Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A - GCaMP6s imaging was performed in ex-vivo transverse pontine slices containing the LC, with recordings of basal activity followed by a high potassium aCSF (50mM). GCaMP6s fluorescence was visible using two-photon excitation under basal conditions for the majority of cells, indicating that LC neuron ‘pacemaker’ firing may result in sufficient intracellular calcium levels at rest to produce visible GCaMP6s signal. Image shows a time-lapse Z-projection of basal fluorescence in a slice (over initial 200 seconds of recording). White dashed lines indicate the approximate boundary of the anatomical LC, and the boundary of the 4 th ventricle. B – Segmentation of individual GCaMP6s expressing LC neurons allowing extraction of time-series data. Segmented neuronal profiles are shown by yellow lines, white arrow indicates the neuron from which the traces in panel C were derived. Ci – Application of high potassium aCSF (50mM, onset at blue dashed vertical line) produced a striking increase in GCaMP6s fluorescence. Slow changes in GCaMP6s signal were also observed, which could represent either z-plane drift of the slice relative to the focal plane, or bleaching of GCaMP6s fluorescence over time in the case of gradual decay in signal. ii – Such slow drifts were corrected by fitting and subtracting a 3 rd order polynomial to the basal portion of the recording. iii – For measurement of transient responses, GCaMP6s timeseries data was processed using a Savitsky-Golay filter.

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Imaging, Ex Vivo, Activity Assay, Fluorescence, Expressing, Extraction, Derivative Assay, Produced

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – 2-photon recordings finished with a 50mM K + aCSF wash (period indicated by black box), to identify healthy GCaMP6s expressing cells (as compared to dead cells or autofluorescent objects). Raw (unfiltered) data is shown, overlaid by the low pass filtered (Savitsky-Golay method) trace used for transient detection. B – A Gaussian model was fitted to the first 200 seconds of baseline imaging data for each cell, to estimate confidence intervals for detection of significant responses. C – Algorithmic detection of an excitatory response to high potassium aCSF, defined as the dF/F 0 trace exceeding the 90% C.I. (centred around the baseline fluorescence value for the preceding 50 seconds) for at least 30 seconds, within a time frame of 50-250 seconds following the entry of high potassium aCSF into the perfusion system (shown by black arrow). D – In another cell from the same recording, this same method showed no significant response to high potassium aCSF (period indicated by black box). In the left panel, raw (unfiltered) data is shown, overlaid by the low pass filtered (Savitsky-Golay method) trace used for transient detection in the right panel. The black arrow indicates the time of high potassium aCSF entry into the perfusion system.

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Expressing, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – CAV-PRS-GCaMP6s was injected directly targeting the LC. This resulted in a transduction efficacy of 64.3±2.5% of dopamine-beta-hydroxylase (DBH) positive LC neurons and a specificity of 86.3±10.1% (mean±SD, n=5 sections from 1 rat). Right panels show a representative section, with GCaMP6s expression in DBH positive neurons within the boundaries of the LC (white dashed line). 4V = 4 th ventricle. B - Clonidine (1μM) produced a decrease in GCaMP6s signal in a majority of LC neurons. Left panel shows recording from a single slice with multicoloured traces showing individual neuronal responses (n=36 cells, blue line mean ± 95% confidence interval shaded). The magnitude of the clonidine inhibition for each neuron correlated with basal fluorescence (F 0 , n=96 neurons, 4 slices from 2 rats). Line and shaded region show regression line and 95% CI, fitted to clonidine responsive cells (n=67). C - TTX (1μM) resulted in a decrease in GCaMP6s signal in the majority of LC neurons (42/63, 6 slices from 3 rats). Left panel shows data for a single recording with multicoloured traces showing individual neural responses (n=21 cells, blue line indicates mean with 95% confidence interval shaded). The magnitude of the TTX inhibition for each neuron correlated with the basal fluorescence (F 0 , n=42). Linear regression and 95% CI shaded for model, fitted to TTX responsive cells.

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Injection, Transduction, Expressing, Produced, Inhibition, Fluorescence

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A - Direct injection of CAV-PRS-GCaMP6s and CAV-PRS-PSAM-eGFP targeting the LC resulted in slices with both excitatory (red box) and inhibitory (blue box) responses to increasing doses of the PSAM-specific ligand PSEM308. (Gray trace shows drift corrected data, black trace shows Savitsky-Golay filtered trace used for subsequent analysis). B – Excited/inhibited/non-responsive neurons per slice showing cell numbers (left) and percentage (right) in each response category (6 slices from 3 rats). C – Dose-response for PSEM308 excitation (left panel, n=24/112 cells) and evoked inhibition (right panel, n=49/112 cells). Dashed lines show responses for individual neurons, thick line shows mean ± 95% CI shaded. D – Lower doses of PSEM308 evoked fewer significant responses, but both excitatory and inhibitory responses persisted at 3uM. Plot shows percentage of neurons in each category at different PSEM308 doses for the whole dataset, n=112 neurons.

