Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity
- Department of Oral Physiology, Graduate School of Dentistry, Osaka University, Japan
- Department of Physiology, School of Dentistry, Aichi Gakuin University, Japan
- Department of Neurobiology & Physiology, School of Dentistry and Dental Research Institute, Seoul National University, Republic of Korea
- Department of Cell Biology, Aging Science, and Pharmacology, Faculty of Dental Science, Kyushu University, Japan
- ADA Forsyth Institute, United States
- Department of Behavioral Sciences, Graduate School of Human Sciences, Osaka University, Japan
Figures
Figure 1 with 1 supplement
Atipamezole abolishes spike-frequency adaptation and blocks cytosolic noradrenaline (NA)-induced GIRK-I in locus coeruleus (LC) neurons.
(A, B) Sample traces of spike trains induced in an LC neuron by injection of current pulses at 75 and 100 pA. Note the spike-frequency adaptation and pulse-afterhyperpolarization (pulse-AHP). (C) Plotting of the cumulative spike numbers vs the elapsed time from the onset of the current pulses every 50 ms at 75 pA (blue circles) or 100 pA (red circles) (n=7). Two-way RM ANOVA, *p<0.001. The saturation level (a) and the half saturation constant (b) were measured by fitting the saturation function defined as y = (a * x)/(b+x) to the data points. The values of a at 75 and 100 pA current pulses were 44.6±16.2 and 52.5±12.3, respectively (paired t-test, p=0.010). The values of b at 75 and 100 pA current pulses were 2.1±0.7 and 2.1±0.4, respectively (paired t-test, p=0.823). (D, E) Sample traces of spike trains induced in an LC neuron evoked by current pulses at 100 pA under control condition and after application of 10 μM atipamezole. Note that atipamezole suppresses spike-frequency adaptation and pulse-AHP (blue and red stars). (F) Plotting of the cumulative spike numbers vs the elapsed time during the current pulses every 50 ms before and after application of atipamezole (blue and red circles, respectively) (n=10). Two-way RM ANOVA, *p<0.001. (G) Plotting of a against b, which were measured by fitting the saturation function to the data points in F. Both the values of a and b obtained by curve fitting to the data points after application of atipamezole (open and filled red circles) were significantly shifted to larger values than those obtained under control condition (open and filled blue circles) (paired t-test, a and b, ‡p=0.003 and ‡p=0.002, respectively), and there was a significant difference in the relationship between a and b (Wilk’s lambda, §p=0.002) (n=10). (H) Top, Voltage command pulse. Lower panel, Superimposed current traces obtained under control condition (1), after application of 100 μM NA (2), and after application of 10 μM atipamezole (Atip) in addition to NA (3). The amplitudes of inward and outward components in NA-induced GIRK-I were –70±22 and 36±11 pA, respectively (paired t-test, ‡p=0.005, n=6). (I) A graph showing the effects of atipamezole on amplitudes of the inward and outward components of NA-induced GIRK-I at –130 and –60 mV, obtained by subtraction of currents recorded under control condition from those recorded after application of NA (2–1) and from those recorded after application of NA and atipamezole (3–1). Inward component, paired t-test, ‡p<0.001; outward component, paired t-test, ‡p<0.001 (n=6).
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Figure 1—source data 1
Data used for graphs presented in Figure 1C, F, G, and I.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Spike-frequency adaptation and A-like K+ current in a male juvenile mouse and a female adult mouse.
(A) A locus coeruleus (LC) neuron of a male mouse at postnatal days 22 (P22) displayed spike-frequency adaptation in response to a 100 pA current pulse at –63 mV while it displayed a late spiking due to the presence of A-like K+ current in response to a 150 pA current pulse at –88 mV. (B) An LC neuron of a female mouse at P70 displayed spike-frequency adaptation in response to a 100 pA current pulse at –59 mV while it displayed a late spiking due to the presence of A-like K+ current in response to a 150 pA current pulse at –78 mV. There were no apparent differences in spike-frequency adaptation and A-like K+ current between juvenile and adult mice and also between male and female mice.
Figure 2 with 6 supplements
Ca2+-dependent rundown of noradrenaline (NA)-induced GIRK-I and its suppression by barbadin.
