Sustained Hypoxia Alters nTS Glutamatergic Signaling and Expression and Function of Excitatory Amino Acid Transporters

Abstract

Glutamate is the major excitatory neurotransmitter in the nucleus tractus solitarii (nTS) and mediates chemoreflex function during periods of low oxygen (i.e. hypoxia). We have previously shown that nTS excitatory amino acid transporters (EAATs), specifically EAAT-2, located on glia modulate neuronal activity, cardiorespiratory and chemoreflex function under normal conditions via its tonic uptake of extracellular glutamate. Chronic sustained hypoxia (SH) elevates nTS synaptic transmission and chemoreflex function. The goal of this study was to determine the extent to which glial EAAT-2 contributes to SH-induced nTS synaptic alterations. To do so, male Sprague-Dawley rats (4-7 weeks) were exposed to either 1, 3, or 7 days of SH (10% O₂, 24 h/day) and compared to normoxic controls (21% O₂, 24 h/day, i.e., 0 days SH). After which, the nTS was harvested for patch clamp electrophysiology, quantitative real-time PCR, immunohistochemistry and immunoblots. SH induced time- and parameter-dependent increases in excitatory postsynaptic currents (EPSCs). TS-evoked EPSC amplitude increased after 1D SH which returned at 3D and 7D SH. Spontaneous EPSC frequency increased only after 3D SH, which returned to normoxic levels at 7D SH. EPSC enhancement occurred primarily by presynaptic mechanisms. Inhibition of EAAT-2 with dihydrokainate (DHK, 300 µM) did not alter EPSCs following 1D SH but induced depolarizing inward currents (I_hold). After 3D SH, DHK decreased TS-EPSC amplitude yet its resulting I_hold was eliminated. EAAT-2 mRNA and protein increased after 3D and 7D SH, respectively. These data suggest that SH alters the expression and function of EAAT-2 which may have a neuroprotective effect.

Keywords: astroglia; autonomic nervous system; chemoreflex; glutamate; respiration; synaptic transmission.

Figure 1.. One day SH increased TS-EPSC amplitude.

A. Example of TS-EPSCs evoked following normoxia (0D SH, blue) and 1D SH (red). Note the increased amplitude after 1D SH. Cells were held at −60 mV and the TS was stimulated at arrow. B. Quantification of TS-EPSC amplitude following SH. Compared to 0D SH (n= 6 neurons), 1D SH (n=7 neurons) increased current amplitude (p = 0.023). Amplitude at 3D (n=8 neurons) and 7D (n=8 neurons) SH was comparable to normoxia. C. The inverse of coefficient of variation (1/CV²) was also increased after 1D of SH vs. normoxic-exposed nTS neurons (p = 0.0034). D. TS-EPSC failure rate decreased after 1 and 3D SH. Rate determined from 20 stimuli. E. TS-EPSC amplitude evoked at 20Hz stimulation (10 events). Note the increase in the first TS-EPSC amplitude after 1D SH, and the decrease after 7D SH (two-way RM ANOVA with Fisher LSD). Following the first event, the amplitude of currents decreased and were comparable among groups. F. Paired pulse ratio (PPR, amplitude ratio of the second-to-first TS-EPSC) demonstrates reduction at 1 and 3D SH. G. Synaptic throughput (sum of 10 events shown in panel E) was not significantly different between durations of SH (one-way ANOVA). For box-and-whisker plots, median indicated by solid line, mean by “+”, quartiles (25 and 75%) by boxes, and the range by the whiskers. For all panels except E: *, p < 0.05 vs. Normoxia (0D SH). Panel E: *, p < 0.05 1D vs. 0D SH; †, p < 0.05 3D vs. 0D SH, ‡, p < 0.05 7D vs. 0D SH.

Figure 2.. Three day SH enhances spontaneous EPSCs.

A. Example of spontaneous currents after 0D (top, blue) and 3D (bottom, green) SH. Cells were held at −60 mV. Note the increase sEPSC frequency after 3D SH. B, C. sEPSC frequency was elevated after 3D SH (p = 0.025), but amplitude was not altered after SH (C). Number of neurons (n) = 6 (0D), 7 (1D), 8 (3D) and 8 (3D). Panels B, C: *, p < 0.05 vs. Normoxia (0D SH), One-way ANOVA with LSD.

Figure 3.. Block of EAAT-2 inhibits TS-EPSCs after 3D SH.

