Dataset DOI: 10.5061/dryad.18931zd7t

Description of the data and file structure

File: Lefton_et_al_Data.xlsx

Description: The data file contains the raw and analyzed data for each figure (and supplemental figures) of the manuscript. Each tab corresponds to a separate figure and in each tab the data are organized by figure panel. The main metric, variable, or value measured, and their unit, are clearly stated (in bold) and reflect the corresponding figure panel. The numerical values listed represent the raw data from which the figures were compiled.

Figure1: NE and LC-NE activity inhibit presynaptic efficacy: (A) Schematic of the recording conditions. (B) Left: time course of the effect of 20 μM NE applied at time t = 0 (gray area) on fEPSP slope and PPF. Right: representative traces showing the effect of NE on the slope of the first fEPSP (top) and on the PPF of the second fEPSP (bottom) from the same recording. Stimulation artifacts were cropped for clarity. (C) Pairwise quantification of the effect of NE on fEPSP slope and PPF for the experiments shown in (B). (D) Correlation between NE-induced fEPSP decrease and PPF increase in experiments shown in (B) and (C). (E) Representative recording of EPSC amplitude in response to minimal stimulations, showing failures (gray) and successes (black). (F) Left: averaged time course (per minute) of minimal-stimulation experiments showing the effect of 20 μM NE on synaptic efficacy (gray) and potency (black). Right: representative traces illustrating the effect of NE on efficacy (gray traces, failures; black traces, successes) and potency (average of successes) over 1-min epochs. (G) Quantification and pairwise comparison of the effect of NE on synaptic efficacy, potency, and strength for individual experiments shown in (F). (H) Schematic illustration of the procedure for expressing ChR2 in LC-NE fibers. (I) Immunohistochemistry (IHC) images of ChR2-EYFP expression in the LC (left) and hippocampal CA1 (right) at 12 weeks, and quantification of efficiency and specificity of ChR2-EYFP expression in the LC (center). (J) Schematic of the recording and optogenetic stimulation conditions. (K) Time course of the effect of the optical stimulation of LC-NE fibers on fEPSPs and PPFs and representative traces. (L) Pairwise quantification of the effect of light (1 Hz, 10 min) on fEPSP slope and PPF for the experiments shown in (K) and in EYFP-control slices. (M) Plot summarizing the effect of light in ChR2-positive slices, EYFP-control slices, and ChR2-positive slices in the presence of silodosin (silo.) (50 nM). (N) Plot summarizing the effect of NE on synaptic strength in the presence of blockers of α2-AR (yohimbine, 500 nM), β-AR (propranolol, 1 μM), α1B-AR (LY746-314, 1 μM), α1-AR (prazosin, 1 μM), α1A-AR (silodosin, 50 nM), or the α1A-AR agonist A61603 (70 nM). n = 5 to 10 mice. (O and P) Time course of the effect of 20 μM NE on fEPSP and PPF in the presence of silodosin (50 nM), representative traces, and pairwise quantification.

Figure 2: Astrocyte Ca2+ dynamics gate the effect of NE on synapses: (A) Approach for astrocyte Ca2+ silencing with iβARK. (B) Representative IHC images of iβARK-mCherry expression in the hippocampal CA1, along with quantification of efficiency and specificity (n = 5 sections). (C) Plot of the stimulation intensity/fEPSP slope relationship (left, unpaired Student’s t test on slope/stimulation ratio) and summary bar graphs of PPF values (right) in RFP-control and iβARK slices at baseline. (D) Kymograph, in which each row shows the average fluorescence across regions of activity (ROAs) of an individual astrocyte, and five representative ΔF/F0 traces (from individual ROAs) of spontaneous Ca2+ transients in RFP-control and iβARK slices. Horizontal time axis applies to the kymograph and representative traces. (E) Plots of the peak amplitude, frequency, and kinetics of spontaneous Ca2+ transients in RFP-control and iβARK slices. (F) Kymographs, in which each row shows the whole-cell fluorescence of an individual astrocyte, and average ΔF/F0 traces (±SEM) across all astrocytes in response to 20 μM NE application in RFP-control and iβARK slices. (G) Plot of the peak ΔF/F0 response in RFP-control and iβARK conditions for experiments shown in (F). (H and I) Time courses of the effect of 20 μM NE on fEPSP slope and PPF and representative traces in RFP-control and iβARK slices. (J) Pairwise quantifications of the effect of NE on fEPSP slope and PPF for the experiments shown in (H) and (I). (K) Plots summarizing the effect of NE on fEPSP slope in RFP-control and iβARK slices. (L) Correlation between the effect of 20 μM NE on astrocyte peak Ca2+ responses and fEPSP slope across three methods of astrocyte silencing and RFP controls (figs. S4 and S5 and table S1). 