(A) Upper panel, Ramp command pulse. Lower panel, Superimposed current traces obtained under control condition (1) and after application of 100 μM NA for 5 min (2). (B) Upper panel, A combined command pulse applied every minute; five trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an interval of 2 s in the presence of extracellular 30 mM TEA and intracellular 0.2 mM EGTA, which were followed by the ramp pulse after an interval of 25 s. Lower panel, Representative Ca2+ currents in response to positive pulses in the presence of NA and 30 mM TEA. (C) Upper panel, Ramp command pulse. Lower panel, Superimposed current traces obtained after application of NA for 5 min (2) and in response to application of the 20th combined pulse in the presence of NA and TEA (3). (D) The I-V relationship of NA-induced GIRK-I obtained by subtraction of currents recorded under control condition from that recorded after application of NA for 5 min (red trace, 2–1) and that obtained by subtraction of the control current from that recorded in response to application of the 20th combined pulse in the presence of NA and TEA (blue trace, 3–1). (E) Plotting of amplitudes of inward components at –130 mV (blue circles) and outward components at –60 mV (red circles) against time. (F) Plotting of normalized amplitudes of inward components at –130 mV and outward components at –60 mV before and 20 min after application of positive pulse trains in the presence of NA and TEA. The amplitudes of inward components at –130 mV and those of outward components at –60 mV were normalized by those recorded in the presence of NA before applying positive pulse trains. Inward component, paired t-test, ‡p<0.001; outward component, paired t-test, ‡p<0.001 (n=8). (G) Upper panel, Ramp command pulse. Lower panel, Superimposed current traces obtained under control condition (1) and after application of 100 μM NA for 5 min (2) in LC neurons dialyzed with 100 μM barbadin (β-arrestin/AP2 blocker). (H) Upper panel, A combined command pulse applied every minute; five trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an inter-train interval of 2 s in the presence of extracellular 30 mM TEA and intracellular 0.2 mM EGTA, which were followed by the ramp pulse after an interval of 25 s. Lower panel, Superimposed current traces obtained after application of NA for 5 min (2) and in response to application of the 20th combined pulse in the presence of NA and TEA (3) in LC neurons dialyzed with barbadin. (I) The I-V relationship of NA-induced GIRK-I obtained by subtraction of currents recorded under control condition from that recorded after application of NA for 5 min (red trace, 2–1) and that obtained by subtraction of the control current from that recorded in response to application of the 20th combined pulse in the presence of NA and TEA (blue trace, 3–1). (J) Plotting of normalized amplitudes (left vertical axis) of inward components at –130 mV (filled blue circles) and outward components at –60 mV (filled red circles) against time (n=8). Amplitudes of GIRK-I were normalized to the values obtained before applying pulse trains. Right vertical axis refers to the original amplitudes. Open blue and red circles represent the normalized amplitudes of GIRK-I shown in E. (K) Plotting of normalized amplitudes of inward components at –130 mV and outward components at –60 mV before and 20 min after application of positive pulse trains in the presence of NA and TEA (n=8). The amplitudes of inward components at –130 mV and those of outward components at –60 mV were normalized to those recorded in the presence of NA before applying positive pulse trains. Open light pink circles represent results obtained under the control condition in F. Inward component, unpaired t-test, *p<0.001; outward component, unpaired t-test, *p<0.001.
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Figure 2—source data 1
Data used for graphs presented in Figure 2E, F, J, and K.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Noradrenaline (NA)-induced GIRK currents show no apparent agonist-dependent rundown but show a moderate rundown following application of a train of positive voltage pulses.
(A) Upper panel, Ramp command pulse applied every minute. Lower panel, Superimposed current traces obtained under control condition (1), in response to 5th ramp pulse (2) and 25th ramp pulse (3) in the presence of 100 μM NA. (B) The I-V relationship of NA-induced GIRK currents obtained by subtraction of currents recorded under control condition from that recorded in response to the 5th ramp pulse (red trace, 2–1) and that obtained by subtraction of the control current from that recorded in response to the 25th ramp pulse (blue trace, 3–1). (C) Plotting of amplitudes of inward components at –130 mV (blue circles) and outward components at –60 mV (red circles) against time. (D) Pooled data showing no appreciable changes in normalized amplitudes of inward components at –130 mV and those of outward components at –60 mV during 20 times repetition of ramp pulses (n=5). Inward component, paired t-test, p=0.075; outward component, paired t-test, p=0.065. (E) Upper panel, Ramp command pulse applied every minute. Lower panel, Superimposed current traces obtained under control condition (1) and in response to 5th ramp pulse in the presence of NA (2). (F) Upper panel, Ten trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an inter-spike interval of 2 s (one pulse train) were applied only once in the presence of extracellular 30 mM TEA and intracellular 0.2 mM EGTA. Lower panel, Representative Na+ current followed by small Ca2+ current in response to positive pulses. (G) Upper panel, Following the train of positive pulses, ramp command pulse applied every minute. Lower panel, Superimposed current traces obtained in response to 5th ramp pulse in the presence of NA (2) and 20 min following one pulse train in the presence of NA (3). (H) The I-V relationship of NA-induced GIRK currents obtained by subtraction of currents recorded under control condition from that recorded in response to the 5th ramp pulse in the presence of NA (red trace, 2–1) and from that recorded 20 min following one pulse train in the presence of NA (blue trace, 3–1). (I) Plotting of amplitudes of inward components at –130 mV (filled blue circles) and outward components at –60 mV (filled red circles) against time (n=5). (J) Pooled data showing significant decreases in normalized amplitudes of inward components at –130 mV and those of outward components at –60 mV before and after 20 times repetition of ramp pulses in the presence of NA (n=5). Inward component, paired t-test, ‡p=0.001. Outward component, paired t-test, ‡p=0.001.
