Plasticity of astroglial networks in olfactory glomeruli

Abstract

Several recent findings have shown that neurons as well as astrocytes are organized into networks. Indeed, astrocytes are interconnected through connexin-formed gap junction channels allowing exchanges of ions and signaling molecules. The aim of this study is to characterize astrocyte network properties in mouse olfactory glomeruli where neuronal connectivity is highly ordered. Dye-coupling experiments performed in olfactory bulb acute slices (P16-P22) highlight a preferential communication between astrocytes within glomeruli and not between astrocytes in adjacent glomeruli. Such organization relies on the oriented morphology of glomerular astrocytes to the glomerulus center and the enriched expression of two astroglial connexins (Cx43 and Cx30) within the glomeruli. Glomerular astrocytes detect neuronal activity showing membrane potential fluctuations correlated with glomerular local field potentials. Accordingly, gap junctional coupling of glomerular networks is reduced when neuronal activity is silenced by TTX treatment or after early sensory deprivation. Such modulation is lost in Cx30 but not in Cx43 KO mice, indicating that Cx30-formed channels are the molecular targets of this activity-dependent modulation. Extracellular potassium is a key player in this neuroglial interaction, because (i) the inhibition of dye coupling observed in the presence of TTX or after sensory deprivation is restored by increasing [K(+)](e) and (ii) treatment with a K(ir) channel blocker inhibits dye spread between glomerular astrocytes. Together, these results demonstrate that extracellular potassium generated by neuronal activity modulates Cx30-mediated gap junctional communication between glomerular astrocytes, indicating that strong neuroglial interactions take place at this first relay of olfactory information processing.

Fig. 1.

Compartmentalized astrocyte morphology and connexin expression in the glomerular layer. (A) Typical morphology of glomerular astrocytes observed at P20 in hGFAP/EGFP mice. Glomerular boundaries (dotted line) are defined by a Nissl staining (blue). Amplification provided by anti-GFP antibodies reveals fine astrocytic processes that are mostly located within OG. (Scale bar: 25 μm.) (B and C) Cx30 (C; red) and Cx43 (B; green) immunoreactivity was studied in the GL at P20 on single confocal sections. (B) Example of Cx43 immunostaining. (C1 and C2) Example of Cx30 immunostaining with (C1) or without (C2) Nissl staining (blue). Nissl-defined OG borders are indicated by dotted lines in C1. (Scale bar: 30 μm.) (D) Quantitative analysis of mean fluorescence intensity (MFI) for Cx43 (green) and Cx30 (red) immunostainings at the OG border, performed on 109 and 67 OG, respectively (SEM are indicated by light colors surrounding curves) (SI Appendix, SI Methods). To combine data obtained from different experiments, results were expressed in Z scores. Horizontal gray lines above the graph correspond to significant difference between the two Cx levels of expression (P < 0.05). Horizontal green and red lines indicate an expression significantly different from the mean for Cx43 and Cx30, respectively. Note the significant decrease in Cx expression measured outside OG compared with the glomerular area (P < 0.01 and P < 0.001 for Cx43 and Cx30, respectively). This differential expression was significantly larger for Cx30 than Cx43 (P < 0.001, two-way ANOVA).

Fig. 2.

Astroglial networks are confined within glomeruli. (A1 and A2) Injections of sulforhodamine B (SrB, red) were performed in glomerular astrocytes from GLT-1/EGFP BAC reporter mice in which astrocytes that could potentially receive the dye were visualized (green in A1). 3D confocal analysis of SrB⁺ and GFP⁺ astrocytes, associated with a Nissl staining (blue) to define OG limits, allowed us to determine the distance of SrB⁺ and GFP⁺ cells from the injected cell (arrow), taking into account their location [within the injected glomerulus (Inj) or in adjacent OG (Adj)]. Examples illustrated in A1 and A2 were obtained from projections of 12 confocal planes (1 μm each). (Scale bar: 50 μm.) (B) For each location (Inj or Adj), the number of GFP⁺ (green) and SrB⁺ (red) cells was obtained for a defined range of distances from the injection site (10-μm large concentric rings from 0–10 to 90–100 μm). Results obtained from six injections were pooled (SI Appendix, SI Methods) and indicate a preferential dye coupling between astrocytes from the same OG. χ² tests were applied for each distance, to compare the percentage of coupled candidates between the two locations (Inj or Adj). *P < 0.05, **P < 0.01, and ***P < 0.001. (C) The number of GFP⁺ astrocytes located either in the injected or in an adjacent OG was determined within a fixed volume around the injected cell (SI Appendix, SI Methods). Within the injected OG, 76 ± 6% of these potential “receiver” cells for the dye are coupled, whereas only 16 ± 5% of the astrocytes located in adjacent OG receive the dye (n = 6 injections, **P < 0.01, χ² test; see SI Appendix, Fig. S5 for complementary information).