Figure 3: Astrocytic, but not neuronal, α1A-ARs are required for NE to affect synapses: (A) Approach for the neuronal deletion of Adra1a. (B) Representative IHC images of Cre-GFP expression in hippocampal CA1 and CA3 neurons, along with quantification of efficiency and specificity (n = 4 sections from 2 mice). (C) Quantification of bulk Adra1a gDNA polymerase chain reaction (PCR) band intensity, normalized to Atcb (β-actin), in Cre-injected Adra1a+/+ and Adra1afl/fl mice. (D) Quantification of Adra1a gDNA, normalized to Atcb (β-actin), in GFP-positive and GFP-negative cells from Cre-injected Adra1afl/fl mice (n = 5) relative to Adra1a+/+ mice (n = 5) (left), and representative PCR gels (right). (E) Time course of the effect of 20 μM NE on fEPSPs and PPFs in N-Adra1aKO slices and representative traces. (F and G) Pairwise quantification of the effect of NE on fEPSP slope and PPF in N-Adra1aKO and N-Adra1aCre-GFP control slices and summary plot of the effect of NE on fEPSPs in both conditions. (H) Approach for the astrocytic deletion of Adra1a. (I) Representative IHC images of Cre-GFP expression in hippocampal CA1 astrocytes, along with quantification of efficiency and specificity (n = 6 sections from 2 mice). (J) Quantification of bulk Adra1a gDNA levels, normalized to Actb (β-actin), in Cre-injected Adra1afl/fl mice and Cre-injected Adra1a+/+ controls (left) and quantification of Adra1a gDNA, normalized to Atcb (β-actin), in GFP-positive and GFP-negative cells from Cre-injected Adra1afl/fl mice (n = 4) relative to Adra1a+/+ mice (n = 3) (right). Representative PCR gels are shown. (K and L) Kymographs, in which each row shows the whole-cell fluorescence of an individual astrocyte; average ΔF/F0 traces (± SEM) across all astrocytes; and quantification of the peak Ca2+ signal in response to 20 μM NE application in A-Adra1aCre-GFP and A-Adra1aKO slices. (M to P) Time courses of the effect of NE on fEPSPs and PPFs and representative traces in A-Adra1acontrol (M) and A-Adra1aKO slices (N), pairwise quantification of the effect of NE on fEPSP slope and PPF (O), and summary plot of the effect of NE in both conditions (P). (Q) Summary plot of the inhibitory effect of NE on fEPSPs in A-Adra1aKO and N-Adra1aKO slices relative to their respective controls.

Figure 4: NE leverages ATP-adenosine-A1R signaling to modulate synaptic efficacy: (A) Left: plot summarizing the effect of 20 μM NE in the presence of A1R antagonists (CPT, 200 nM, or DPCPX, 100 nM), an adenosine scavenger (ADA, 1U/ml), a cocktail of P2X/P2Y (PPADS, 10 μM), A2A (ZM241385, 50 nM) and A2B (PSB603 50 nM) receptor antagonists, a cocktail of mGluR inhibitors (CPPG, 5 μM; MPEP, 3.6 μM; YM298198, 2 μM), or in slices from NT5eKO mice (n = 4 to 9 mice). Right: schematic of the ATP-adenosine-A1R pathways showing different points of genetic or pharmacological intervention. (B) Time course of the effect of NE on fEPSPs and PPFs in the presence of the A1R antagonist CPT and representative traces. (C) Pairwise quantification for the experiments shown in (B). (D) Approach for the deletion of Adora1 in CA3 or CA1 neurons. (E) Representative IHC images of Cre-GFP expression in CA1- and CA3-injected animals, along with quantification of regional specificity (n = 4 sections from 2 mice). (F and G) Time course, representative traces, and quantification of the effect of 5 μM adenosine on fEPSPs in CA1-Ado1KD and CA3-Ado1KD slices. (H to J) Time course, representative traces, pairwise quantification, and summary plot of the effect of 20 μM NE on fEPSPs in CA1-Ado1KD and CA3-Ado1KD slices. (K and L) Time course, representative traces, and summary plot of the effect of 20 μM NE on fEPSPs and PPFs in NT5eKO slices. (M and N) Time course, representative traces, and quantification of the effect of adenosine on fEPSPs in NT5eKO and control slices. 

