Journal: Methods in Molecular Biology

Article Title: Toxoplasma gondii

doi: 10.1007/978-1-4939-9857-9

Figure Lengend Snippet: Fig. 2 Characterization of clones in a plate reader. A suspension of 5 106 tachyzoites/ml in Ringer’s buffer with 100 μM of EGTA is dispensed into each well of a 96 well plate after shaking for 3 min the fluorescence is measured. Reagents are added at 60 s (Subheading 3.3). (a) Fluorescence changes before and after the addition of the indicated reagents with two Toxoplasma clones (3 and 5). DMSO is used as a control. (b) Kinetic measurements for clones 3 and 5 showing changes in the fluorescence of GCamP6f in function of time. IO Ionomycin, TG Thapsigargin, Zap Zaprinast, Ca2+ Extracellular Ca2+, 1.8 mM. (c) Same experiment to the one presented in A with clones 7 and 9 that express GCamP6s and compare with the reference cell line expressing GCamp6f. (d) Kinetic measurements with clones 7 and 9. Same conditions as (b)

Article Snippet: GECI plasmids: GCaMP6f (Addgene 40755), GCaMP6m (Addgene 40754), GCaMP6s (Addgene 40753), R-GECO (Addgene 32462), B-GECO (Addgene 32448), jRGECO1a (Addgene 61563), jRCaMP1a (Addgene 61562), jRCaMP1b (Addgene 63136), ER-LAR-GECO1 (Addgene 32444), mitoLAR-GECO1.2 (Addgene 32461).

Techniques: Clone Assay, Suspension, Fluorescence, Control, Expressing

Journal: Methods in Molecular Biology

Article Title: Toxoplasma gondii

doi: 10.1007/978-1-4939-9857-9

Figure Lengend Snippet: Fig. 3 Ratiometric measurements with a clone expressing GCaMP6s-mScarlet. (a) GCaMP and mScarlet fluorescence changes in function of time measured in the F-7000 fluorescence spectrophotometer. (a) Parasites at a concentration of 2 107 tachyzoites/ml were resuspended in Ringer’s buffer with 100 μM of EGTA at a final volume of 2.5 ml. 1.8 mM of Ca2+ is added at 400 s. (b) Ratio calculation of the fluorescence tracings shown in (a). (c) Western blots showing the expression of GCaMP6f-Ty1 and mScarletI-3xHA. Antibodies: Mouse ⍺-Ty1 1:2000 (a generous gift from Dr. Etheridge) and Rat ⍺-HA 1:200 (Roche). Secondary: goat ⍺-mouse IRDye 800 (LI-COR) and goat ⍺-rat IRDye 680 (LI-COR)

Article Snippet: GECI plasmids: GCaMP6f (Addgene 40755), GCaMP6m (Addgene 40754), GCaMP6s (Addgene 40753), R-GECO (Addgene 32462), B-GECO (Addgene 32448), jRGECO1a (Addgene 61563), jRCaMP1a (Addgene 61562), jRCaMP1b (Addgene 63136), ER-LAR-GECO1 (Addgene 32444), mitoLAR-GECO1.2 (Addgene 32461).

Techniques: Expressing, Spectrophotometry, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Amygdala GABA Neurons: Gatekeepers of Stress and Reproduction

doi: 10.1101/2025.01.06.631361

Figure Lengend Snippet: Functional connectivity of GABAergic neurons in posterodorsal medial amygdala (MePD) reconfigures in response to optical stimulation of urocortin-3 (UCN3) neurons or acute restraint stress. (A) Schematic diagram of the experimental design for optically stimulating MePD UCN3 neurons and simultaneously imaging calcium activity from MePD GABAergic neurons. Heatmaps of calcium activity (ΔF/F) in response to (left) optical stimulation of UCN3 neurons and (right) during restraint-induced stress. (B-G) Representative photomicrographs of the MePD from a UCN3-Cre::VGAT-Flpo mouse injected with pAAV-Syn-Flex-rc+pAAV-EF1a-fDIO-GCaMP6s. (B&E) Red fluorescence (tdTomoato) labels UCN3 neurons expressing Chrimson-tdTomato, (C&F) green fluorescence labels GABAergic neurons, and (D&G) red and green signals merged. (H&I) Functional connectivity in the GABA populations was quantified using signed lagged cross-correlation (SLxCorr). (J) Functional connectivity matrices show a statistically significant difference between the baseline period and subsequent UCN3 stimulation or restraint stress across animals, assessed via Kolmogorov-Smirnov tests for each animal trial and using Fisher’s method for combining p-values across different animals (see Methods). Distribution of combined p-value obtained from different combinations of trials (one from each animal) is consistently below the 0.05 confidence level. Scale bars: B-D, 200µm; E-G, 50µm.