Figure 2—figure supplement 2
Differential rundown of noradrenaline (NA)-induced GIRK currents following application of various types of positive voltage pulse trains in the presence of TEA.
(A) Upper panel, Ramp command pulse applied every minute. Lower panel, Superimposed current traces obtained under control condition (1) and in response to 5th ramp pulse in the presence of NA (2). (B) Upper panel, Ten trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an inter-spike interval of 2 s (one pulse train) were applied only once in the presence of extracellular 30 mM TEA and intracellular 0.2 mM EGTA. Following one pulse train, ramp command pulses were applied every minute. Lower panel, Superimposed current traces obtained in response to 5th ramp pulse in the presence of NA (2), 1 min following pulse trains in the presence of NA and TEA (3) and 20 min following pulse trains in the presence of NA and TEA (4). (C) The I-V relationship of NA-induced GIRK currents obtained by subtraction of currents recorded under control condition from that recorded in response to the 5th ramp pulse in the presence of NA (red trace, 2–1), from that recorded 1 min following one pulse train in the presence of NA and TEA (blue trace, 3–1), and from that recorded 20 min following one pulse train in the presence of NA and TEA (green trace, 4–1). (D) Plotting of normalized amplitudes (left vertical axis) of inward components at –130 mV (filled blue circles) and outward components at –60 mV (filled red circles) against time (n=5). Amplitudes of GIRK-I were normalized to the values obtained before applying pulse trains. Right vertical axis refers to the original amplitudes. Open blue and red circles represent the normalized amplitudes of GIRK-I, the original amplitudes of which are shown in Figure 2—figure supplement 1I. (E) Pooled data showing appreciable changes in normalized amplitudes of inward components at –130 mV and those of outward components at –60 mV before and after 20 times repetition of ramp pulses in the presence of NA and TEA (n=5). Inward component, paired t-test, ‡p<0.001; outward component, paired t-test, ‡p<0.001. (F) Upper panel, Ramp command pulse. Lower panel, Superimposed current traces obtained under control condition (1) and after application of 100 μM NA for 5 min (2). (G) Upper panel, A combined command pulse applied every minute; one train of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) in the presence of extracellular 30 mM TEA and intracellular 0.2 mM EGTA, which was followed by the ramp pulse after an interval of 19 s. Lower panel, Superimposed current traces obtained after application of NA for 5 min (2) and in response to application of the 20th combined pulse in the presence of NA and TEA (3). (H) The I-V relationship of NA-induced GIRK currents obtained by subtraction of currents recorded under control condition from that recorded after application of NA for 5 min (red trace, 2–1) and that obtained by subtraction of the control current from that recorded in response to application of the 20th combined pulse in the presence of NA and TEA (blue trace, 3–1). (I) Plotting of amplitudes of inward components at –130 mV (blue circles), outward components at –60 mV (red circles), and baseline currents (black circles) against time. (J) Pooled data showing decreases in amplitudes of inward components at –130 mV and those of outward components at –60 mV at respective conditions (2–1 and 3–1) (n=8). Inward component, paired t-test, ‡p<0.001; outward component, paired t-test, ‡p<0.001. (K) Plotting of normalized amplitudes of inward components at –130 mV (filled blue circles) and outward components at –60 mV (filled red circles) against time before and during application of positive pulse trains in the presence of NA and TEA (n=8). The amplitudes of inward components at –130 mV and those of outward components at –60 mV were normalized by those recorded in the presence of NA before applying positive pulse trains. Open blue and red circles represent results obtained in Figure 2E.
Figure 2—figure supplement 3
Ca2+ transients in response to current pulse injections in locus coeruleus (LC) neurons.
(A, B) Nomarski and fura-2 images of a fura-2 loaded LC neuron (arrowhead). (C, D) Spike trains at 14 and 21 Hz evoked in response to depolarizing current pulses. The maximal firing frequencies of LC neurons in response to depolarizing current pulses were usually lower than 20–25 Hz (n>40). (E) Ca2+ transients in response to current pulse injections which induce spike trains at 14 and 21 Hz in the same LC neuron. Note a long-lasting tail of Ca2+ transient (downward arrowheads) in response to stronger activation (21 Hz) of LC neurons. (F) Relationship between action potential frequency and the peak amplitude of Ca2+ transients (n=5). (G, H) Differential summation of Ca2+ transients induced by the two different sets of five trains of spike firings at 14–16 and at 19–21 Hz, respectively, evoked by two different current pulses with intensities of 75 and 100 pA. Note an emergence of long-lasting slow Ca2+ transients (downward arrowheads) in response to stronger activation (19–21 Hz) of LC neurons, suggesting an involvement of CICR in the generation of such long-lasting slow Ca2+ transients.