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Injection, Inhibition

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Plots of PSEM308 perfusion in 2 slices from a rat injected with CAV-PRS-GCaMP6s targeting the LC. In both plots, the large majority of GCaMP6s signals remain stable, with larger dF changes occurring in only a small number of cells, without temporal alignment to the PSEM308 bolus or other fluctuating cells (n=13 top panel, n=16 bottom panel)

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Injection

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Slice 2P recordings were made following direct transduction of LC with CAV-PRS-GCaMP6s and CAV-PRS-PSAM. Slices were perfused with boluses of PSEM308 (10μM) with/without atipamezole (2μM), then repeated after a 20-minute washout period. Right trace shows the inhibitory responses of a neuron to PSEM308 which was reversibly attenuated in the presence of atipamezole. B –Atipamezole (2μM) significantly reduced inhibitory responses (n=41/151 cells, 27.2%) to 10μM PSEM308. (Boxes show median ± IQR, whiskers 1.5x IQR, mean indicated by white circles, RM-ANOVA, F stat = 15.11, df=2, p < 0.00005, Tukey HSD, ** - P<0.01) C - PSEM308 excitation was not attenuated by atipamezole (n=22/151 cells, 14.6%, RM-ANOVA, F stat = 3.13, df=2, p = 0.0542). (Boxes show median ± IQR, whiskers 1.5x IQR, mean indicated by white circles, Two outlier responses have been cut off to better illustrate the data). D – Paired neuron simulation showing transient activation of a ‘PSAM transduced neuron’ (top trace shows time course of PSAM conductance) was sufficient to generate lateral inhibition in the non-transduced neuron, via reciprocal noradrenaline release and activation of α2Rs.

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Transduction, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Slice recordings were made following direct transduction of LC with CAV-PRS-GCaMP6s and CAV-PRS-PSAM. Slices were perfused with a bolus of 10μM PSEM308 with/without AAT block, then allowed to recover for a 20 minute washout period. Right panel shows time series data for a single neuron whose lateral inhibition was not sensitive to AAT blockade. B & C – AAT blockade suggested a significant change in inhibitory (B, n=10 neurons, RM-ANOVA F stat = 7.474, df=2, p-value =0.0043, 3 slices, 1 rat) but not excitatory (C, n=4 neurons, RM ANOVA F stat = 0.111, df=2, p-value =0.9, 3 slices, 1 rat) responses following 10μM PSEM308. The only significant difference found was a decrease in inhibition between the initial inhibitory response to PSEM308 and the final inhibitory response to PSEM308 following AAT block washout (Tukey HSD adjusted P-value = 0.015). (Boxes show median + IQR, whiskers show 1.5x IQR).

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Transduction, Blocking Assay, Inhibition

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – LC neurons that were either excited or inhibited following bolus PSEM308 perfusion (grey box, left panel) appeared to be clustered in space (subpanels 1-4 on the right show heatmaps of GCaMP6s signal in individual neurons at time points 1-4 (dashed vertical lines) as shown on the left panel) B – graphical representation of the distances between the centres of pairs of cells. For each PSEM308 non-responsive cell (A) and each PSEM308 inhibited cell (B), the distance to the nearest PSEM308 excited cell was found (in this case a1 & b3 respectively). C - Comparison of the distance between non-responsive→excited vs inhibited→excited nearest neighbour pairs. Inhibited→excited pairs were significantly closer together than non-responsive→excited pairs (n=47 non-responsive→excited pairs, n=44 inhibited→excited pairs, 9 slices from 5 animals, two tailed t-test stat = - 2.811, p value = 0.0061)

