Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor

Abstract

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice.

Keywords: energy metabolism; fluorescent biosensor; glucose metabolism.

Figure 1.. Sensor design and characterization.

(A) Top, schematic representation of the glucose sensor. Abbreviations indicate the N-terminal pRSET affinity tag sequence (Nt), the glucose-binding protein (TtGBP) and the circularly-permuted T-Sapphire (cpmTS). Bottom, the full sequence of the sensor as reported in Díaz-García et al. (2017), highlighting each domain with the same color code from the schematic representation. (B) Excitation and emission spectra of the purified sensor protein. (C) Dose-response curves at different pH values. Data points and whiskers represent the mean and the range of duplicate measurements, respectively. The curves represent the best fit to a Hill equation (see Eq. 1 in Methods). Experiments were performed at room temperature of approximately 22°C.

Figure 2.. Calibration of the lifetime sensor iGlucoSnFR-TS.

(A) Representative images of IAA-treated, permeabilized HEK293T cells visualized by 2p-FLIM. The cells were incubated at different glucose concentrations after permeabilizing with β-escin. Cells exposed to higher glucose concentrations in the bath perfusion exhibited higher fluorescence lifetimes of the sensor. Note that permeabilization with β-escin caused HEK293T cells to adopt a round shape. (B) Dose-response curve obtained in permeabilized HEK293T cells, at 34°C. Points represent the mean ± SD of 133 – 258 cells from three independent experiments (N = 3, each for a particular passage/transfection). The solid line corresponds to the curve obtained using averaged parameters from three different experiments (see Table 1). The dashed line represents the in vitro calibration of the purified sensor, performed under similar conditions. Points represent the mean ± SD of three independent experiments (N = 3, each for a particular protein preparation). (C) Low pH increases the lifetime of the sensor in permeabilized cells at 34°C. The cells were bathed with a solution containing 1 mM glucose, with the pH adjusted to 7.35 (N_cells = 238, imaged in different fields from three coverslips, each from a different passage/transfection) or 7.00 (N_cells = 471, imaged in different fields from three different coverslips, from the same passage/transfection). Box plots indicate the median and middle half of the data, with whiskers spanning the 5 – 95 percentiles of the population. The notch represents the 95% confidence interval around the median, and the horizontal line indicates the mean. The raw data is overlapped to the box plots. Significance levels were obtained using an unpaired non-parametric Mann-Whitney test.

Figure 3.. Determination of the intracellular glucose concentration in HEK293T cells.

(A) Top: Representative images of non-permeabilized cells exhibit the typical flat, adherent appearance of HEK293T cells. The fluorescence lifetime of iGlucoSnFR-TS was sensitive to changes in the extracellular glucose concentration. Bottom: Cells exhibit higher lifetime values after glycolytic inhibition with 0.5 mM of IAA for 10 min at room temperature. (B) Glucose sensor measurements on intact HEK293T cells at 34°C, as a function of [glucose]_o. Box plots indicate the median and middle half of the data, with whiskers spanning the 5 – 95 percentiles of the population. The horizontal line indicates the mean. The notch represents the 95% confidence interval around the median, and the horizontal line indicates the mean. The raw data is overlapped to the box plots. The right-hand axis indicates the calculated intracellular glucose concentration from the permeabilized-cell calibration. Data were collected from 374 cells in two independent experiments (each for a particular passage/transfection) for the control condition (black box plot). The dataset for the treatment with IAA (depicted in red) comprises 248 cells imaged in several fields from three coverslips of two independent passages/transfections. Data from cells treated with IAA, and permeabilized with β-escin (shown in gray), are included for the conditions of 5 and 10 mM glucose (N_cells = 219 and 212 respectively, imaged in different fields from three coverslips, each from a different passage/transfection). Experiments were performed at 34°C. For comparisons in the groups at 2 mM glucose, an unpaired non-parametric Mann-Whitney test was used. Significance levels for multiple comparisons at 5 and 10 mM glucose were obtained using an unpaired non-parametric Kruskal-Wallis test with a Dunn’s post hoc test.

Figure 4.. Neuronal glucose concentrations in mouse brain slices.