Article Snippet: Separate mice cohorts received the following viral injection into the MePD: (i) a mixture of pAAV-Syn-Flex-rc[Chrimson-tdTomato] (2.0×10 gc/mL, Serotype:5, #62723, Addgene, Massachusetts, USA) + pAAV-EF1a-fDIO-GCaMP6s (2.3×10 gc/mL; Serotype:8; #105715, Addgene, Massachusetts, USA; final volume 400nl); (ii) a mixture of pAAV-Syn-Flex-rc[Chrimson-tdTomato] (2.0×10 gc/mL, Serotype:5, #62723, Addgene, Massachusetts, USA) + pAAV.CAG.Flex.GCaMP6s.WPRE.SV40 (4.3×10 gc/mL; Serotype:9; # 100842, Addgene, Massachusetts, USA; final volume 400nl); (iii) a mixture of control virus AAV-Ef1a-DIO-EYFP (250nl, 4.4 × 10 gc/mL, Serotype:9, #27056, Addgene, Massachusetts, USA) + pAAV-EF1a-fDIO-GCaMP6s (2.3×10 gc/mL; Serotype:8; #105715, Addgene, Massachusetts, USA; final volume 250nl).

Techniques: Functional Assay, Imaging, Activity Assay, Injection, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca 2+

doi: 10.1016/j.jbc.2023.105063

Figure Lengend Snippet: A novel experimental approach to study DA release combined with selective expression of the calcium sensor jRGECO1a in cultured DArgic neurons . The experiments suggest that AMPH-induced DA efflux occurs in the absence of change in intracellular Ca 2+ . A , graphical illustration of the approach with the numbering of each step as follows: (1), midbrain neurons from postnatal rat pups are seeded on a glia monolayer (2), a dual viral approach is exploited where one AAV mediates tyrosine hydroxylase promoter-driven Cre expression and a second AAV mediates the Cre-dependent expression of a gene of interest (3), this allows targeted expression of the gene in DArgic neurons (4), one to two days before imaging, HEK293 cells stably expressing the DA sensor GRAB DA1H are seeded on top of the culture (5), the DA sensor expressing cells have attached to the culture the day after being seeded and stay well attached during live imaging with constant flow (6), the DA sensor in the HEK293 cells allows for a good signal-to-noise ratio and makes it possible to easily separate the signal of the DA sensor from the sensor expressed in the neurons. B , change in DA signal from GRAB DA1H ( left ) and Ca 2+ signal from jRGECO1a ( middle ) upon application of 10 μM AMPH as indicated. Data shown are an average trace of three independent experiments ± SEM. The bar graphs ( right ) represent the average change in fluorescence during the last minute of AMPH from baseline. Data show that AMPH causes a change in the fluorescent DA signal, but not the Ca 2+ signal. Data are mean of ΔF/F 0 in % ± SEM, n = 3 for GRAB DA1H and n = 6 for jRGECO1a. C , change in DA signal from GRAB DA1H ( left ) and Ca 2+ signal from jRGECO1a ( middle ) upon applying 20 μM NMDA and 10 μM glycine as indicated. Data shown are an average trace of three independent experiments ± SEM. The bar graphs ( right ) represent the average change in fluorescence during the last minute of NMDA treatment from baseline. Data are mean of ΔF/F 0 in % ± SEM, n = 3 for GRAB DA1H and n = 9 for jRGECO1a. ns, p > 0.05; ∗ p ≤ 0.05; ∗∗∗∗ p < 0.0001; one-sample t test. AAV, adeno-associated virus; AMPH, amphetamines; DA, dopamine; NMDA, N-methyl-D-aspartate.

Article Snippet: AAV1 particles from Cre-dependent constructs containing the Ca 2+ sensor jRGECO1a (pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40) or the axonally targeted Ca 2+ sensor axon-GCaMP6s (pAAV-hSynapsin1-FLEx-axon-GCaMP6s) were obtained from Addgene (cat. #100853 and #11201, respectively).