Figure S1: Effect of NE on the AMPAR-mediated fEPSP: (A) Pairwise quantification of the effect of the AMPAR antagonist NBQX (10μM) on fEPSPs and representative traces. n = 2 mice. (B) Dose-response curve of the inhibitory effect of NE on fEPSP slope (n = 5 to 7 slices per dose). A 4-parameter logistic non-linear symmetrical sigmoid fit was used to determine the IC50. (C) Coefficient of variation analysis showing 1/CV2 of the slope of fEPSPs in the NE condition against the mean, normalized to baseline. All but one recording give rise to a data point below the diagonal line (slope = 1), and the slope of the line from the baseline coordinate (1,1) to the average coordinate in NE (0.73,0.44) is greater than 1, indicative of a presynaptic locus of action. (D) Time course and pairwise quantification of the effect of NE on fEPSP and PPF with stimulations of Schaffer collaterals paused for five minutes at the onset of NE application. (E) Pairwise quantification of the effect of 20μM NE on fEPSP and PPF in the presence of the NMDAR antagonist D-AP5 (50μM, n = 4 mice). (F) Plots of the inhibitory effect of NE as a function of the initial fEPSPs slope (left) or initial PPF (right) across control conditions. The naïve and vehicle condition (DMSO, 0.01%) are denoted separately, but the linear regression, correlation coefficient and p-values are shown for the control condition as a whole. (G) Quantification and representative images of ChR2-eYFP expression of LC-NE projections in the hippocampal CA1 at 7, 10, 11, 12 and 13 weeks post-injection (n = 6-12 slices, from 1-2 animals). (H) Time course and representative traces showing the effect of the optical stimulation of LC-NE fibers on fEPSPs and PPF in EYFP-control slices. (I,J) Time course, representative traces and pairwise quantification showing the effect of the optical stimulation of LC-NE fibers on fEPSPs and PPF in the presence of silodosin (50nM). (K-Q) Pairwise quantification of the effect of 20 μM NE on fEPSP slope and PPF in prazosin (α1-AR antagonist, (K,L)), yohimbine (α2-AR antagonist, (M,N)), propranolol (β antagonist, (O)), yohimbine and propranolol (P), and LY746-314 (α1B-AR antagonist, (Q)) at indicated concentrations. n = 5-10 mice. (R,S) Pairwise quantification showing the inhibitory effect of the α1A-AR agonist A61603 (70nM) on fEPSP and PPF (R) and its blockade by silodosin (S). n = 9-3 mice respectively. (T) Pairwise quantification showing the potentiating effect of silodosin perfusion (50nM) on fEPSP and inhibitory effect on PPF (n = 3 mice).

Figure S2: Presynaptic Gq signaling enhances presynaptic efficacy and synaptic strength:  (A) Schematic of micro-injections to express the excitatory hM3D(Gq) DREADD actuator in CA3 (presynaptic) neurons. (B) IHC of DREADD expression (in green) in CA3 but not CA1 neurons (NeuN, in red) and quantification of regional specificity. (C) Time courses and representative traces of the effect of 10μM of the hM3D(Gq) agonist CNO, on fEPSP and PPF in control slices. (D) Time courses and representative traces of the effect of 10μM CNO, on fEPSP and PPF in slices expressing hM3D(Gq) in the CA3. Note the increase in synaptic strength (fEPSP) and release probability (decreased PPF). (E) Pairwise quantification of the effect of CNO on fEPSP and PPF in DREADD and control slices. (F) Summary plots comparing the effect of NE on fEPSP and PPF in DREADD and control slices. n=3 biological replicates each.

