A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope

Study of the relationship between mental tension and risk factors for developing cardiovascular diseases

conducted a study of the influence of mental stress on risk factors for developing cardiovascular diseases, which revealed a statistically significant tendency to worsen blood pressure, heart rate and blood glucose levels after 3-hour fasting with an increase in the level of mental stress in 3rd-year students. The largest part of the subjects with high levels of stress had a bad habit – Smoking.провели исследование влияния психической напряжённости на факторы риска развития сердечно-сосудистых заболеваний, которое выявило статистически достоверную тенденцию к ухудшению показателей артериального давления, частоты сердечных сокращений и уровня глюкозы в крови после 3-часового голодания при увеличении уровня психической напряженности у студентов 3 курса. Наибольшая часть обследуемых с высоким уровнем стресса имели вредную привычку – курение

Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics

Background The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca²⁺-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca²⁺ ions while maintaining a higher fluorescence brightness at the basal level of Ca²⁺ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca²⁺/F⁺Ca²⁺) and slow Ca²⁺ dissociation kinetics. Results Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca²⁺ (i.e. becoming dimmer with an increase of Ca²⁺ concentration). In the presence of Mg²⁺ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca²⁺ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca²⁺ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca²⁺ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. Conclusions We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca²⁺ ions buffering capacity as compared with commonly used GECIs