EASI-FISH reveals spatial and axonal projection patterns of molecularly defined cell types in the central amygdala (CEA)

We combined Expansion-Assisted Iterative Fluorescence in situ hybridization (EASI-FISH) with retrograde tracer labeling to reveal the spatial and axonal projection patterns of molecularly defined neuronal types in the central amygdala (CEA).  Here, 29 marker-genes were measured with EASI-FISH in 100µm thick brain slices in the mouse CEA, after retrograde tracer labeling in 5 downstream brain targets: the bed nucleus of the stria terminalis (BNST), lateral substantia nigra (lateral SN), periaqueductal gray   (PAG), parabrachial nucleus (PBN), and parvocellular reticular formation (PCRt).  'CEA_mFISH_data.csv' contains spot count for all marker genes, axonal projections, spatial locations (z, y, x) and cell body size (area) and shape (solidity), and cell_cluster for each neuron. Each row represents measurement from one neuron. Data collected in six samples from two animals are included here. </p

DP can occur without alterations in γ (DP) but only when preceded by LTP that does not involve a change in γ (LTP)

<p><b>Copyright information:</b></p><p>Taken from "Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity"</p><p>BMC Neuroscience 2004;5():44-44.</p><p>Published online 11 Nov 2004</p><p>PMCID:PMC535344.</p><p>Copyright © 2004 Lüthi et al; licensee BioMed Central Ltd.</p> (A) Plot of amplitude . time from a representative experiment. Black bar represents LTP pairing, grey bar DP pairing protocol. Throughout the figure, green represents baseline, red LTP and blue DP. (B) Current-variance relationship for baseline, LTP and DP (γ values for this cell (pS): baseline = 6.5, LTP = 6.7, DP = 6.0). (C) Mean EPSCs (average of all responses used for non-SFA) superimposed (left) and peak scaled (right). (D) Amplitude histogram (bin width = 2 pA; N = 151 for baseline, N = 211 for LTP, N = 513 for DP) for baseline, LTP and DP. Inset: 10 consecutive responses for baseline, LTP and DP

Bi-directional modification of AMPA receptor conductance (γ) during LTP (LTPγ) and DP (DP)

<p><b>Copyright information:</b></p><p>Taken from "Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity"</p><p>BMC Neuroscience 2004;5():44-44.</p><p>Published online 11 Nov 2004</p><p>PMCID:PMC535344.</p><p>Copyright © 2004 Lüthi et al; licensee BioMed Central Ltd.</p> (A1,2) Plot of amplitude . time from two representative experiments. Black bar represents LTP pairing, grey bar DP pairing protocol. Throughout the figure, green represents baseline, red LTP and blue DP. (B1,2) Current-variance relationship for baseline, LTP and DP (γ values for the same cells (pS): cell 1:baseline = 2.2, LTP = 6.5, DP = 3.7; cell 2: baseline = 4.5, LTP = 7.4, DP = 3.2). For this figure and in Figure 3, lines are parabolic fits of the data (see Methods). (C1,2) Inset. Mean EPSCs (average of all responses used for non-SFA) superimposed (left) and peak scaled (right). Amplitude histograms (bin width = 2 pA; number of trials: cell 1: N = 130 for baseline, N = 245 for LTP, N = 533 for DP; cell 2: N = 150 for baseline, N = 267 for LTP, N = 337 for DP; frequency normalised to the data set with the smallest number of observations for baseline, LTP and DP)

cAMP production, P-CREB analysis, and ventricle sizes in the presence and absence of coronin 1.