Figure S3: Imaging and analysis of astrocyte Ca2+ responses to NE and α1A-AR pharmacology:  (A) Schematic of micro-injections for lck-GCaMP6f expression in astrocytes in the s. radiatum of the CA1. (B) IHC of lck-GCaMP6f expression in CA1 astrocytes and quantification of efficiency and specificity. (C) Schematic of the 2-PSLM conditions for astrocyte lck-GCaMP6f imaging in hippocampal slices. (D) Overview of the STARDUST analysis workflow for ROA and cell-based timeseries analysis. (E) Left, Kymographs of pseudo-colored astrocyte Ca2+ signals (each row represents an individual cell) and average ΔF/F0 traces (± s.e.m.) across all astrocytes, in responses to the application of NE at indicated concentrations. Right, Dose-response curve showing the peak amplitude of the astrocyte Ca2+ response as a function of NE concentration. (F) Left, Kymograph of pseudo-colored astrocyte Ca2+ signals (each row represents a single cell) and average ΔF/F0 traces (± s.e.m.) across all astrocytes, in response to the application of 20μM NE in control conditions and in the presence of silodosin (50nM, α1A-AR antagonist). Right, Quantification of the peak astrocyte Ca2+ response to 20μM NE in control and silodosin. (G) Kymographs (each row shows the average fluorescence across ROAs of a single astrocyte) and 5 representative ΔF/F0 traces (from individual ROAs) showing spontaneous astrocyte Ca2+ transients in control and silodosin conditions. (H) Quantification of the peak amplitude, frequency, and kinetics of spontaneous astrocyte Ca2+ transients in control conditions and in the presence of silodosin. 

Figure S4: CalEx blocks the effect of NE on astrocyte Ca2+ and synapses:  (A) Schematic of micro-injections to express the CalEx actuator PMCA2 (plasma membrane Ca2+ pump) in CA1 astrocytes. (B) IHC of CalEx expression in CA1 s. radiatum astrocytes and quantification of specificity and efficiency. (C) Plot of the stimulation intensity/fEPSP slope relationship (left, unpaired Student’s t-test on slope/stim ratio) and summary bar graphs of PPF values (right) in RFP-control and CalEx slices at baseline. (D,E) Kymograph (each row shows the average fluorescence across ROAs of an individual astrocyte), 5 representative ΔF/F0 traces (from individual ROAs), and quantification of the peak amplitude, frequency, and kinetics of spontaneous Ca2+ transients in RFP-control and CalEx slices. (F,G) Kymographs (each row represents an individual cell), average ΔF/F0 traces (± s.e.m.) across all astrocytes, and quantification of the peak amplitude, in response to 20μM NE application in RFP-control and CalEx slices. (H) Time course and representative traces of the effect of adenosine on fEPSP and PPF in CalEx slices. (I) Pairwise quantification of the effect of adenosine on fEPSP and PPF in CalEx slices. (J,K) Time course, representative traces, and pairwise quantifications of the effect of NE on fEPSP and PPF in CalEx slices. (L) Summary bar graphs showing the inhibitory effect of NE and adenosine on fEPSP in RFP-control and CalEx slices. The p-values in (L) are from ANOVAs across iβark, CalEx, and RFP-control conditions followed by Tukey’s post-hoc test, reflecting the fact that the RFP-control condition for the experiments shown in (L) is the same across iβark (Fig.2) and CalEx (this figure) experiments.