Techniques: Expressing, Cell Culture, Imaging, Stable Transfection, Fluorescence, Virus

Journal: The Journal of Biological Chemistry

Article Title: Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca 2+

doi: 10.1016/j.jbc.2023.105063

Figure Lengend Snippet: Dose-dependent AMPH-induced efflux still occurs in the presence of the voltage-gated sodium-channel blocker TTX and is not accompanied by an increase in cytosolic Ca 2+ . The absence of spontaneous firing of the neuron allows for the isolation of the DA signal solely from the contribution of AMPH. In the presence of TTX, the effect of AMPH on the DA signal from GRAB DA1H is dose-dependent, while no change in intracellular the Ca 2+ signal from jRGECO1a is detected. A , representative trace of three independent experiments illustrating the effect of TTX on extracellular DA and intracellular Ca 2+ . The jRGECO1a trace is the average signal ± SEM of 5 somas in the recording. First, upon application of AMPH in the absence of TTX, an increase is seen in the DA signal, while it is unclear if it causes any change in the Ca 2+ signal due to spontaneous fluctuation. Then, in the presence of 1 μM TTX the Ca 2+ signal drops and becomes a flat line, and the DA signal decreases. Application of AMPH in the presence of TTX causes no change in the Ca 2+ signal. Still, it induces an increase in the DA signal with a clearer signal than the signal in the absence of TTX. Finally, 10 μM DA is added to saturate GRAB DA1H and thereby visualize the dynamic range of the fluorescent DA signal. B , ( top ) representative trace of dose-response experiment in the presence of TTX with 1, 10, and 30 μM AMPH followed by 20 μM NMDA and 10 μM glycine. The jRGECO1a trace is the average signal ± SEM of 3 somas in the recording. ( Bottom ) Representative images of the change in DA and Ca 2+ signal from baseline during the last minutes of each of the drug applications as indicated by the arrows . The scale bar represents 50 μm. C , the bar graph shows the average change in DA signal compared to vehicle. Data are mean of ΔF/F 0 in % ± SEM, with n = 4 for 1, 10, and 30 μM AMPH responses and n = 3 for NMDA response. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, one-way ANOVA with Holm–Šídák multiple comparison test (F (2, 6) = 18.6, p = 0.0027); ## p < 0.01, one-sample t test. D , the bar graph shows the average change in Ca 2+ signal from baseline. While there was no change in Ca 2+ signal for the doses of AMPH tested, there was a clear change upon applying 20 μM NMDA + 10 μM glycine. Data are mean of ΔF/F 0 in % ± SEM, n = 11.; ns, nonsignificant ( p > 0.05), one-way ANOVA with Holm–Šídák multiple comparison test (F(2, 20) = 2.071, p = 0.15); #### p < 0.0001, one-sample t test. AMPH, amphetamine; DA, dopamine; NMDA, N-methyl-D-aspartate; TTX, tetrodotoxin.

Article Snippet: AAV1 particles from Cre-dependent constructs containing the Ca 2+ sensor jRGECO1a (pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40) or the axonally targeted Ca 2+ sensor axon-GCaMP6s (pAAV-hSynapsin1-FLEx-axon-GCaMP6s) were obtained from Addgene (cat. #100853 and #11201, respectively).

Techniques: Isolation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca 2+

doi: 10.1016/j.jbc.2023.105063

Figure Lengend Snippet: AMPH does not induce an intracellular Ca 2+ increase in the neurites of DA neurons . A , representative image of a DA neuron transduced with the axonally targeted Ca 2+ sensor AAV-hSynapsin1-FLEx-axon-GCaMP6s expressed in Cre-expressing neurons, driven by the AAV-TH-Cre vector. The scale bar represents 100 μm. B , illustration of the individual ROIs selected for discrete Ca 2+ measurements in the neurites compared to the soma. C , representative Ca 2+ traces from the neurites and soma, plotted separately, of a neuron stimulated with 10 μM AMPH and 20 mM KCl. The neurites trace is the average signal ± SEM of the 86 neurite ROIs indicated as #2 to 87 in Figure 3 B . D , enlarged traces from 3B during AMPH application. ( Top ) Plots of individual traces of the segmented neurites shown as ROI #2 to 87 in 3B. ( Bottom ) Plot of the soma trace shown as ROI #1 in B . E , comparison of mean responses to AMPH and KCl compared to baseline in individual neurite fractions (n = 199) and somas (n = 5). ∗ p < 0.05, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, one-way ANOVA with Holm-Šídák multiple comparison test (F (5, 606) = 22.22, p < 0.0001). AAV, adeno-associated virus; AMPH, amphetamine; DA, dopamine; ROIs, regions of interest.