Figure 2—figure supplement 4
Weakening of spike-frequency adaptation and decrease in pulse-AHP accompany GIRK rundown.
(A–D) Sample traces of spike trains induced in a locus coeruleus (LC) neuron evoked by current pulses at 75 and 100 pA under control conditions and after rundown of GIRK-I as shown in Figure 4A–F. Note the abolishment of spike-frequency adaptation and reduction of pulse-AHP after rundown of GIRK-I. (E) Plotting of the cumulative spike numbers vs the elapsed time during the current pulses every 50 ms obtained under control condition (pink circles: 75 pA; blue squares: 100 pA) and those obtained after rundown of GIRK-I (green circles: 75 pA; red squares: 100 pA). 75 pA current pulse, two-way RM ANOVA, *p=0.002; 100 pA current pulse, two-way RM ANOVA, *p=0.004 (n=7). (F) Plotting of the saturation level (a) vs the half saturation constant (b), which were measured by curve fitting to the data points in E. The values of a and b obtained after rundown of GIRK-I (green circles, 75 pA; red squares, 100 pA) were significantly larger than those obtained under control condition (pink circles, 75 pA; blue squares, 100 pA) (75 pA current pulse, paired t-test, a and b, ‡p=0.002 and ‡p=0.002, respectively; 100 pA current pulse, paired t-test, a and b, ‡p=0.004 and ‡p=0.007, respectively), and there was a significant difference in the relationship between a and b (75 pA current pulse, Wilk’s lambda: §p=0.028; 100 pA current pulse, Wilk’s lambda: §p=0.018) (n=7).
Figure 2—figure supplement 5
Atipamezole blocks rundown of noradrenaline (NA)-induced GIRK currents, waning of spike-frequency adaptation, and suppression of pulse-ADP caused by repetitive application of positive pulse trains.
(A) Upper panel, Voltage command pulse. Lower panel, Superimposed current traces obtained under control condition (1), after application of 100 μM NA for 5 min (2) and after application of NA and 10 μM atipamezole for 5 min (3). (B) Upper panel, A combined command pulse applied every minute; five trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an inter-train interval of 2 s in the presence of extracellular 30 mM TEA, NA, atipamezole, and intracellular 0.2 mM EGTA, which were followed by the ramp pulse after an interval of 25 s. Lower panel, Superimposed current traces obtained before and after 20 times application of positive pulse trains in the presence of NA and atipamezole (3 and 4, respectively) and after washout of atipamezole but still in the presence of NA (5). (C) The I-V relationship of NA-induced GIRK currents obtained by subtraction of the currents recorded under the control condition from those recorded after application of NA for 5 min (red trace, 2–1), that obtained by subtraction of the control current from those recorded after application of atipamezole in addition to NA (blue trace, 3–1), and that obtained by subtraction of the currents recorded after 20 times application of positive pulse trains in the presence of NA and atipamezole from those recorded 10 min after washout of atipamezole but still in the presence of NA (green trace, 5–4). (D) Pooled data showing protective effects of atipamezole on the rundown of GIRK currents; amplitudes of inward components at –130 mV and those of outward components at –60 mV at respective conditions (2–1, 3–1, and 5–4). Inward component, one-way RΜ ANOVA, *p<0.001; outward component, *p<0.001 (n=6). (E–H) Sample traces of spike trains induced in an LC neuron evoked by current pulses at 75 and 100 pA under control conditions and after repetitive application of positive pulse trains in the presence of atipamezole under voltage-clamp condition as shown in A–C. Note that atipamezole prevented the waning of spike-frequency adaptation and reduction of pulse-AHP which could have been caused by [Ca2+]i increases in response to repetitive application of positive pulse trains. (I) A plot of the cumulative spike numbers vs the elapsed time during the current pulses every 50 ms obtained under control condition (pink circles: 75 pA; blue squares: 100 pA) and after repetitive application of positive pulse trains in the presence of atipamezole (green circles: 75 pA; red squares: 100 pA). 75 pA current pulse, two-way RM ANOVA, p=0.181; 100 pA current pulse, two-way RM ANOVA, p=0.958 (n=5). (J) A plot of the saturation level (a) vs the half saturation constant (b), which were measured by curve fitting to the data points in i. The values of a and b, which were obtained after repetitive application of positive pulse trains in the presence of atipamezole (pink circles, 75 pA; blue squares, 100 pA) were not significantly different from those obtained under control condition (green circles, 75 pA; red squares, 100 pA) (75 pA current pulse, paired t-test, a and b, p=0.963 and p=0.236, respectively; 100 pA current pulse, paired t-test, a and b, p=0.188 and p=0.645, respectively), and there was no significant difference in the relationship between a and b (75 pA current pulse, Wilk’s lambda, p=0.276; 100 pA current pulse, Wilk’s lambda, p=0.340) (n=5).