Figure  S5: The SERCA pump inhibitor thapsigargin blocks the effect of NE on astrocyte Ca2+ and synapses: (A,B) Kymograph (each row shows the average fluorescence across ROAs of an individual astrocyte), 5 representative ΔF/F0 traces (from individual ROAs), and quantification of the peak amplitude, frequency, and kinetics of spontaneous Ca2+ transients in TTX alone (RFP-control) and TTX + thapsigargin (1μM) conditions. All slices were obtained from animals with RFP-transduced astrocytes for cell-segmentation purposes. Thapsigargin was bath applied 20min prior to the start of recording. (C,D) Kymographs (each row represents a cell), average ΔF/F0 traces (± s.e.m.) across all astrocytes, and quantification of the peak amplitude, in response to 20μM NE application in RFP-control and thapsigargin conditions. (E,F) Time course, representative traces, and pairwise quantification of the effect of 5μM adenosine on fEPSP and PPF in thapsigargin-treated slices. (G,H) Time course, representative traces, and pairwise quantifications of the effect of 20μM NE on fEPSP and PPF in thapsigargin-treated slices. (I) Summary of the inhibitory effect of NE and adenosine on fEPSP in control and thapsigargin-treated slices.

Figure S6: Ca2+ and synaptic recordings in A- AdraKO and N-AdraKO slices:  (A) Schematic of the experimental design and qPCR analysis of the expression of astrocyte markers (Kcnj10: Kir4.1 and Slc1a2: GLT-1) and neuronal markers (Snap25: SNAP25 and Dlg4: PSD95) in GFP-sorted cells from AAV5-gfaABC1D::Cre-GFP (n = 9 mice) and AAV5-hSyn::Cre- GFP injected mice (n = 12 mice). Statistical analysis was done by z-scoring ΔΔCt values and averaging them for a ‘marker type’ (i.e. astrocyte, or neuron) within samples, followed by a paired Student’s t-test. (B) Adra1a mRNA levels measured by qPCR in GFP-positive and GFP-negative cells from hSyn::Cre-GFP injected Adra1a+/+ (n = 4) and Adra1afl/fl mice (n = 3). (C) Same as (B) in gfaABC1D::Cre-GFP injected mice (n=3 biological replicates each). (D) Schematic of microinjections of AAV5-hSyn::Cre or AAV5-gfaABC1D::Cre in LSL-tdTomato mice (Ai14 mice), and strategy for validation of cell-specificity. (E) IHC of Cre-dependent tdTomato expression in neurons (NeuN), oligodendrocytes (Olig2), microglia (Iba1), and astrocytes (GFAP) in the hippocampus of Cre-injected Ai14 mice. Cell markers are shown in green and tdTomato in red. Owing to the CAG promoter, expression of tdTomato appears stronger in recombined neurons than in recombined astrocytes. Scale bar: 40μm. (F) Quantification of tdTomato expression in Creinjected Ai14 mice (n = 3 mice, 4 sections/mouse), in % of maker-positive cells that are tdTomatopositive and marker-positive. Numbers indicate the number tdTomato-positive cells over the total number of cells counted for each cell-type. (G) Same as (A) for tdTomato-positive and negative cells from Cre-injected Ai14 mice (n = 3 mice). (H) Pairwise quantifications of the effect of 20μM NE on the holding current in whole-cell patch-clamp recordings (V-clamp) of CA1 pyramidal neurons in slices from control mice, in the absence or presence of the α1A-AR antagonist silodosin (100nM), and in slices from N-AdraKO mice (GFP-positive neurons), and illustrative traces. Note the inward current (downward shift) caused by NE, which is completely absent in the presence of silodosin and in N-AdraKO neurons. Numbers in grey in the traces indicate the holding current (I(h)) at the start and end of the recording. (I) Summary plot of the effect of NE on the change in holding current in control conditions, in the presence of silodosin and in GFP-positive N-AdraKO neurons. n = 2-3 mice. (J) Plot of the stimulation intensity/fEPSP slope relationship (left, unpaired Student’s t-test on slope/stim ratio) and summary bar graphs of PPF values (right) in N-AdraKO and N-AdraCre-GFP slices at baseline. (K) Time course and representative traces of the effect of 20μM NE on fEPSP and PPF in N-AdraCre-GFP slices. (L) Plot of the stimulation intensity/fEPSP slope relationship (left, unpaired Student’s t-test on slope/stim ratio) and summary bar graphs of PPF values (right) in A-AdraKO and A-AdraCre-GFP slices at baseline. (M,N) Kymograph (each row shows the average fluorescence across ROAs of a single astrocyte), 5 representative ΔF/F0 traces (from individual ROAs), and quantification of the peak amplitude, frequency, and kinetics of spontaneous Ca2+ transients in A-AdraKO and A-AdraCre-GFP slices. (O) Time course and representative traces of the effect of 5μM adenosine on fEPSP and PPF in A-AdraKO slices. (P) Pairwise quantification of the effect of 5μM adenosine on fEPSP and PPF in A-AdraKO and AAdraCre-GFP slices. (Q) Summary plot of the inhibitory effect of adenosine on fEPSP in A-AdraKO and A-AdraCre-GFP slices. (R) Summary plot of the inhibitory effect of adenosine on fEPSP in NAdraKO and N-AdraCre-GFP slices. 