Article Snippet: AAV1 particles from Cre-dependent constructs containing the Ca 2+ sensor jRGECO1a (pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40) or the axonally targeted Ca 2+ sensor axon-GCaMP6s (pAAV-hSynapsin1-FLEx-axon-GCaMP6s) were obtained from Addgene (cat. #100853 and #11201, respectively).

Techniques: Transduction, Expressing, Plasmid Preparation, Comparison, Virus

Journal: The Journal of Biological Chemistry

Article Title: Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca 2+

doi: 10.1016/j.jbc.2023.105063

Figure Lengend Snippet: AMPH increases spontaneous Ca 2+ spikes upon inhibition of the D2 autoreceptor . Still, AMPH-induced DA efflux is not affected by increasing or chelating cytosolic Ca 2+ , blockade of voltage-gated Ca 2 + channels or inhibition of PKC. A , 10 µM AMPH causes an increase in jRGECO1a Ca 2+ spikes when coincubated with 20 nM of the D2 receptor antagonist haloperidol, while the opposite effect is seen in the presence of the D2 receptor agonist quinpirole (50 μM). ( Left ) Bar graph of the change in the Ca 2+ -dependent fluorescent signal of jRGECO1a from baseline during coapplication of AMPH with either haloperidol or quinpirole. Data are mean of ΔF/F 0 in % ± SEM obtained from 14 neurons from four independent experiments. ( Right ) Representative trace of the effect of haloperidol and quinpirole on AMPH-induced increase in intracellular Ca 2+ with colored backgrounds indicating drug treatments. B , preincubation with 20 μM NMDA and 10 μM glycine boost intracellular Ca 2+ before application of AMPH did not affect AMPH-induced DA efflux. ( Left ) Bar graph showing the change in the DA-dependent fluorescent signal of GRAB DA1H following application of AMPH with and without pre-incubation with NMDA. Data are mean of ΔF/F 0 in % ± SEM, n = 3. ( Right ) Representative trace of the effect of NMDA on AMPH-induced DA signal from GRAB DA1H and Ca 2+ from jRGECO1a with colored backgrounds indicating drug treatments. The jRGECO1a trace is the average signal ± SEM of 3 somas in the recording. C , preincubating the neurons with 50 μM BAPTA-AM for 15 min to chelate intracellular Ca 2+ did not affect DA efflux. ( Left ) The bar graph shows the change in the DA-dependent fluorescent signal of GRAB DA1H following the application of AMPH with and without preincubation with BAPTA-AM. Data are mean of ΔF/F 0 in % ± SEM, n = 3. ( Right ) representative trace of the effect of BAPTA-AM on AMPH-induced DA efflux. D , preincubation with 30 μM nifedipine for 5 min to block the L-type calcium channel did not affect DA efflux. ( Left ) The bar graph shows the change in DA fluorescent signal following the application of AMPH with and without preincubation with nifedipine. Data are mean of ΔF/F 0 in % ± SEM, n = 3. ( Right ) Representative trace of the effect of nifedipine on AMPH-induced DA efflux. E , pre-incubation with 100 nM ω-conotoxin for 5 min to inhibit N-type calcium channels did not affect DA efflux. ( Left ) The bar graph shows the change in DA fluorescent signal following application of AMPH with and without preincubation with ω-conotoxin. Data are mean of ΔF/F 0 in % ± SEM, n = 3. ( Right ) Representative trace of the effect of ω-conotoxin on AMPH-induced DA efflux. F , preincubation with the PKC inhibitor 1 μM Go 6976 for 3 min did not affect DA efflux. ( Left ) The bar graph shows the change in DA fluorescent signal following application of AMPH with and without preincubation with Go 6976. Data are mean of ΔF/F 0 in % ± SEM, n = 4. ( Right ) Representative trace of Go 6976 on AMPH-induced DA efflux. ns, p > 0.05, ∗∗ p ≤ 0.01; paired t test. AMPH, amphetamines; DA, dopamine; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; NMDA, N-methyl-D-aspartate.

Article Snippet: AAV1 particles from Cre-dependent constructs containing the Ca 2+ sensor jRGECO1a (pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40) or the axonally targeted Ca 2+ sensor axon-GCaMP6s (pAAV-hSynapsin1-FLEx-axon-GCaMP6s) were obtained from Addgene (cat. #100853 and #11201, respectively).

