Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway

Abstract

The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

Figure 1. Expression of GCaMP3 in astrocytes

A. The cartoon illustrates the procedure to inject AAV2/5 capable of expressing GCaMP3 in s.l. astrocytes of P56 mice. The right hand image shows fluorescence signal for GCaMP3 detected by IHC in the CA3 region. B. GCaMP3 and GFAP expression in an astrocyte from the s.l. region. The GCaMP3 expressing astrocyte was GFAP positive and 85 ± 2 % of all GFAP positive astrocytes in the s.l. expressed GCaMP3 (n = 4 mice). C. Maximal projection territory area for GFAP, GCaMP3, Lck-GCaMP3 and Lck-GFP. D-E. GFAP maximal projection territory areas (D) and intensity (E) for astrocytes. F-G. Traces and average data for astrocyte current-voltage relationships (-120 to +40 mV) from control mice or those microinjected with GCaMP3. H. The image shows a representative astrocyte with circles drawn radially (5 μm spacing). Such circles were used to measure the intensity of GCaMP3 expression at increasing distances from the center of the soma (in the graph). The highest intensity was in the soma, which has the largest volume and intensity fell with distance. Average data are shown as mean ± S.EM.

Figure 2. Properties of Ca²⁺ signals in s.l. astrocytes

A. Image of a single s.l astrocyte expressing GCaMP3 with 11 ROIs indicated (Supp movie 1). B. Distributions of astrocyte Ca²⁺ signal properties (blue bars are for branches and the red bars are for somata). C. Traces for 11 ROIs from Fig 2A. D. Plots Ca²⁺ signal amplitude (left axis) and number (right axis) in 5 μm bins as a function of distance from the soma. The number of Ca²⁺ signals in 5 μm bins is shown by the black line. E. Image of an s.l. astrocyte with a yellow line indicating the approximate position of the region chosen for 200 Hz line scan imaging. F. Image of line scan data for the line shown in E, with an expanded region corresponding to a branchlet shown below. In these images, the x-axis is time and the y-axis is distance along the scanned line. G. The trace for the selected region shown in F. H. Distributions showing Ca²⁺ signal half widths from line scan experiments for somatic and branchlet regions. I-J. Bar graphs summarize Ca²⁺ signal properties such as dF/F and half width for line scan data. Average data are shown as mean ± S.EM.

Figure 3. Stratum lucidum region astrocyte spontaneous Ca²⁺ signals are not affected when action potentials or glutamate receptors are blocked, but are markedly reduced in number when intracellular Ca²⁺ stores are depleted or disrupted (IP3R2 KO mice)

A. Four superimposed traces for branchlets under control conditions and then in the presence of 0.5 μM TTX, which had no effect. B-D. As in A, but for experiments when the brain slices were treated with antagonists of mGluR2/3 (10 μM LY341495), mGluR5 (50 μM MPEP) and NMDA receptors (50 μM APV). E. As in A, but for slices treated with cyclopiazonic acid (20 μM CPA) to deplete intracellular Ca²⁺ stores. F. Representative traces for spontaneous Ca²⁺ signals recorded from IP3R2 KO mice and their WT littermates. The average data are shown in Supp Fig. 1 & 2

Figure 4. Activation of P2X2-YC channels in the mossy fiber pathway of SPRAE mice increases glutamate release onto CA3 pyramidal neurons

A. P2X2-YC expression (green), from anti-GFP primary antibody and Alexa 488-conjugated secondary antibody, in the mossy fiber pathway. B. Five 1 s traces superimposed showing mEPSCs recorded from a CA3 pyramidal neuron from a WT mouse before, during and after 100 μM ATPγS application. C. As in B, but for recordings from SPRAE^hom mice. D. Quantification of experiments such as those shown in B and C. The y-axis plots the approximate number of quanta in 2 s bins (Q_mini was measured from mEPSCs before the application of ATPγS, Q_noise was measured from silent periods and Q_total was measured in 2 s bins). E. Traces and graphs show an exemplar neuron, showing an increase in frequency in the presence of ATPγS, but no increase in amplitude. Measuring all mEPSCs in 7/11 cells showed no increase in mEPSC amplitude (F), whereas frequency was increased (G). Experiments for CA3 interneurons are reported in Supp Fig. 5. Average data are shown as mean ± S.EM.