Figure S7: Adenosine signaling in control conditions, in NT5eKO animals and across astrocyte Ca2+ interventions: (A,B) Time course, representative traces, and pairwise quantifications of the effect of 5μM adenosine on fEPSP and PPF in slices from naïve animals. (C) Time courses and representative traces of the effect of 5μM adenosine on fEPSP and PPF in slices in which astrocytes express RFP (RFP-control). (D-E) Pairwise quantification of the effect of 20μM NE on fEPSP and PPF in DPCPX (100nM, D), and a cocktail of P2X/P2Y (PPADS, 10μM), A2A (ZM241385, 50nM) and A2B (PSB603, 50nM) receptor antagonists (E). (F) Time courses and representative traces of the effect of 20μM NE and 5μM adenosine on fEPSP and PPF in slices from CA3-Ado1KD that received 2 bilateral AAV5-hSyn::Cre-GFP injections in the CA3 (4 total) (G) Pairwise quantification of the effect of 20μM NE and 5μM adenosine on fEPSP experiments shown on (F). n = 2 mice. (H) Pairwise quantification of the effect of 20μM NE on fEPSP and PPF in the presence of a cocktail of mGluR antagonists (CPPG, 5μM; MPEP, 3.6μM and YM298198, 2μM). n = 4 mice. (I) Pairwise quantification of the effect of 20μM NE on fEPSP and PPF in adenosine deaminase (ADA, 1U/mL, n = 9 mice). (J,K) Time course, representative traces, and pairwise quantification of the effect of 5μM adenosine on fEPSP and PPF in NT5eKO slices (paired Student’s t-tests). (L) Time courses and representative traces of the effect of 5μM adenosine on fEPSP and PPF in iβARK slices. (M) Pairwise quantification of the effect of adenosine on fEPSP and PPF in iβARK and RFP-control slices (N) Summary plot of the inhibitory effect of adenosine on fEPSP in iβARK and RFP-control slices. The p-value in (N) is from an ANOVA across iβARK, CalEx and RFP-control conditions followed by Tukey’s post-hoc test, reflecting the fact that the RFP-control condition for fEPSP recordings experiments is common to CalEx (SupFig.4) and iβark (Fig.2).

Variables

• fEPSP slope: slope of the field excitatory post-synaptic potential. Either raw value (mV/ms) or normalized
• PPF: paired-pulse facilitation. Either raw value (no dimension) or normalized
• PPR: paired-pulse ratio. Either raw value (no dimension) or normalized
• Efficacy: rate of synaptic transmission successes (percentage or probability)
• Potency: amplitude (in pA) of successful synaptic events
• Strength: a product of potency and efficacy (in pA)
• Number of cells counted
• Efficiency: percent of a cell type expressing the genetic reporter
• Specificity: precent of reporter-expressing cells that are of desired cell-type
• Spontaneous calcium activity (dF/F0). Activity observed in the absence of stimulation
• Evoked calcium activity (dF/F0). Activity observed in response to stimulation (e.g., NE application)
• Amplitude of calcium events. Peak amplitude (dF/F0) of calcium events
• Frequency of calcium events (transients/cell/min)
• Duration of calcium events. Time (in s) from event onset to return to baseline
• Rise time of calcium event. Time (in s) from 10% or peak amplitude to 90% of peak amplitude
• gDNA levels. Genomic DNA intensity band 
• mRNA levels. Log2 ratio of mRNA expression relative to that of housekeeping gene

Code/software

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Other publicly accessible locations of the data:

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Data was derived from the following sources:

• https://www.biorxiv.org/content/10.1101/2024.05.21.595135v1