Techniques: Inhibition, Incubation, Blocking Assay

Journal: Nature Communications

Article Title: Locomotion-dependent auditory gating to the parietal cortex guides multisensory decisions

doi: 10.1038/s41467-025-57347-y

Figure Lengend Snippet: a Top, schematic of in vivo calcium imaging of PPC excitatory neurons in a task-performing mouse. Bottom left, imaging planes across imaged mice (scale bar: 1 mm); bottom right, immunohistochemical confirmation of CaMKIIα expression in GCaMP6s-expressing neurons (scale bars: 20 μm). b Calcium activity from four example neurons (green) and locomotion speed (gray) in a moving mouse during the task. c Response types during stationary (left) and moving (right) sessions. Top, percentages of neurons by response type. Bottom, percentages of unisensory and multisensory neurons. d Stimulus-evoked amplitudes of PPC excitatory neurons during correct unisensory trials in stationary vs. moving sessions. Red, visual-selective neurons; blue, auditory-selective neurons; black, multisensory neurons. Magenta (visual and auditory-selective neurons) and black (multisensory neurons) cross-lines indicate means ± SD. e Schematic of dPCA decomposing PPC activity into PC axes representing task-relevant variables. Stim, stimulus; Deci, decision. f Explained variances for the top 20 demixed PCs. Asterisks denote statistical comparisons between stimulus PCs and others. g Stimulus PC trajectories averaged across unisensory correct trials. Circles mark 1 s after stimulus onset. Projection distance, Euclidean distance between points at 0 s and 1 s; Go/No-go distance, Euclidean distance between Go and No-go trajectories at 1 s. h Projection distances of unisensory trajectories. i Go/No-go distances in auditory or visual trials. j Schematic of the dPCA classifier predicting trial types based on Euclidean distances to trial-averaged trajectories. k dPCA classifier accuracy for sorting unisensory trials (left, actual data; right, shuffled trial labels). Solid lines, stationary sessions; dashed lines, moving. For shuffled data, lines represent 95% confidence intervals from 500 repetitions. Colors, unisensory trial types. Magenta, stimulus onset (solid) and offset (dashed). l Mean classifier accuracy during 0.5 ~ 1 s post-stimulus period. Data are presented as mean ± SEM except for d . Gray lines represent individual data. Source data are available in Source Data File. Sample numbers and statistical information are listed in Supplementary Data . NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For in vivo calcium imaging of AC STR neurons, we injected ~0.5 μl of retrograde AAV expressing GCaMP6s (AAVrg-hSyn1-GCaMP6s-P2A-nls-dTomato, Addgene) into the right STR (posterior 1.5 mm, lateral 3.45 mm from bregma; depth 2.8 mm) of the WT mice.

Techniques: In Vivo, Imaging, Immunohistochemical staining, Expressing, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prospective and retrospective values integrated in frontal cortex drive predictive choice.

doi: 10.1073/pnas.2206067119

Figure Lengend Snippet: Fig. 3. Two-photon calcium imaging in ALM layer 5. (A) Experimental procedure of two-photon calcium imaging. Mice received the AAV2/9-CaMKIIa-GCaMP6s injection at least 3 wk before the start of imaging. Over the course of learning, calcium activities of ALM layer 5 neurons were imaged with a two-photon microscope. Cell bodies were detected with custom-written software (HDBCellSCAN). (B) An example of fluorescence (F) and deconvolved fluorescence (dF) traces from a representative ramping-up cell in a novice mouse. (C) An example trace of a ramping-up cell from an expert mouse. (D) (Left) Grayscale image showing the single-trial activities (dF) in the four trial types; right reward (red), left reward (blue), right no-reward (purple), and left no-reward (cyan). Each row represents a single trial. (Right) Trial-type averaged activity plotted in the same color scheme. Same cell as shown in (B). (E) Same analysis for the cell shown in (C).

Article Snippet: The plasmid carrying the CaMKIIa promoter and GCaMP6s sequence (pAAV-CaMKIIa-GCaMP6s) was derived from pAAV-CaMKIIa-GCaMP6sP2A-nls-dTomato (addgene plasmid #51086) by deleting the P2A-nls-dTomato sequence.

Techniques: Imaging, Injection, Microscopy, Software, Fluorescence, Activity Assay