TRPA1 Channels Are Regulators of Astrocyte Basal Calcium Levels and Long-Term Potentiation via Constitutive d-Serine Release

Functional evidence that astrocyte branches express TRPA1 channels that mediate spotty Ca²⁺ signals. A, Representative traces of spontaneous spotty Ca²⁺ signals recorded from branches of astrocytes expressing Lck-GCaMP3 (Shigetomi et al., 2013). B, As in A, but in the presence of HC 030031 (80 μm). C, Cumulative probability plots for Ca²⁺ signal peak heights (dF/F), duration (T_0.5), and frequency. The cumulative probability plots are shifted to smaller amplitudes and lower frequencies in the presence of HC 030031. Note the two distributions are significantly different on the basis of a Kolmogorov–Smirnov test. Average data are presented in the text. D, Scatter plot shows how the number of spotty Ca²⁺ events measured per astrocyte with Lck-GCaMP3 decreases in the presence of HC 030031. E, Plots the number of spotty Ca²⁺ events measured as a function of distance from the soma. Note there is a consistent decrease in the number of Ca²⁺ events measured in the presence of HC 030031 throughout astrocyte territories, implying that the significant decrease shown in E does not occur at any specific distance from the soma. Rather, the HC 030031-sensitive Ca²⁺ events appear to be drawn from entire astrocytes.

Blocking TRPA1 channels reduces astrocyte basal Ca²⁺ levels. A, Graphs plot dF/F over time from astrocytes loaded with Fluo-4 in hippocampal slices from adult mice. Application of HC 030031 (80 μm) reduced the baseline fluorescence of astrocyte somata. Note that in these traces, some of the peaky Ca²⁺ transients have been clipped to more clearly show the drop in baseline Ca²⁺. B, Cumulative probability plot of dF/F evoked by HC 030031 applications to astrocytes in acute hippocampal slices from WT and TRPA1^−/− mice. Note the two distributions are significantly different on the basis of a Kolmogorov–Smirnov analysis. The bar graph summarizes the finding that HC 030031 reduces resting Ca²⁺ levels in astrocytes from WT hippocampal slices.

Blocking TRPA1 channels reduces LTP. A, fEPSP traces were recorded from the stratum radiatum before and after high-frequency stimulation (HFS; 2 × 100 Hz, intertrain interval 10 s). Traces are shown for several experiments at various time points before and after HFS. B, HFS-induced robust LTP in control experiments (n = 7). HC 030031 (80 μm, n = 6) suppressed LTP in a manner that was rescued by 10 μm d-serine (n = 6). C, d-Serine (10 μm) did not affect LTP (n = 6, p = 0.876). D, The “glycine site” antagonist DCKA (10 μm; n = 6) suppressed LTP E, AITC (100 μm) did not affect LTP (n = 7). F, G, HC 030031 did not affect AMPAR-mediated fEPSPs (F) or presynaptic fiber volleys (G; n = 7).

Reduced LTP in TRPA1^−/− mice. A, Traces for fEPSPs recorded from hippocampal slices harvested from WT and TRPA1^−/− mice. The relationship between fEPSP slope and presynaptic fiber volley for WT and TRPA1^−/− mice is also shown. The inset shows an average fEPSP. B, LTP was suppressed in TRPA1^−/− mice relative to WT controls. C, Suppressed LTP in TRPA1^−/− mice was restored by bath applications of 10 μm d-serine. D, Summary bar graph showing the degree of LTP in control recordings and under the various conditions indicated. HC 030031 (80 μm) failed to suppress LTP in TRPA1^−/− mice and LTP was equivalent in IP3R2^−/− and WT littermate mice.

LTP was not altered when GAT-3 was blocked or when GABA was applied in the bath. A–C, LTP was normal and indiscernible from WT controls in slices treated with (A) the GAT-3 blocker β-alanine (100 μm), (B) GABA (10 μm), and (C) both β-alanine and GABA together. D, Summary bar graph of the findings shown in A–C.

Extracellular d-serine levels are regulated by TRPA1 channel function. A, Photograph of a hippocampal slice from a 2-month-old mouse with biosensor electrodes placed in the stratum radiatum. This diagram illustrates how the biosensors work. B, Representative traces and average data for biosensor electrode calibration. C, Representative traces showing measurement of [d-serine] with biosensor electrodes when the biosensor was positioned above the slice, moved into the slice, and then withdrawn from the slice. This protocol results in two measurements of [d-serine] indicated in blue and red text. D, As in C, but for recordings from a TRPA1^−/− mouse. E, The bar graph summarizes the peak [d-serine] for three experimental conditions. There were no significant differences. F, The bar graph summarizes the steady-state [d-serine] within the slice for three experimental conditions. Note that [d-serine] levels are reduced in WT slices treated with HC 030031 and in slices from TRPA1^−/− mice.