Fig. 3.

Astrocyte GJC is regulated by neuronal activity within olfactory glomeruli. (A) Typical IR-differential interference contrast image of simultaneous recordings of local field potential (LFP) and V_m of glomerular astrocyte in the same glomerulus (delimited by dotted line) with the two recording pipettes. (B) V_m in glomerular astrocytes show fluctuations as indicated by whole-cell recordings. (C) Simultaneous recordings of LFP (blue) and astrocyte V_m (black) in the same glomerulus showed correlated fluctuations (zero-lag correlation, r = −0.7, P < 0.01; see cross-correlogram analysis in SI Appendix, Fig. S5) silenced in presence of 0.5 μM TTX (horizontal bar). Rectangle shows the region presented in B. (D) Averaged cross-correlogram obtained for 18 recordings, filtered in the 0.05- to 0.2-Hz frequency band, showed similar correlation (r = −0.45 ± 0.05, P < 0.01). The lag corresponding to the maximal negative value of the correlation coefficient (indicated by dotted line in the Inset) indicates that astrocyte V_m is delayed by 722 ± 143 ms compared with LFP. (Inset) High magnification of the curve around zero lag (SI Appendix, Fig. S5). (E Left) For dye injections in OG astrocytes, treatment with TTX resulted in a significant reduction of dye coupling (n = 26 and 12 in control and TTX conditions, respectively; P < 0.0001). (E Right) Unilateral naris occlusion (Occ) performed at P1 led to a reduced GJC at P20 between glomerular astrocytes (n = 11 and 12 for control and occluded conditions, respectively; P < 0.0001).

Fig. 4.

Cx30 is the molecular target for the activity dependence of GJC between glomerular astrocytes. (A) In KO Cx43 mice, dye coupling was diminished by 66% in the presence of TTX when injections were performed in glomerular astrocytes (n = 6 for both control and TTX conditions; P < 0.001), whereas in KO Cx30 animals, dye coupling was not affected by TTX treatment (78% of control, n = 10 and n = 6 for control and TTX conditions, respectively; P = 0.1462). Note that in the control condition, the number of coupled cells was reduced by 42% (n = 6, P < 0.001) and 29% (n = 10, P < 0.001) in KO Cx43 and KO Cx30 mice, respectively, compared with wild type (n = 26). (B) Dye coupling was also strongly inhibited by early olfactory deprivation in P20 KO Cx43 mice (n = 4 and n = 5 for control and occluded conditions, respectively; P < 0.01), whereas in P20 KO Cx30 animals, dye coupling at P20 was not affected by olfactory deprivation (n = 4 and 5 for control and occluded conditions, respectively; P = 0.72).

Fig. 5.

The activity dependence of glomerular astroglial involves extracellular potassium. (A1 and A2) For glomerular injections, dye-coupling inhibition by TTX treatment (43%, n = 26 and 12 in control and TTX conditions, respectively; P < 0.0001) and by early unilateral naris occlusion (56% inhibition, n = 11 and 12 for control and occluded conditions, respectively; P < 0.0001) was reversed in the presence of high [K⁺]_e (6 instead of 3 mM in control; 108% of control, n = 6, P = 0.42 and 96% of control, n = 5, P = 0.67, for TTX and occlusion, respectively). (B) Treatment with barium 200 μM decreased by 17% (n = 6, P < 0.05) the number of coupled cells, whereas this parameter was increased by 39% (n = 4, P < 0.001) in high [K⁺]_e. (C) In contrast, high [K⁺]_e had no effect in KO Cx30 mouse (110% of control, n = 8, P = 0.44).