Figure 5. Stratum lucidum region astrocyte Ca²⁺ responses during glutamate release from mossy fibers in SPRAE^hom mice

A. IHC for P2X2-YC in the mossy fibers (green) along with staining for astrocytes using GFAP (red); higher magnification views shown in panel B. C. Three representative traces superimposed for ROIs from astrocyte branches from a WT mouse before, during and after 100 μM ATPγS applications. D. As in C, but for astrocytes located in the s.l. region and imaged from SPRAE^hom mice. E. Properties of astrocyte Ca²⁺ signals before and during ATPγS applications in WT mice. F. Properties of astrocyte Ca²⁺ signals before and during ATPγS applications in SPRAE mice. However, there was no significant difference in the duration of the spontaneous Ca²⁺ transients in WT and SPRAE mice before ATPγS. For somata, in WT mice their T_0.5 was 6.2 ± 0.9 s and in SPRAE mice the T_0.5 was 4.3 ± 1.1 s (n = 27 and 15; p = 0.16 with an unpaired t test). For branches, in WT mice their T_0.5 was 3.5 ± 0.2 s and in SPRAE mice the T_0.5 was 3.4 ± 0.4 s (n = 213 and 246; p = 0.83 with an unpaired t test). G. S.l. region astrocyte cell surface glutamate imaging with iGluSnFR in WT and SPRAE^hom mice. Average data are shown as mean ± S.EM.

Figure 6. EFS evokes Ca²⁺ signaling in astrocytes during bursts of stimuli

A. Traces from astrocyte somata located in the s.l. region during local EFS of the mossy fiber pathway. The red traces are averages of the individual black traces. Signals were seen only for greater than two stimuli. B. As in A, but for traces from branch ROIs; blue is an average of the individual black traces. The numbers above each set of traces indicate the success rate for each stimulus. C. Summary graph from experiments such as those shown in A and B. D. The graph shows the increase in cell surface iGluSnFR fluorescence as a function of the number of local stimuli delivered to the mossy fiber terminals with EFS. E. Plots the branchlet signals for Ca²⁺ and iGluSnFR: note the two plots do not overlap. In this plot, a indicates a threshold shift, b indicates that the relationship between Ca²⁺ signals in astrocyte branches and the number of stimuli was approximately linear. F. Plots the area of the astrocyte Ca²⁺ signals as a function of EFS stimuli and in relation to the territory of an s.l. astrocyte (Fig. 1C). G. As in F, but for iGluSnFR signals. H. Image of an s.l. astrocyte expressing GCaMP3 taken at the peak of 15 stimuli, showing Ca²⁺ elevation in most of its territory. Average data are shown as mean ± S.EM.

Figure 7. Evaluations of astrocyte responses to 1 and 15 stimuli in IP3R2 KO mice and with various GECIs

A-D. Ca²⁺ imaging traces for somata and branches before, during and after EFS with 1 or 15 stimuli under the various conditions indicated. In the case of IP3R2 KO mice, comparative measurements were made with WT littermates (B). E. Bar graphs summarize average data from the experiments shown in A-D. Average data are shown as mean ± S.EM.

Figure 8. Electrical field stimulation evokes Ca²⁺ signals in astrocytes that are mediated by glutamate and GABA

A.Traces show the protocol: two local EFS stimuli were delivered to the mossy fiber terminals 8 min apart. B. Application of TTX before the second EFS stimulation abolished the astrocyte Ca²⁺ signals. C. As in B, but for applications of the mGluR2/3 receptor antagonist LY341495. D. As in B, but for applications of the mGluR2/3 and GABA_B receptor antagonists together (LY341495 and CGP52432). E. Summary bar graph for astrocyte branches from experiments such as those shown in A-D, and for additional evaluations as indicated. The differences were analyzed using a Dunnett's ANOVA test, whereby all the treatments were compared to the control 2^nd stimulation response. F. Representative traces (black) and average data (red) for agonist evoked Ca²⁺ signals in astrocyte branches. The area under the curve is shown in blue. G. Summary data for experiments such as those in F. Average data are shown as mean ± S.EM.

Figure 9. Glutamate clearance regulates EFS-evoked and spontaneous Ca²⁺ signals in astrocytes

A. Traces for EFS-evoked astrocyte Ca²⁺ signals in branches before and during applications of TBOA (0.3 μM). The bar graphs to the right show average data, indicating that TBOA increased and prolonged EFS-evoked signals. B. Traces for spontaneous Ca²⁺ signals in astrocyte branches before and during TBOA. C. As in B, but for iGluSnFR glutamate signals. Note in the presence of TBOA the baseline increases and iGluSnFR transients are observed. D-F. Quantification of experiments such as those shown in B. G-I. Bar graphs show quantification of experiments such as those shown in C. J-K. Images of the arrangement of cellular elements around boutons located in the s.r. (J) and s.l. (K). Postsynaptic spines (blue) form a larger, more complex synaptic complex with boutons (yellow) in the stratum lucidum. Red dots indicate location of a PSD. Astrocyte processes (green) are generally more distal from PSDs in stratum lucidum than in stratum radiatum. Neighboring axons not forming boutons also surround synapse in both areas (purple). Scale bars = 1 micron. L. Graph summarizes the shortest distances from a PSD to an astrocyte branchlet for the stratum radiatum and stratum lucidum. Average data are shown as mean ± S.EM.