed: 36934897
DOI: 10.1016/j.jviromet.2023.114714 -
STAR Protocols Mar 2023Direct electrical recordings from conventional boutons in the mammalian central nervous system have proven challenging due to their small size. Here, we provide a...
Direct electrical recordings from conventional boutons in the mammalian central nervous system have proven challenging due to their small size. Here, we provide a protocol for direct whole-cell patch-clamp recordings from small presynaptic boutons of primary dissociated cultured neurons of the rodent neocortex. We describe steps to prepare primary neocortical cultures and recording pipettes, followed by identifying boutons and establishing a whole-cell bouton recording. We then provide details on precise pipette capacitance compensation required for high-resolution current-clamp recordings from boutons. For further details on the use and execution of this protocol, please refer to Ritzau-Jost et al..
PubMed: 36920913
DOI: 10.1016/j.xpro.2023.102168 -
Frontiers in Pharmacology 2023ATP release from the lens hemichannels has been explained as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent...
ATP release from the lens hemichannels has been explained as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and connexin hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. The response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K current. The presence of this outward current in control conditions appeared to be a reliable predictor of a cell's response to GSK treatment. In some studies, recordings were obtained from single cells by combining cell-attached and whole-cell patch clamp configurations. This approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of Cx43 hemichannels.
PubMed: 36909173
DOI: 10.3389/fphar.2023.1101498 -
Molecules (Basel, Switzerland) Feb 2023The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE...
The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06-9.78 ng/mL and 0.17-29.64 ng/mL, respectively). The recovery was in the range of 79.88-120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10-9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption.
Topics: Humans; Chromatography, Liquid; Antihypertensive Agents; Chromatography, High Pressure Liquid; Tandem Mass Spectrometry; Solvents
PubMed: 36903457
DOI: 10.3390/molecules28052213 -
NPJ Regenerative Medicine Feb 2023Neonatal mouse heart can regenerate after left ventricle (LV) apical resection (AR). Since current AR rodent method is accomplished by resecting LV apex until exposure...
Neonatal mouse heart can regenerate after left ventricle (LV) apical resection (AR). Since current AR rodent method is accomplished by resecting LV apex until exposure of LV chamber, it is relatively difficult to operate reproducibly. We aimed to develop a modified AR method with high accuracy and reproducibility and to investigate whether cardiac regenerative capacity could be replicated in neonatal rats. For 15% AR of whole heart weight in 1-day-old (P1) neonatal mice, a modified 10 μL pipette tip cut to 0.48 mm in internal diameter was connected to a vacuum pump working at 0.06 ± 0.005 MPa and gently kept in touch with LV apex for nearly but no more than 12 s. LV apex was resected by a single incision adjacent to the pipette tip. The modified AR method in P1 mice achieved cardiac structural and functional recovery at 21 days post resection (dpr). Data from different operators showed smaller variation of resected LV apex and higher reproducibility using the modified AR method. Furthermore, we showed that 5% AR of whole heart weight in P1 neonatal rats using a modified 200 μL pipette tip cut to 0.63 mm in internal diameter led to complete regeneration of LV apex and full preservation of cardiac function at 42 dpr. In conclusion, the modified AR rodent model leads to accurate resection of LV apex with high homogeneity and reproducibility and it is practically convenient for the study of structural, functional, and molecular mechanisms of cardiac regeneration in both neonatal mice and rats.
PubMed: 36806296
DOI: 10.1038/s41536-023-00284-5 -
Animals : An Open Access Journal From... Jan 2023Erythrocyte sedimentation rate (ESR) is a hematological test that can detect inflammatory activity within the body. Although not specific for any particular disease, ESR...
Erythrocyte sedimentation rate (ESR) is a hematological test that can detect inflammatory activity within the body. Although not specific for any particular disease, ESR is often used as a screening "sickness indicator" due to its reliability and low cost. The Westergren method is a manual ESR technique commonly used but requires special graduated pipettes and over 1mL of whole blood, precluding its use in smaller patients where limited sample volumes can be obtained. A modified micro-ESR technique has been described using hematocrit capillary tubes but is used less commonly. ESR has been reported to be a useful inflammatory indicator in gopher tortoises () and box turtles ( spp.) but not in Florida cottonmouth snakes (). Having an inexpensive screening test for inflammation can help guide medical decisions within conservation efforts of imperiled species. This study evaluated the correlation between these two ESR methodologies in threatened eastern indigo snakes (, EIS) and found a very strong correlation ( = 0.897), without constant or proportional biases and a reference interval of 0 (90% CI -1-1)-9 mm/h (90% CI 8-11) was defined. Additionally, a significant difference was found between healthy EIS and EIS in mid-ecdysis ( = 0.006) and EIS with gastric cryptosporidiosis ( = 0.006), indicating ESR as a useful inflammatory indicator in EIS.
PubMed: 36766352
DOI: 10.3390/ani13030464