<p>(A) Hippocampal neurons (7 d culture) were fixed and stained with antibodies against coronin 1 (Alexa 488) and cAMP-, PKA substrate-, or P-CREB antibodies (568). Scale bar, 20 µm. (B) Wild-type or coronin 1–deficient brain lysates were immunoblotted using anti-P-CREB antibodies and reprobed using anti–coronin 1 and anti-actin antibodies. (C) Sections from wild-type or coronin 1–deficient brain were immunolabeled using anti–P-CREB antibodies, anti–coronin 1, and anti-histone antibodies. Scale bar, 10 µm. (D) Amygdalary regions of wild-type and coronin 1–deficient mice were analyzed for cAMP levels by ELISA. The values shown are normalized to total protein amounts (pmol cAMP/mg total protein). The data shown are from three independent experiments; <i>n</i> = 10 mice per genotype, <i>p</i><0.01, Student's <i>t</i> test, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s018" target="_blank">Table S1</a>. (E) Basal (Control) and cAMP-stimulated PKA activity was determined in brain lysates from wild-type (WT) and coronin 1–deficient (KO) mice using a Peptag assay. PKA activity was monitored by the negative charge increase of the PKA substrate-containing peptide. (Upper panel) Electrophoretic pattern. (Lower panel) Quantitation (<i>n</i> = 3), <i>p</i> = 0.01. (F, G) Ventricle sizes in the presence and absence of coronin 1 as analyzed by MRI on live animals. H, mean ventricular size. <i>N</i> = 6, left <i>p</i><0.01, right <i>p</i><0.05, and middle <i>p</i><0.01, Student's <i>t</i> test, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s018" target="_blank">Table S1</a>.</p

Rescue of fear conditioning in coronin 1–deficient mice by in vivo 8-Br-cAMP infusion into amygdala.

<p>(A) Scheme of the in vivo preparation. (B) Cannula implantation loci in both animal cohorts: single injection mice (left) and double injection mice (right). (C) Contextual learning was tested before (Ctrl) and 12 h later following single (<i>n</i> = 10 WT and 11 coronin 1 −/−) and double cAMP injections (<i>n</i> = 10 for both genotypes). Single injections, <i>p</i><0.01. Double injections, <i>p</i>>0.05, RM-ANOVA, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s018" target="_blank">Table S1</a>.</p

ELISA and FRET-based analysis of cAMP production in coronin 1–positive and coronin 1–negative cells in the presence and absence of F-actin.

<p>(A) Coronin 1–expressing or control Mel JuSo cells were left untreated or stimulated with isoproterenol (5 µM, 4 min) and processed for cAMP analysis using ELISA (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#s4" target="_blank">Materials and Methods</a>). The values were normalized to total protein amounts (pmol cAMP/mg protein). The data shown are a representative of three independent experiments. Data represent mean and SEM. (B, C) FRET-based analysis of cAMP production upon stimulation of wild-type or coronin 1–expressing Mel JuSo cells that had been transfected with pICUE3 with isoproterenol (10 µM). (B) the normalized emission ratio (CFP/YFP) time course of coronin 1–expressing (gray) or wild type (red) Mel JuSo cells shown in C (mean, <i>n</i> = 50). (C) Stills from movies representing the normalized CFP/YFP emission ratios. Arrows indicate the time of isoproterenol addition. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s014" target="_blank">Movies S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s015" target="_blank">S2</a>. (D–F) FRET-based analysis of cAMP production upon stimulation of Latrunculin B–treated (4 µM, 30 min) wild-type or coronin 1–expressing Mel JuSo cells (transfected with pICUE3) with isoproterenol (5 µM, at T = 25 s). (D) The normalized emission ratio (CFP/YFP) time course of coronin 1–expressing (grey) or wild-type (red) Mel JuSo cells (mean, <i>n</i> = 30). (E) Coronin 1 and F-actin staining in untreated and Latrunculin B–treated cells. (F) Stills from movies representing the normalized CFP/YFP emission ratios. Arrows indicate the time of isoproterenol addition. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s016" target="_blank">Movies S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s017" target="_blank">S4</a>.</p

Decreased E/I synapse ratio and loss of cortico-LA synaptic plasticity in the absence of coronin 1.

