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. 2019 May 22;102(4):745-761.e8.
doi: 10.1016/j.neuron.2019.02.037. Epub 2019 Mar 25.

A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine

Jiesi Feng  1 , Changmei Zhang  2 , Julieta E Lischinsky  3 , Miao Jing  4 , Jingheng Zhou  5 , Huan Wang  6 , Yajun Zhang  7 , Ao Dong  1 , Zhaofa Wu  6 , Hao Wu  8 , Weiyu Chen  2 , Peng Zhang  9 , Jing Zou  10 , S Andrew Hires  10 , J Julius Zhu  11 , Guohong Cui  5 , Dayu Lin  12 , Jiulin Du  2 , Yulong Li  13
Affiliations

A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine

Jiesi Feng et al. Neuron. .

Abstract

Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRABNE) sensors with a 230% peak ΔF/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral- or transgenic-mediated expression of GRABNE sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes.

Keywords: GPCR; GRABNE; neurotransmitter; norepinephrine; sensor.

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Figures

Figure 1.
Figure 1.. Design and optimization of genetically encoded NE sensors. See also Figure S1.
(A) Selection of a candidate sensor scaffold by screening several NE-binding GPCRs. Shown at the right are example images of the indicated chimeric GPCR-cpEGFP candidates expressed in HEK293T cells. Yellow arrows indicate robust membrane trafficking, and red arrows indicate impaired membrane trafficking. See also Fig. S1. (B) Identification of the most responsive NE sensor, NE0.5m (indicated by the black square) by screening the cpEGFP insertion site in ICL3 of the α2AR. ΔF/F0 refers to the peak change in fluorescence intensity in response to 100 μM NE. (C) Optimizing the GRABNE sensors by mutational screening of the insertion linker. NE0.5m was used as a template, and the indicated amino acids on N-terminal and C-terminal sides of the cpEGFP insert were mutated individually. Sensor GRABNE1m (indicated by the pink squares) was identified due to having the strongest response (ΔF/F0) and brightness relative to the original NE0.5m sensor (indicated by the dashed line at 1.0). (D) Tuning the sensor’s affinity for NE by introducing mutations in the GPCR. Magnified views of the ligand-binding pocket view from the cytosol are shown; key residues involved in ligand binding and inducing a conformational change upon ligand binding are indicated. The middle panel shows example images of HEK293T cells expressing the indicated GRABNE sensors; EGFP fluorescence is shown in the left column, and the fluorescence response in the presence of 100 μM NE is shown in the right column. Shown at the right are the normalized dose-response curves for the three GRABNE sensors, with C50 values (top), and the average fluorescence change in response to 100 μM NE (bottom); n = 21-67 cells from 3-5 cultures for each sensor. The scale bars in (A) and (D) represent 10 μm. Unless noted, values with error bars indicate mean ± SEM. ***p < 0.001 (Student’s t-test).
Figure 2.
Figure 2.. Characterization of GRABNE sensors in cultured cells. See also Figure S2.
(A-C) HEK293T cells were loaded with NPEC-NE, which was uncaged by photolysis with a pulse of 405-nm light. Uncaging caused a rapid increase in GRABNE1h fluorescence, which was blocked in the presence of 10 μM yohimbine (YO). The data in B represent 3 trials each, and the data in C represent 7 cells from 3 cultures. The white dotted square indicates the image region and the purple square indicates the illumination region. (D-F) NE was applied to HEK293T cells expressing GRABNE1m or GRABNE1h to measure Ton. Yohimbine (YO) was then applied in order to measure Toff; The white dotted line indicates the line-scanning region. n ≥ 6 cells from 6 cultures. (G) The indicated compounds were applied to GRABNE1m and GRABNE1h, and the change in fluorescence relative to NE is plotted. (H)Dose-response curves for GRABNE1m, GRABNE1h, and wild-type α2AR for NE and DA, with EC50 values shown; n ≥ 3 wells with 100-300 cells each. (I) Fast-scan cyclic voltammetry measurements in response to increasing concentrations of NE and DA. The insets show exemplar cyclic voltammograms of NE and DA at 100 μM, with peak current occurring at ~0.6 V. (J) Time course of ΔF/F0 for GRABNE sensors measured over a 2-h time frame; note that the fluorescent signal remained at the cell surface even after 180 min, indicating no measurable internalization or desensitization, n = 2 wells with 100-300 cells each. (K) A TANGO assay was performed in order to measure β-arrestinμmediated signaling by GRABNE1m, GRABNE1h, and wild-type α2AR in the presence of increasing concentrations of NE; n = 4 wells with ≥105 cells each. (L,M) GRABNE sensors do not couple to downstream G protein signaling pathways. Wild-type α2AR, but not GRABNE1m or GRABNE1h, drives Gμi signaling measured using a luciferase complementation assay (L). Disrupting of G protein activation with pertussis toxin does not affect the NE-induced fluorescence change in GRABNE1m or GRABNE1h (M). n = 3 wells with ≥105 cells each. The scale bars in (A), (D), and (J) represent 10 μm. *p < 0.05, **p < 0.01, and ***p < 0.001; n.s., not significant (Student’s t-test).