Figure 2—figure supplement 6
Tertiapin-Q did not block Ca2+-dependent rundown of noradrenaline (NA)-induced GIRK currents.
(A) Upper panel, Voltage command pulse. Lower panel, Superimposed current traces obtained under control condition (1), after application of 100 μM NA for 5 min (2), after application of NA and 200 nM tertiapin-Q (Ter-Q) (3), and after application of NA following washout of Ter-Q (4). (B) The I-V relationship of NA-induced GIRK-I obtained by subtraction of currents recorded under control condition from those recorded after application of NA for 5 min (red trace, 2–1), that obtained by subtraction of the control current from those recorded after application of Ter-Q in the presence of NA (blue trace, 3–1), and that obtained in the presence of NA by subtraction of currents recorded after application of Ter-Q from those recorded after washout of Ter-Q (green trace, 4–3). (C, D) Pooled data showing that Ter-Q reversibly decreased the amplitudes of inward components at –130 mV and those of outward components at –60 mV at respective conditions (2–1, 3–1, and 4–3) (n=6). Inward component, one-way RM ANOVA, *p<0.001; outward component, one-way RM ANOVA, *p<0.001. (E) Upper panel, Voltage command pulse. Lower panel, Superimposed current traces obtained under control condition (1), after application of NA for 5 min (2), and after application of NA and Ter-Q for 5 min (3). (F) Upper panel, A combined command pulse applied every minute; five trains of 20 positive pulses (5 ms duration to 0 mV at 20 Hz) at an inter-train interval of 2 s in the presence of extracellular 30 mM TEA, NA, Ter-Q, and intracellular 0.2 mM EGTA, which were followed by the ramp pulse after an interval of 19 s. Lower panel, Superimposed current traces obtained before and after 20 times application of positive pulse trains in the presence of NA and Ter-Q (3 and 4, respectively) and after washout of Ter-Q but still in the presence of NA (5). (G) The I-V relationship of NA-induced GIRK-I obtained by subtraction of currents recorded under the control condition from those recorded after application of NA for 5 min (red trace, 2–1), that obtained by subtraction of the control current from those recorded after application of NA and Ter-Q (blue trace, 3–1), and that obtained by subtraction of the currents recorded after 20 times application of positive pulse trains in the presence of NA and Ter-Q from those recorded 10 min after washout of Ter-Q but still in the presence of NA (green trace, 5–4). (H) Pooled data showing no protective effects of Ter-Q on the rundown of GIRK-I; amplitudes of inward components at –130 mV and those of outward components at –60 mV at respective conditions (2–1, 3–1, and 5–4) (n=7). Inward component, one-way RM ANOVA, *p<0.001; outward component, one-way RM ANOVA, *p<0.001. (I–L) Sample traces of spike trains induced in an LC neuron evoked by current pulses at 75 and 100 pA under control conditions and after washout of Ter-Q after the rundown protocol in Ter-Q as shown in E–H. Note the abolishment of spike-frequency adaptation and reduction of pulse-AHP after rundown of GIRK currents. (M) Plotting of the cumulative spike numbers vs the elapsed time during current pulses every 50 ms obtained under control condition (blue circles, 75 pA; pink squares, 100 pA) and those obtained after rundown of GIRK currents (green circles, 75 pA; red squares, 100 pA) (n=6). 75 pA current pulse, two-way RM ANOVA, *p<0.001; 100 pA current pulse, two-way RM ANOVA, *p<0.001. (N) Plotting of the saturation level (a) vs the half saturation constant (b), which were measured by curve fitting to the data points in M. The values of a and b, which were measured by curve fitting to the data points obtained after rundown of GIRK currents (green circles, 75 pA; red squares, 100 pA) were significantly larger than those obtained under control condition (pink circles, 75 pA; blue squares, 100 pA) (75 pA current pulse, paired t-test, a and b, ‡p=0.015 and ‡p=0.010, respectively; 100 pA current pulse, paired t-test, a and b, ‡p=0.004 and ‡p<0.001, respectively), and there was a significant difference in the relationship between a and b (75 pA current pulse, Wilk’s lambda: §p=0.020; 100 pA current pulse, Wilk’s lambda: §p=0.010) (n=6).
Figure 3 with 1 supplement
Restraint stress (RS) reduces expression of α2A-ARs in locus coeruleus (LC) neurons.
