The effect of GIRK4 and RGS6 ablation on APD heterogeneity, μ.

(A) Average APD heterogeneity, μ, as a function of BCL in WT, Girk4-/-, and Rgs6-/- hearts. (B-D) The effect of CCh on μ at different BCL in WT, Girk4-/-, and Rgs6-/- hearts. n = 8, 5, 8 for WT, Rgs6-/-, and Girk4-/- respectively. Statistics performed using 1-way ANOVA.</p

The effect of GIRK4 and RGS6 ablation on APD₈₀.

<p>(A) Change in average APD₈₀ with decreasing BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts. (‘#’ denotes statistical significance of p < 0.05 between WT and <i>Girk4</i>^<i>-/-</i>. ‘$’ denotes statistical significance of p < 0.05 between WT and <i>Rgs6</i>^<i>-/-</i>). (B) Representative 2D APD₈₀ maps from WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts, constructed at BCL = 120 ms both at baseline and post-CCh injection. Representative action potential traces are shown at baseline (top panel, pixels marked by *) and post-CCh (bottom panel, pixels marked by Δ). (C-E) The effect of CCh on APD₈₀ at decreasing BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts. (‘*’ denotes statistical significance of p < 0.05 between baseline and 300nM CCh; ‘&’ denotes statistical significance of p < 0.05 between baseline and 3uM CCh). n = 8, 5, 8 for WT, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i>_, respectively. All statistics performed using 1-way ANOVA.</p

The influence of M₂R-I_KACh signaling on in vivo HR and HRV.

<p>Summary of baseline and post-CCh (300nM CCh) in-vivo HR (A) and HRV (B) in WT, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i> mice. (‘*’ denotes statistical significance of p < 0.05 between baseline and CCh within the same genotype. ‘&’ denotes a statistically significant (p < 0.05) difference for both baseline and CCh when comparing between two genotypes). ‘#’ denotes statistical significance of p < 0.05 between WT and <i>Rgs6</i>^<i>-/-</i> mice post-CCh. ‘$’ denotes statistical significance of p < 0.05 between <i>Girk4</i>^<i>-/-</i> and <i>Rgs6</i>^<i>-/-</i> mice post-CCh. Statistics performed using 1-way ANOVA.) (C) Quantification of the total number of mice that exhibited arrhythmias post CCh. (‘*’ denotes statistical significance of p < 0.05 between <i>Girk4</i>^<i>-/-</i> and <i>Rgs6</i>^<i>-/-</i>. Statistics performed using Fisher’s exact test). (D) Representative examples of ECG data during control and demonstrating episodes of arrhythmia in WT and <i>Rgs6</i>^<i>-/-</i>, and no arrhythmia in <i>Girk4</i>^<i>-/-</i> mice post CCh. n = 8, 8, 6 for WT, <i>Rgs6</i>^<i>-/</i>-, and <i>Girk4</i>^<i>-/-</i>_, respectively.</p

The effect of GIRK4 and RGS6 ablation on CV.

<p>(A) Average CV as a function of BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts. (B) Representative 2D activation maps from WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i> hearts, constructed at BCL = 150 ms both at baseline and post-CCh injection. (C-E) The effect of CCh on CV at different BCL in WT, <i>Girk4</i>^<i>-/-</i>, and <i>Rgs6</i>^<i>-/-</i>. n = 8, 5, 8 for WT, <i>Rgs6</i>^<i>-/-</i>, and <i>Girk4</i>^<i>-/-</i>_, respectively. Statistics performed using 1-way ANOVA.</p

The effect of GIRK4 and RGS6 ablation on S_max.

<p>Summary of the baseline and post-CCh maximum slopes of APD restitution, S_max, in WT (n = 8), <i>Girk4</i>^<i>-/-</i> (n = 8) and <i>Rgs6</i>^<i>-/-</i> (n = 5) mice. (‘*’ denotes statistical significance of p < 0.05 between post-CCh and baseline for the same genotype. Statistics performed using 1-way ANOVA).</p

AAV9 and AAV2retro-mediated transduction following intracolonic injections.

A and B, tdTomato labeling in L6 DRG and sacral spinal cord was more abundant in AAV9-injected compared to AAV2retro injected animals from cohort 1. The images in A1 and B1 represent a maximum intensity projection of four stitched 3D stacks spanning the entire ganglion (collected with a 3 μm z-step). Scale bars: A1 and B1, 150 μm; A2 and B2, 100 μm. C, Quantitative analysis of tdTomato expressing L6 DRG neurons from cohort 1 (circles) and cohort 2 (squares). D-E, Sacral spinal cords from cohort 2 showed labelling of afferent fibers and a few neurons in the spinal cord, consistent with the previously observed pattern (D, E). Scale bar = 100μm. F-G, The presence of tdTomato labelled fibers and cells in cervical spinal cord as well as cells of the liver provides evidence for systemic distribution of AAV9 (F, G). Scale bars: F, 100μm; G, 150μm.</p

Intraspinal delivery of AAV9 and AAV2retro viral vectors leads to effective transduction of both spinal and primary afferent neurons.