Figure 3.
Figure 3.. Characterization of GRABNE sensors in cultured neurons. See also Figure S2.
(A-C) GRABNE1m is expressed in various plasma membrane compartment of cultured neurons. Cultured cortical neurons were co-transfected with GRABNE1m and RFP-CAAX to label the plasma membrane, and the fluorescence response induced by bath application of NE was measured in the cell body, dendritic shaft and spine, and axon (C). n > 10 neurons from 4 cultures. (D,E) Cultured cortical neurons expressing GRABNE1m and GRABNE1h, but not GRABNEmut, respond to application of NE (10 μM). EGFP fluorescence and pseudocolor images depicting the response to NE are shown in (D), and the time course and summary of peak ΔF/F0 are shown in (E). n > 15 neurons from 3 cultures. (F) Dose-response curve for GRABNE sensors expressed in cultured cortical neurons in response to NE and DA. n > 10 neurons from 3 cultures. (G) Example trace (top) and summary (bottom) of cultured neurons transfected with GRABNE1m and treated with the indicated compounds at 10 μM each. n = 9 neurons from 3 cultures. (H,I) The fluorescence change in GRABNE1m induced by 100 μM NE is stable for up to 2 h. Representative images taken at the indicated times are shown in (H). An example trace and summary data are shown in (I). Where indicated, 10 μM yohimbine (YO) was added. n = 5 neurons from 3 cultures. The scale bars in (A), (B) and (H) represent 10 μm; the scale bars in (D) represents 25 μm. ***p < 0.001; n.s., not significant (Student’s t-test).
Figure 4.
Figure 4.. Release of endogenous NE measured in mouse brain slices. See also Figure S4.
(A) Left, schematic illustration of the slice experiments. An AAV expressing hSyn-GRABNE1m was injected into the LC; two weeks later, acute brain slices were prepared and used for electric stimulation experiments. Right, exemplar 2-photon microscopy images showing the distribution of GRABNE1m in the plasma membrane of LC neurons. (B) Left and middle, representative pseudocolor images and corresponding fluorescence changes in GRABNE1m-expressing neurons in response to 2, 20, and 100 pulses delivered at 20 Hz. The ROI (50-μm diameter) for data analysis is indicated in the images. Right, summary of the peak fluorescence change in slices stimulated as indicated; n = 5 slices from 5 mice. (C) Exemplar traces and summary data of GRABNE1m-expressing neurons in response to 20 electrical stimuli delivered at 20 Hz in ACSF, 4-AP (100 μM), or 4-AP with Cd2+ (100μM); n = 4 slices from 4 mice. (D) Kinetic properties of the electrically evoked fluorescence responses in GRABNE1m-expressing LC neurons. Left, image showing a GRABNE1m-expressing LC neuron for line scan analysis (red dashed line). Middle and right, example trace and summary of the responses elicited in GRABNE1m-expressing neurons before, and after 10 pulses delivered at 100Hz; n = 4 slices from 4 mice. (E) The norepinephrine transporter blocker desipramine (Desi, 10 μM; red) increases the effect of electrical stimuli (20 pulses at 20 Hz) or two trains of stimuli with a 1-s interval compared to ACSF (black traces). n = 5 slices from 5 mice. (F) The fluorescence response in GRABNE1m-expressing neurons is stable. Eight stimuli (20 pulses at 20 Hz) were applied at 5-min intervals, and the response (normalized to the first train) is plotted against time. n = 5 slices from 5 mice. (G) Traces and summary data of the fluorescence response measured in neurons expressing GRABNE1m (the same plot in (E, left, gray curve)), GRABNEmut, or GRABDA1m in response to 20 pulses delivered at 20 Hz in the presence of ACSF or 20 μM YO; n = 3-7 slices from 3-7 mice. (H) Traces and summary data of the fluorescence response measured in neurons expressing GRABNE1m or GRABDA1m. Where indicated, 50 μM NE, 50 μM DA, 20 μM yohimbine (YO), and/or 20 μM haloperidol (Halo) was applied to the cells. n = 3-5 slices from 3-5 mice. The scale bars represent 10 μm. *p < 0.05, **p < 0.01, and ***p < 0.001; n.s., not significant (Student’s t-test).
Figure 5.
Figure 5.. GRABNEcan be used to measure noradrenergic activity in vivo in transgenic zebrafish. See also Figure S5.