(A, B) Confocal images of LC neurons showing immunoreactivities for TH and α2A-ARs, together with a merged one in non-stress and 3-day RS mice. Arrowheads indicate the membrane regions of TH-positive neurons, along which α2A-ARs were differentially expressed between the control and the RS mice. Asterisks indicate LC neurons. (C) Western blotting analyses showing the expression of α2A-ARs in non-stress and 3-day RS mice (n=6 and 7, respectively). Unpaired t-test, †p<0.001. (D, E) Confocal images of LC neurons in non-stress and RS mice. Upper panels from left to right showing the respective immunoreactivity for TH, α2A-ARs (with rabbit anti-α2A-AR) and Na+-K+-pump, and an enlarged image of the region enclosed with a rectangle (arrowheads) in its immediate left panel. Lower panels from left to right showing the merged image of TH and α2A-ARs, that of TH, α2A-ARs, and Na+-K+-pump, that of α2A-ARs and Na+-K+-pump, and an enlarged image of the region enclosed with a rectangle in its immediate left panel. (F, G) Western blotting analyses showing expressions of α2A-ARs and Na+-K+-pump in membrane fraction and those of α2A-ARs and β-actin in cytosol fraction in non-stress and 5-day RS mice (n=3 and 3 samples, respectively). Each sample represents the analysis result in the LC tissues obtained from two to three mice. Membrane; unpaired t-test, †p=0.016. Cytosol; unpaired t-test, †p=0.083.
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Figure 3—source data 1
Original files for the western blot analysis in Figure 3C, F, and G.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig3-data1-v1.zip
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Figure 3—source data 2
PDF containing the original blots in Figure 3C, F, and G with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig3-data2-v1.pdf
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Figure 3—source data 3
Data used for graphs presented in Figure 3C, F, and G.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Reliability of anti-α2A-AR antibody revealed by differential expression of α2A ARs between locus coeruleus (LC) and MTN.
(A–C) Confocal images of LC and MTN neurons showing the immunoreactivities for TH and α2A ARs, together with a merged one. Filled arrowheads indicate MTN neurons, in which TH was not expressed, but α2A ARs were more extensively expressed than in LC neurons. Open arrowheads indicate oval-shaped neurons. Open double arrowheads indicate rhombus-like shaped neurons. Differential staining of α2A-ARs between MTN and LC neurons verifies the reliability of the antibody against α2A-ARs.
Figure 4
Restraint stress (RS) reduces noradrenaline (NA)-induced GIRK-I in locus coeruleus (LC) neurons.
(A–E) Representative traces of NA-induced GIRK-I obtained from LC neurons in non-stress and 1-day, 2 day, 3-day, and 5-day RS mice. (F, G) Amplitudes of inward and outward components of NA-induced GIRK-I obtained from LC neurons in non-stress and 1-day, 2-day, 3-day, and 5-day RS mice (n=8, 7, 5, 6, and 8, respectively) decreased with the increase in the period of RS, in a way that can be described by a saturation function (red interrupted lines). The saturation level (a+b) and the half saturation constant (c) were determined by fitting the saturation function, defined as y=a + (b* x)/(c+x), to the data points. The values of a, b, and c for the inward component of GIRK-I were –151.1, 108, and 0.9, respectively, and those for the outward component of GIRK-I were 81.1, –67, and 1.6, respectively. Inward component: one-way ANOVA, p<0.001, post hoc Fisher’s PLSD, 1 day; #p<0.001 vs non-stress and *p <0.05 vs 3 day and 5 day, 2 day; #p<0.001 vs non-stress, 3 day; #p<0.001 vs non-stress, 5 day; #p<0.001 vs non-stress. Outward component: one-way ANOVA, p<0.001, post hoc Fisher’s PLSD, 1 day; p=0.004 vs non-stress and *p<0.05 vs 3 day and 5 day, 2 day; p=0.015 vs non-stress, 3 day; p<0.001 vs non-stress, 5 day; p<0.001 vs non-stress. (H) Relative expressions of α2A and α2C mRNAs, normalized to GAPDH in LC neurons (n=5). Paired t-test, ‡p=0.014. (I) Relative expressions of GIRK1, GIRK2, and GIRK3 mRNAs, normalized to GAPDH in LC neurons (n=5). One-way RM ANOVA, p<0.001, post hoc Fisher’s PLSD; *p=0.008 for GIRK1 vs GIRK2, *p<0.001 for GIRK1 vs GIRK3, *p=0.008 for GIRK2 vs GIRK3. (J, K) Relative expressions of α2A and GIRK1 mRNAs, respectively, normalized to GAPDH in LC neurons in non-stress mice (n=8) and 3-day RS mice (n=6). α2A-AR: unpaired t-test, †p=0.020; GIRK1: unpaired t-test, †p=0.013. (L) Normalized relative expressions of GIRK2 mRNA in LC neurons in non-stress mice (n=8) and 3-day RS mice (n=6), normalized to the ratio of the mean value of the relative expressions of GIRK1 mRNA to that of GIRK2 mRNA in LC neurons in non-stress mice (I). Unpaired t-test, †p=0.037.