A. Effect of EAAT-2 block with DHK (300 μM). DHK did not appreciably alter TS-EPSC amplitude at Normoxia (0D) or after 1 and 7D SH. By contrast, DHK significantly decreased TS-EPSC amplitude from its aCSF baseline (#, p= 0.041, paired t-test). An example of the reduction in TS-EPCS amplitude by DHK is shown in the inset; solid green = aCSF, dashed grey = DHK. Average of 20 events. The magnitude of amplitude change by DHK at 3D SH did not reach significance compared to normoxia (p = 0.06, one-way ANOVA). B. Paired pulse ratio (PPR) during aCSF baseline, DHK, and its washout following 3D SH. C. Magnitude of change in synaptic throughput (as Fig 1E). DHK did not alter throughput. In panels A & C dashed line represents aCSF baseline to which events after DHK within each group was tested. # p < 0.05 DHK vs. its aCSF baseline, paired t-test.

Figure 4.. sEPSCs following SH are not altered by EAAT-2 block.

A, B. The magnitude of response to EAAT-2 block with DHK (300 μM) on sEPSC frequency (A) and amplitude (B) relative to aCSF baseline. DHK did not affect sEPSC frequency or amplitude after SH. In panels A & B dashed line represents aCSF baseline to which events after DHK within each group was tested.

Figure 5.. Depolarizing currents following DHK are blunted following 3D SH.

A. Representative traces of spontaneous activity showing the inward current produced in response to DHK after 0, 3 and 7D SH. Cells were voltage clamped at −60 mV. Note the downward deflection indicative of a depolarizing shift in I_hold seen after 0 and 7D SH is reduced with 3D SH. B. Group data showing the total change in holding current across SH. DHK induced depolarization after 0, 1 and 7D SH. Conversely, 3D SH did not change I_hold. p = 0.06 vs. Normoxia (0D SH), One-way ANOVA with LSD; # p < 0.05 DHK vs. aCSF baseline, paired t-test.

Figure 6.. SH alters EAAT expression.

A. Immunoreactivity (top, greyscale) of normoxic nTS demonstrating differential EAAT-1 and EAAT-2 expression. Note that EAAT-2 protein is more highly expressed in nTS compared to EAAT-1. Scale, 100 μm. AP, area postrema; CC, central canal; DmnX, dorsal motor nucleus of the vagus. Bottom, co-labeling of EAAT-1 or EAAT-2 (green, arrow) with the astrocyte markers s100B (red) and GFAP (blue). Merged image is also shown. Scale bar, 10 μm. B. Relative mRNA expression of EAAT-2 (E2) to EAAT-1 (E1) in nTS in normoxia (0D SH) and following 1, 3 and 7 SH. Note EAAT-2 is expressed more than EAAT-1 at 0, 1 and 3 D SH. After 7D SH EAAT-1 and −2 are expressed at relatively similar amounts. * p < 0.05, paired t-test. C. EAAT-1 mRNA (left) expression at 1, 3 and 7D SH relative to normoxia (N, blue line). EAAT-1 mRNA does not significantly change compared to its normoxic controls. By contrast, EAAT-2 mRNA (right) expression at 3 D SH is increased relative to normoxia (N, blue line). * p < 0.05 t-test. For RT-PCR “n” refers to the number of rats, 0 vs. 1D SH, n=5 each; 0 vs. 3D SH, n=3 each, 0 vs. 7D SH, n=4 each. D. Representative example of 0D and 7D immunoblot (IB) for EAAT-2 with bands at μ 60 kDa. Note the increase in EAAT-2 nTS expression after 7D SH. E. EAAT-1 (left) and EAAT-2 (right) protein expression at 1, 3 and 7D SH relative to its normoxic control (N, blue line). Note the 7D SH increased EAAT-1 &2 protein in nTS. For IB analysis “n” refers to the number of rats: EAAT-1; 0 vs. 1D SH, n=6 & 7; 0 vs. 3D SH, n=4 & 6, 0 vs. 7D SH, n=5 each. EAAT-2; 0 vs. 1D SH, n=6 each, 0 vs. 3D SH, n=4 & 6, 0 vs. 7D SH, n=3 & 5 each. For C&D, dashed line represents normoxic mRNA or protein within each group to which 1, 3 or 7D SH was tested. *, p < 0.05 vs. their normoxic (0D SH) controls.