<p>(A, B) Analysis of synapses in brain tissue from wild-type or coronin 1–deficient mice (<i>n</i> = 3) by serial block face scanning by electron microscopy. Scale bar, 2 microns. Data shown are mean values ± SD, <i>p</i><0.01. (C) Hippocampal neurons were labeled using anti-vGLUT1 and anti-vGAT antibodies, followed by Alex Fluor 488 or 568 labeled secondary antibodies, respectively. The average number of inhibitory and excitatory synapses per 10 µm, as well as the E/I synapse ratio from wild-type or coronin 1–deficient neurons were determined. Quantitation (right panel) was done from 60 neuron terminals from four mice per genotype. For vGLUT1, <i>p</i><0.01; for vGAT, <i>p</i>>0.05; and for vGLUT/vGAT ratio, <i>p</i><0.001, Student's <i>t</i> test. (D) Scheme of the experimental preparation (LA, lateral amygdala) and pairing protocols used to induce LTP. (E) Coronin 1–deficient animals (Cor1 −/−, red symbols) exhibited normal thalamo-LA LTP [<i>p</i><0.05 versus baseline; <i>p</i>>0.05 versus wild-type (WT, gray symbols), Student's <i>t</i> test]. Scale bars, 2 mV and 10 ms. (F) Cortico-LA LTP is completely absent in coronin 1–deficient mice (red symbols). Strong cortico-LA LTP was induced in wild-type [WT (grey symbols), <i>n</i> = 12, <i>p</i><0.05 versus baseline, <i>p</i><0.05 versus Cor 1 −/− (red symbols), Student's <i>t</i> test), but not in coronin 1–deficient mice [Cor 1 −/− (red), <i>n</i> = 7, <i>p</i>>0.05 versus baseline, Student's <i>t</i> test]. Scale bars, 50 pA and 20 ms. (G) Synaptic transmission and PPR in wild-type (left) and coronin 1–deficient mice (right) in the presence of forskolin. Forskolin enhances synaptic transmission and decreases PPR at cortico-LA synapses in WT (<i>n</i> = 12, <i>p</i><0.05 versus pre-forskolin baseline), but not in Cor 1 −/− (<i>n</i> = 7, <i>p</i>>0.05 versus pre-forskolin baseline, Student's <i>t</i> test) mice. Scale bars, 100 pA and 20 ms. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s018" target="_blank">Table S1</a> for additional statistics.</p

Analysis of coronin 1–Gα interaction in cells and in vitro by surface plasmon resonance.

<p>(A, B) Cells were transfected with the different constructs indicated, stimulated with 10 µM isoproterenol (A, 4 min; B, 10 min) followed by lysis and analyzed for cAMP. Panel B shows the results of immunoprecipitating Gα followed by immunoblotting for coronin 1 (upper panels). The lower panels in B show the immunoblots for Gα and coronin 1 following SDS-PAGE of the transfected cell lysates. (C) Purified Gαs was covalently immobilized on an NTA sensor chip surface through EDC/NHS chemistry. The indicated concentrations of coronin 1 or coronin 1 mutant were sequentially injected into the SPR sensor chip. The kinetic data were collected and analyzed. The Relative Response Units (RUs) of stable binding under each different concentration is shown in the lower panel.</p

Stimulus-dependent association of coronin 1 with Gαs.

<p>(A, B) Coronin 1–expressing Mel JuSo cells were left untreated or stimulated with isoproterenol (10 µM, 10 min), lysed, and Gα molecules were immunoprecipitated with the indicated antibodies as described, followed by separation on SDS-PAGE and immunoblotting using anti–coronin 1 (A) or anti-Gα antibodies (B). The appearance of Gα as a doublet may be a result of posttranslational modifications and/or partial cleavage. (C) Coronin 1–expressing Mel JuSo cells were treated with Latrunculin B (4 µM, 30 min; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio-1001820-g005" target="_blank">Figure 5D–F</a>), lysed, and proteins immunoprecipitated using anti-Gα antibodies, followed by SDS-PAGE and immunoblotting. (D) NIE-115 cells were stimulated with isoproterenol (10 µM, 10 min), lysed, and immunoprecipitated using anti–coronin 1 antibodies. Protein complexes were separated by SDS-PAGE and immunoblotted for coronin 1 and the Gα molecules indicated. (E) Coronin 1–expressing Mel JuSo cells or control cells were starved in rolipram (100 µM, 1 h) and stimulated with cholera toxin (1 µg/mL) for 1 h at 37°C prior to stimulation with isoproterenol (10 µM, 4 min). cAMP production was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#s4" target="_blank">Materials and Methods</a>.</p