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Figure 4—source data 1
Data used for graphs presented in Figure 4F–L.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Expressions of TH, monoamine oxidase A (MAO-A), dopamine-β-hydroxylase (DBH), asparagine endopeptidase (AEP), and tau proteins in locus coeruleus (LC) neurons.
(A) Western blotting of TH and MAO-A in LC neurons obtained from non-stress and 5-day RS mice. (B, C) Western blot analysis revealing no significant change in TH expression (n=10 samples) but a significant increase in MAO-A expression (n=6 samples) in 5-day RS group compared to non-stress group. Unpaired t-test, †p=0.029. (D) Western blotting of pro-AEP and active AEP in LC neurons obtained from non-stress and 5-day RS mice. (E, F) Western blot analysis revealing significant increases in pro-AEP and active AEP (n=6 and n=6 samples, respectively) in 5-day RS group compared to non-stress group. Unpaired t-test, †p=0.034 (E) and †p=0.047 (F). (G) Western blotting of tau and tau N368 fragment in LC neurons obtained from non-stress and 5-day RS mice. Unpaired t-test, †p=0.025. (H, I) Western blot analyses revealing a significant increase in tau and tau N368 fragment in LC neurons (n=8 and n=6, respectively) in 5-day RS group compared to non-stress group. Each sample represents the analysis result in the LC tissues obtained from two to three mice. Unpaired t-test, †p<0.05.
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Figure 5—source data 1
Original files for the western blot analysis in Figure 5A, D, and G.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig5-data1-v1.zip
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Figure 5—source data 2
PDF containing the original blots in Figure 5A, D, and G with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig5-data2-v1.pdf
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Figure 5—source data 3
Data used for graphs presented in Figure 5B, C, E, F, H, and I.
- https://cdn.elifesciences.org/articles/106362/elife-106362-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Immunoreactivities of TH, monoamine oxidase A (MAO-A), dopamine-β-hydroxylase (DBH), and asparagine endopeptidase (AEP) in locus coeruleus (LC) neurons.
(A) Confocal images of neurons in LC at a low magnification showing the immunoreactivities for TH and MAO-A, together with a merged one. Restraint stress (RS) appeared to cause slight increases in the expressions of TH and MAO-A. Brightness of background in sample image obtained from the control was corrected to be equal to that obtained from the RS. (B) Confocal images of LC neurons at a high magnification showing the immunoreactivities for DBH and AEP, together with a merged one. RS caused increases in the expression of AEP.
Figure 6 with 1 supplement
Presumed cellular mechanisms for the degeneration of locus coeruleus (LC) by stress.
(A, B) Differential free concentrations of noradrenaline (NA) to be metabolized by monoamine oxidase A (MAO-A) into 3,4-dihydroxyphenyl-glycolaldehyde (DOPEGAL) between control and stress conditions: Under the control condition (A), NA in the cytosol is mostly taken up into the cytoplasmic vesicles by VMAT2 (thick arrow 3), rather than being directly metabolized by MAO-A into DOPEGAL (thin interrupted arrow). Vesicular NA is released from cell bodies as autocrine following [Ca2+]i increases caused by action potentials, and the released NA activates α2A-AR-coupled GIRK channels, causing autoinhibition. Subsequently, the autocrine-released NA is slowly taken up into the cytosol of LC neurons by NAT after dissociation from α2A-ARs. Under the stress condition (B), an activation of CRF receptors in LC neurons by stress inhibits leak K+ channels and increases firing activities in LC neurons, subsequently causing a larger [Ca2+]i increase together with Ca2+-induced Ca2+ release (CICR). Impairment of autoinhibition due to Ca2+-dependent internalization of α2A-ARs-coupled GIRK channels leads to the persistent excitation in LC neurons, which enhances autocrine release of NA (thick arrow 1). Subsequently, the excessively autocrine-released NA is taken up directly and rapidly by NAT into the cytosol without binding to α2A-ARs (thick arrow 2). Such a facilitation of reuptake of NA by NAT would increase active NA storage into cytoplasmic vesicles by VMAT2 (thick arrow 3), while the rate of NA leakage from cytoplasmic vesicles would also increase (arrow 4) due to a dynamic equilibrium in cytoplasmic vesicles between active NA storage into cytoplasmic vesicles and passive NA leakage from cytoplasmic vesicles. Subsequently, such an increase in the rate of NA leakage would result in an increase in an MAO-A metabolite, DOPEGAL, and AEP, leading to a production of cleaved tau N368 fragment and an impairment of learning/memory.
Figure 6—figure supplement 1
Restraint stress (RS)-induced impairment of spatial memory.