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Slices were prepared following retrograde targeting of two distinct LC modules with CAV-PRS-GCaMP6s injected into olfactory bulb and CAV-PRS-PSAM or CAV-PRS-fsRED into brainstem. B – This approach resulted in a higher number of transduced LC BS neurons than LC OB projecting neurons, and a subset of co-labelled cell bodies (n=3 sections, 1 rat, 60 cells). (For this assay CAV-PRS-fsRED was injected in the brainstem) C - Slices generated with cross-modular targeting showed predominantly inhibitory responses to PSEM308 in the LC OB (top panel), compared to slices in which CAV-PRS-GCaMP6s and CAV-PRS-PSAM were co-injected (bottom panel). Plots show individual neuron level data (Savitsky-Golay filtered) from a single slice from each dataset. D - The predominance of inhibitory responses is highlighted further when examining the number (top panel) and proportion (bottom panel) of cells in each response category for each neuron per slice (n=8 slices, 4 rats) E – Raster heatmap plot of all neurons in this cross-modular dataset, sorted according to their response to PSEM308 (most excited at the top). The predominance of inhibition and the biphasic nature of responses are both evident. (n=93 neurons, 8 slices, 4 rats). The effect of atipamezole (10µM) attenuating the lateral inhibition is shown and which recovers after washing. F – Pooled data showing a significant block of the lateral inhibition by atipamezole (10µM). RM-ANOVA F stat =23.815, df=2, p-value < 0.00005, Tukey HSD * - P<0.05) (Boxes show median ± IQR, whiskers show 1.5x IQR, mean – white circles).

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Injection, Generated, Inhibition, Blocking Assay

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Slices were prepared following retrograde targeting of two distinct LC modules – CAV-PRS-GCaMP6s targeting LC BS and CAV-PRS-PSAM targeting LC OB . B – GCaMP6s responses of LC BS neurons to PSEM308 activation of LC OB neurons were largely inhibitory - left panel shows number of cells and right panel shows percentage of cells per slice in each response category. C – Raster heathmap plot showing PSEM308 responses for all neurons in this dataset, sorted according to their response from most excited top. (n=17 neurons, 2 slices, 1 rat). D – Pooled data showing the significant blockade by atipamezole (10µM) of the lateral inhibition (RM-ANOVA F stat =12.2667, df=2, p-value = 0.0002, Tukey HSD ** - p<0.01) but having no effect on the excitation (RM ANOVA F stat=0.5947, df=2, p=0.59). (Boxes show median ± IQR, whiskers show 1.5x IQR, mean – white circles).

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Noradrenergic cross-modular reciprocal inhibition within the locus coeruleus

doi: 10.1101/2022.09.07.506929

Figure Lengend Snippet: A – Averaged time series data from a single slice showing PSEM308 excited and PSEM308 inhibitory responses, illustrates the biphasic nature of the inhibitory response, with the GCaMP6s signal diverging both during the initial PSEM308 response, and in the subsequent post-inhibition excitation. (mean +/- 95% C.I., n=11 excited neurons, n=16 inhibited neurons) B – Biphasic responses were more prevalent in neurons showing initial inhibition (29/40 neurons, 72.5%), than for neurons showing initial excitation (4/23 neurons, 17.4%) C – Left panels show the magnitude of initial and rebound responses for excitation- >inhibition and inhibition->excitation biphasic responses. Right panel shows the time between the peak of the initial response and rebound response for both excitation- >inhibition and inhibition->excitation biphasic responses. (Violin plot dashed lines show median and quartiles).

Article Snippet: Illumination from a 470nm LED (Thorlabs) with a 550nm bandpass emission filter was used to visually identify GCaMP6s positive cells and orient the objective lens (Nikon MRP07220, 16x magnification, 0.8 numerical aperture) above the transduced LC.

Techniques: Inhibition

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

Article Title: L-type voltage-gated Ca 2+ channel Ca V 1.2 regulates chondrogenesis during limb development

doi: 10.1073/pnas.1908981116

Figure Lengend Snippet: Ca2+ transients in limb mesenchyme are regulated by voltage-gated L-type Ca2+ channels. (A–O) Live-imaging analyses of Ca2+ transients visualized with GCaMP6s in micromass-cultured chicken limb bud cells. mCherry was co-overexpressed as a control signal. The micromass-cultured cells were treated with K-gluconate (40 mM; C and D), Nifedipine (250 μM; H and I) or both (M and N). At each time point, images were thresholded to visualize GCaMP transients and newly emerged GCaMP signals were counted as GCaMP transients, as marked by yellow arrowheads in (B′, D′, G′, I′, L′, and N′). (E, J, and O) Temporal dynamics of GCaMP transients (n = 3 each). Time-lapse analyses started 5 h after plating. GCaMP flashes were counted every minute for 60 min. (Scale bars: 100 μm in A, F, and K.)

Article Snippet: This resulted in an increase in frequency of the Ca 2+ transients to 5.27% ( P < 0.01 vs. control), implying that VGCCs are activated in response to depolarization ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 2. caption a7 Ca 2+ transients in limb mesenchyme are regulated by voltage-gated L-type Ca 2+ channels. ( A – O ) Live-imaging analyses of Ca 2+ transients visualized with GCaMP6s in micromass-cultured chicken limb bud cells. mCherry was co-overexpressed as a control signal.

Techniques: Imaging, Cell Culture, Control