A-D, tdTomato (red, subscript 1) and GFP (green, subscript 2) immunofluorescence in Ai14 mice injected unilaterally in the L3/L4 region of spinal cord with AAV9 (A and B), or AAV2retro (C and D); scale bars = 150 μm. The discrepancy in the tdTomato and GFP labeling is largely due to the differential subcellular localization of the two reporter proteins, and more specifically to the overwhelming tdTomato labeling in the neuropil of densely transduced dorsal horn regions and in the central processes of transduced DRG neurons. The high fluorescence intensity of these regions required imaging parameters that were not optimal for regions with lower density of transduced neurons. In contrast, the discrete nuclear localization of Cre-GFP allowed clear visualization of individual nuclei in the spinal cord. E and F, tdTomato (red, subscript 1) and GFP (green, subscript 2) immunofluorescence in neurons of the ipsilateral L4 DRG following transduction with AAV9 (E) or AAV2retro (F); not all tdTomato-labeled cells were immunoreactive for nuclear GFP, scale bars = 150μm. G and H, NeuroTrace staining (blue, subscript 1), tdTomato (red, subscript 2) and CGRP (green, subscript 3) immunofluorescence in the ipsilateral L4 DRG following transduction with AAV9 (G) or AAV2retro (H); arrows point to examples of tdTomato and CGRP colocalization, scale bars = 100 μm.</p

Transduction of spinal cord and DRG neurons following intrathecal delivery of AAV9 and AAV2retro.

A and B, tdTomato (red, subscript 1) and GFP (green, subscript 2) immunofluorescence in the lumbar spinal cord of mice injected with AAV9 (A) or AAV2retro (B); scale bars = 200μm. C and D, tdTomato (red, subscript 1) immunofluorescence and NeuroTrace staining (blue, subscript 2) showing transduced neurons in L4 DRG following injection of AAV9 (C) or AAV2retro (D); scale bars = 100μm. E, NeuroTrace (C2, D2) was used to quantify the area of individual L4 DRG neurons. The areas of total (E1), AAV9 transduced tdTomato+ (E2), and AAV2retro transduced tdTomato+ (E3) DRG neurons are shown as frequency histograms to illustrate the number of DRG neurons relative to their area (μm2). F, The cumulative frequency distribution plot of DRG neuron area shows that while the size distribution of AAV9 tdT+ DRG neurons (red line) closely matches that of total NeuroTrace labeled DRG neurons (black line; Kolmogorov-Smirnov test, p>0.05), the size distribution of the AAV2retro tdT+ DRG neuron population (blue line) is skewed toward larger cells compared to both the total DRG neuron population, and the AAV9 tdT+ DRG neuron population, with 66.35% of the total DRG neuron population and 59.84% of the AAV9 tdT+ DRG neuron population comprised of neurons with an area ≤ 500 μm2, while only 33.06% of the AAV2r+ DRG neuron population has an area ≤ 500 μm2 (Kolmogorov-Smirnov test, **** = pG, Each dot represents the mean area of DRG neurons from a single animal (n = animal #). The mean area of AAV2retro tdT+ DRG neurons is significantly larger than the mean area of total, and AAV9 tdT+, DRG neurons (Welch’s one-way ANOVA with Dunnet’s T3 multiple comparisons test ** = p<0.01; **** = p<0.0001).</p

Targeting of spinoparabrachial neurons by AAV9, AAV2retro, and DiI.

A-C, Representative images of the parabrachial complex (A1, B1, C1; scale bars = 500 μm) and dorsal spinal cord (A2, B2, C2; scale bars = 50 μm) following parabrachial complex injections of AAV9 (A), AAV2retro (AAV2r, B), or Fast-DiI (C). The images in B2 and C2 represent a maximum intensity projection of 3D stacks spanning 12 and 16 μm, respectively, and collected with a 4-μm z-step. Arrowheads in A2, B2 and C2 highlight labeled spinal projection neurons in the superficial dorsal horn. Additionally, in B2, the short arrow highlights a plexus of labeled processes in lamina I, and the long arrow indicates labeling of the dorsal corticospinal tract. D, Stacked Bars represent the relative distribution of labeled projection neurons between the dorsal horn (DH) and the lateral spinal nucleus (LSN) in the ipsilateral (left) and contralateral (right) spinal cord following injection with AAV9 (top), AAV2retro (middle), or Fast-DiI (bottom) (** pE, The average labeling of spinal tracts at the level of L3 is shaded for AAV9 (E1) and AAV2r (E2). F, Retrograde labeling of cortical neurons in primary motor cortex (M1) following parabrachial injection of AAV2retro; scale bar = 150μm.</p

Table_1_Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.DOCX

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 μM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 μM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 μM 2-BP or 100 μM glutamate for 24 h, while 300 μM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.</p