(A) Left panel: Y maze to explore two arms, the start (S) and familial (F) arms with the novel (N) arm closed, for 15 min of training. Right panel: Y maze to explore three arms (the S, F, and N arms) for a 5 min test session. (B) Time spent in S, F, and N arms in non-stress mice and in 1-day, 3-day, and 5-day RS mice. Two-way RM ANOVA, #p<0.05. (C) Distance traveled in S, F, and N arms in non-stress mice and in 1-day, 3-day, and 5-day RS mice. Two-way RM ANOVA, #p<0.05.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL6 | Japan SLC | RRID:IMSR_JAX:000664 | Female and male |
| Antibody | Mouse anti-TH (monoclonal) | Santa Cruz Biotechnology | Cat# sc-25269, RRID:AB_628422 | IHC (1:500), |
| Antibody | Rabbit anti-sodium potassium ATPase (monoclonal) | Abcam | Cat# ab76020, RRID:AB_1310695 | IHC (1:1000), WB (1:1000) |
| Antibody | Goat anti-α2A AR (monoclonal) | Abcam | Cat# ab45871, RRID:AB_722745 | IHC (1:200) |
| Antibody | Rabbit anti-α2A AR (monoclonal) | Neuromics | Cat# RA14110, RRID:AB_2225052 | IHC (1:500) |
| Antibody | Cy5 donkey anti-mouse IgG (polyclonal) | Vector Laboratories | Cat# cy-2500, RRID:AB_11099905 | IHC (1:500) |
| Antibody | FITC donkey anti-rabbit IgG (polyclonal) | Jackson ImmunoResearch | Cat# 711-095-152, RRID:AB_2315776 | IHC (1:500) |
| Antibody | Cy3 donkey anti-goat IgG (polyclonal) | Jackson ImmunoResearch | Cat# 711-095-152, RRID:AB_2307351 | IHC (1:500) |
| Antibody | Rabbit anti-MAO-A (monoclonal) | Abcam | Cat# ab126751, RRID:AB_11129867 | IHC (1:2000) |
| Antibody | FITC donkey anti-mouse IgG (polyclonal) | Jackson ImmunoResearch | Cat# 715-545-150, RRID:AB_2340846 | IHC (1:500) |
| Antibody | Cy3 donkey anti-rabbit IgG (polyclonal) | Jackson ImmunoResearch | Cat# 711-165-152, RRID:AB_2307443 | IHC (1:2000) |
| Antibody | Mouse anti-legumain (monoclonal) | Santa Cruz Biotechnology | Cat# sc-133234, RRID:AB_213501 | IHC (1:200) |
| Antibody | Rabbit anti-DBH (polyclonal) | ImmunoStar | Cat# 22806, RRID:AB_572229 | IHC (1:500) |
| Antibody | Anti-rabbit IgG, HRP-linked (polyclonal) | Cell Signaling Technology | Cat# 7074, RRID:AB_2099233 | WB (1:5000) |
| Antibody | Rabbit anti-α2A AR (polyclonal) | alomone | Cat# AAR-020, RRID:AB_10687546 | WB (1:500) |
| Antibody | Rabbit anti-GAPDH (polyclonal) | Abfrontier | Cat# LF-PA0018, RRID:AB_161673 | WB (1:1000) |
| Antibody | Mouse anti-beta actin (monoclonal) | MilliporeSigma | Cat# A5441, RRID:AB_476744 | WB (1:2000) |
| Antibody | Rabbit anti-TH (polyclonal) | GeneTex | Cat# GTX113016 RRID:AB_1952230 | WB (1:1600) |
| Antibody | Rabbit anti-MAO-A (monoclonal) | Abcam | Cat# ab126751, RRID:AB_11129867 | WB (1:6400) |
| Antibody | Mouse anti-legumain (monoclonal) | Santa Cruz | Cat# sc-133234, RRID:AB_213501 | WB (1:400) |
| Antibody | Mouse anti-TAU-5 (monoclonal) | Santa Cruz Biotechnology | Cat# SC-58860, RRID:AB_785931 | WB (1:1500) |
| Antibody | Mouse anti-beta actin (monoclonal) | Proteintec, Rosemont | Cat# 66009–1-Ig, RRID:AB_2687938 | WB (1:1280) |
| Antibody | Rabbit anti-tau AEP-cleaved (N368) (monoclonal) | MilliporeSigma | Cat# ABN1703 | WB (1:5000) |
| Chemical Compound, Drug | Atipamezole | MilliporeSigma | A9611 | Electrophysiology |
| Chemical Compound, Drug | Barbadin | MilliporeSigma | SML3127 | Electrophysiology |
| Chemical Compound, Drug | Tertiapin-Q | Tocris Bioscience | 1316 | Electrophysiology |
| Software; Algorithm | Axograph X | Axograph | RRID:SCR_014284 | Electrophysiology |
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Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity
eLife 14:RP106362.
https://doi.org/10.7554/eLife.106362.4