(A) Representative images of dentate granule neurons located 50 μm deep into a hippocampal slice, exposed to different glucose concentrations in ACSF. (B) Box plots indicate the median and middle half of the data, with whiskers spanning the 5 – 95 percentiles of the population. The notch represents the 95% confidence interval around the median, and the horizontal line indicates the mean. The raw data is overlapped to the box plots (N_neurons = 51, N_slices = 4 and N_mice = 2 for [glucose]_o = 0.5 mM; N_neurons = 69, N_slices = 6 and N_mice = 3 for 2, 5 and 10 mM glucose in the ACSF). Experiments were performed at 34°C. The red dashed line represents the expected sensor lifetime when the intracellular glucose concentration is equilibrated with the nominal glucose concentration in the bath (predicted from the in-cell calibration in Figure 2).

Figure 5.. Glycolytic inhibition increases neuronal glucose concentrations in acute mouse brain slices.

(A) Representative trace of iGlucoSnFR-TS lifetime in an experiment designed to assess intracellular glucose concentration in a dentate granule neuron upon glycolysis inhibition. The bars indicate the times of application of fuels (10 mM glucose and 2 mM lactate), as well as IAA (0.5 mM), which inhibits glycolysis irreversibly. The temperature was lowered (shaded zone) to minimize cell damage during IAA application (The change in the fluorescence lifetime after cooling and heating reflects the temperature sensitivity of the sensor, see Figure 6). To minimize the effects of dysregulated synaptic activity, a cocktail of synaptic blockers was used: 5 μM NBQX, 25 μM D-AP5 and 100 μM Picrotoxin to inhibit AMPA, NMDA and GABA_A ionotropic receptors, respectively. (B) Addition of lactate (Lac) to the ACSF did not affect the [glucose]_i, but subsequent glycolytic inhibition with IAA (Lac+IAA) increased the fluorescence lifetime of the sensor, indicating an elevation in [glucose]_i. Box plots indicate the median and middle half of the data, with whiskers spanning the 5 – 95 percentiles of the population. The notch represents the 95% confidence interval around the median, and the horizontal line indicates the mean. The raw data is overlapped to the box plots (N_neurons = 41, N_slices = 5 and N_mice = 3). Significance levels were obtained using a paired non-parametric Friedman test with a Dunn’s post hoc test for multiple comparisons. One outlier data point was omitted because it was >5 SD below the mean.

Figure 6.. Temperature sensitivity of iGlucoSnFR-TS and quantitative in vivo imaging of neuronal glucose concentrations.

(A) Representative trace of iGlucoSnFR-TS lifetime of a protein sample (black open circles, scale on left axis), imaged in a glass pipette loaded with a solution containing 1 mM glucose. The temperature of the bath (gray dashed line, scale on right axis) was varied over time and the fluorescence lifetime of iGlucoSnFR-TS followed with changes in the opposite direction. (B) A linear regression performed on lifetime values at three different temperatures (29.41 ± 0.04, 34.02 ± 0.05 and 37.0 ± 0.1°C), shows a negative relationship between iGlucoSnFR-TS lifetime and temperature of −0.019 ns per degree Celsius (slope of the curve). Data points represent the mean ± SD of three independent experiments. The dashed line was constructed with the average parameters of individual linear regressions from each experiment. (C) Dose-response curve in permeabilized HEK293T cells at 37°C. Solid symbols represent the mean ± SD of 82 – 146 cells from three independent experiments (N = 3, each for a particular passage/transfection). The solid line corresponds to the curve obtained using averaged parameters from three different experiments (see Table 1). The dashed line represents the in vitro calibration of the purified sensor, performed under similar conditions. Open symbols represent the mean ± SD of three independent experiments (N = 3, each for a particular protein preparation). (D) Left, representative images of neurons from the visual cortex of awake mice expressing the sensor iGlucoSnFR-TS. Right, summary of lifetime measurements in V1 neurons (left axis), converted to glucose concentrations (right axis) using the in-cell calibration obtained at 37°C. The calculated in vivo [glucose]_i in V1 neurons was 1.41 ± 0.45 mM (mean ± SD; N_neurons = 87, N_mice = 5). The box plot represents the median and middle half of the data, with whiskers spanning the 5 – 95 percentiles of the population. The notch represents the 95% confidence interval around the median, and the horizontal line indicates the mean. The raw data is overlapped to the box plot.