(A) In vivo confocal image of a Tg(HuC:GRABNE1m) zebrafish expressing GRABNE1m in neurons driven by the HuC promoter. Larvae at 6 days post-fertilization were used. (B-D) Bath application of NE (50 μM) but not DA (50 μM) elicits a significant increase in fluorescence in the tectal neuropil of Tg(HuC:GRABNE1m) zebrafish, but not in GRABNEmut zebrafish, and this increase is blocked by YO (50 μM), but not ICI 118,551 (50 μM). n = 7. (E-H) Visual looming stimuli evoke the release of endogenous NE in the midbrain of GRABNE1m zebrafish, but not in GRABNEmut zebrafish. The looming stimuli paradigm is shown in the left of (E). Where indicated, YO (50 μM) or ICI 118,551 (50 μM) was applied. Desipramine (Desi, 50 μM) application slowed the decay of looming-induced NE release (H). n = 6 for GRABNEmut, and n = 9 for the others. (I-K) Single-cell labeling of GRABNE1m in the midbrain of zebrafish larva (I), with looming-evoked responses shown in (I and J). The summary data for 6 labeled cells are shown in (K). (L) Looming-evoked calcium responses of optic tectal neurons reported by jRGECO1a show no difference with or without HuC:GRABNE1m overexpression. Exemplar traces of looming-evoked responses of single tectal neurons (L1, left). Responsive neurons sorted as descending amplitudes (L1, right). 20 s before each stimuli as the baseline. Averaged looming-evoked jRGECO1a responses of every neurons shown as gray lines (L2) and the averaged responses of all neurons (L2, red line and black line, respectively). The responsive ratio and averaged amplitude of every fish are shown in (L2). n = 6. The scale bar shown in (A, left) represents 10 μm; the scale bars shown in (A, right), (B) and (E) represent 50 μm. The scale bar shown in (I) represents 5 μm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s., not significant (Wilcoxon matched-pairs signed rank test in panel H, all others were analyzed using the paired or unpaired Student’s t-test).
Figure 6.
Figure 6.. GRABNE1m can be used to measure optogenetically stimulated noradrenergic activity in vivo in freely moving mice.
(A) Schematic illustration depicting the experimental design for recording GRABNE1m and GRABNEmut fluorescence in response to optical stimulation of C1V1 in the locus coeruleus (LC). (B) Representative traces of optogenetically stimulated GRABNE1m (top) and GRABNEmut (bottom) activity in the LC before (baseline, left), 15 min after an i.p. injection of the NET blocker desipramine (10 mg/kg, middle), and 15 min after an i.p. injection of the α2AR antagonist yohimbine (2 mg/kg, right). The vertical tick marks indicate the optogenetic stimuli. Black arrows represent the timing for grabbing and i.p. injection. (C-D) Average traces of GRABNE1m fluorescence (C), summary data (D) and the decay time constant (E) in response to optical stimulation in the LC following treatment with the indicated compounds. n = 15 trials from 3 mice for each condition. (F,G) Schematic illustration (F, left), representative traces (F, right), average fluorescence change (G, left), and summary data (G, right) for GRABNE1m in response to optical stimulation of noradrenergic neurons in the LC and dopaminergic neurons in the SNc. ***p < 0.001 (for D and E, One-Way ANOVA, forG, Student’s t-test).
Figure 7.
Figure 7.. GRABNE1m can be used to measure noradrenergic activity in the hypothalamus during stress, food-related behavior, and social interaction. See also Figure S6.
(A) Schematic diagrams depicting the fiber photometry recording, virus injection, and recording sites. (B) Histology showing the expression of GRABNE1m (green) and placement of the recording; the nuclei were counterstained with DAPI (blue). Scale bar: 500μm. (C1-E6) Representative traces (C1-C6), average per-stimulus histograms (D1-D6), and summary data (E1-E6) showing normalized GRABNE1m fluorescence (ΔF/F) before and during the forced swim test (1), and before, between and during the tail suspension test (2), the hand presentation test (3), social interaction with an intruder of the opposite sex (4) and the same sex (5), and presentation of peanut butter (6). n = 3 animals each. (F) Representative traces of GRABNE1m fluorescence during the tail suspension test 10 minutes after saline injection, 25 minutes after atomoxetine (ATX) or yohimbine (YO) injection, and 15 minutes after GBR 12909 or sulpiride (Sul) injection. (G-I) Average peri-stimulus histograms (H), peak change in GRABNE1m fluorescence, and post-test decay time measured during the tail suspension test after injection of the indicated compounds. n = 3 each. The Shapiro-Wilk normality test was performed; if the test revealed it followed a normal distribution, a paired Student’s t-test or one-way repeated measures ANOVA followed by Tukey’s multiple comparisons was performed. If the values did not follow a normal distribution, a non-parametric ANOVA (Friedman’s test) was performed followed by Dunn’s multiple comparisons test. In (C) and (D), the blue dotted lines represent the start of the stimulus, and the red dotted lines represent the end of the trial. *p < 0.05, **p < 0.01 and ***p<0.001.
See this image and copyright information in PMC

Comment in

  • Sensing norepinephrine.
    Vogt N. Vogt N. Nat Methods. 2019 May;16(5):362. doi: 10.1038/s41592-019-0418-7. Nat Methods. 2019. PMID: 31040428 No abstract available.

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