Gap junction-mediated astrocytic networks in the mouse barrel cortex

Abstract

The barrel field of the somatosensory cortex constitutes a well documented example of anatomofunctional compartmentalization and activity-dependent interaction between neurons and astrocytes. In astrocytes, intercellular communication through gap junction channels composed by connexin 43 and 30 underlies a network organization. Immunohistochemical and electrophysiological experiments were undertaken to determine the coupling properties of astrocyte networks in layer IV of the developing barrel cortex. The expression of both connexins was found to be enriched within barrels compared with septa and other cortical layers. Combination of dye-coupling experiments performed with biocytin and immunostaining with specific cell markers demonstrated that astrocytic networks do not involve neurons, oligodendrocytes or NG2 cells. The shape of dye coupling was oval in the barrel cortex whereas it was circular in layer IV outside the barrel field. Two-dimensional analysis of these coupling areas indicated that gap junctional communication was restricted from a barrel to its neighbor. Such enrichment of connexin expression and transversal restriction were not observed in a transgenic mouse lacking the barrel organization, whereas they were both observed in a double-transgenic mouse with restored barrels. Direct observation of sulforhodamine B spread indicated that astrocytes located between two barrels were either weakly or not coupled, whereas coupling within a barrel was oriented toward its center. These observations indicated a preferential orientation of coupling inside the barrels resulting from subpopulations of astrocytes with different coupling properties that contribute to shaping astrocytic networks. Such properties confine intercellular communication in astrocytes within a defined barrel as previously reported for excitatory neuronal circuits.

Figure 1.

Dye coupling among astrocytes in the barrel cortex studied at different developmental stages. A, Photomicrograph of an acute coronal slice of the somatosensory cortex at P10, showing a field of six barrels observed with Nomarski optics and infrared illumination. B, Photomicrograph taken at a higher magnification showing, at the tip of the patch-clamp pipette, an astrocyte selected for recording on the basis of the size and shape of its somata. C, D, Two examples of dye coupling in the barrel cortex at P5 and P10 obtained after fixation of the slice and peroxidase revelation of biocytin. E, Inhibition of GJC at P10 studied in a slice treated with carbenoxolone (100 μm). F, Diagram of the developmental study (P5 to P20) of GJC assessed by biocytin injection in astrocytes. Dye coupling was quantified by counting the number of biocytin-positive cells. Note the large and statistically significant difference in the levels of coupling observed between P5 and P10. The number of experiments ranged from 6 to 10. *p < 0.05 between P5 and the other developmental ages using one-way ANOVA, and **p < 0.01 between the extent of dye at P10 in control or with carbenoxolone treatment using unpaired t test. Scale bars: A, E (for E, C, D), 100 μm; B, 10 μm.

Figure 2.

Immunostaining identification of dye-coupled cells. Dye coupling was studied with biocytin injection and postfixation revelation with a secondary antibody coupled to fluorophores selected to be processed with immunohistochemical identification of various brain-cell populations. A, B, Dye coupling performed with biocytin (green) and combined with NeuN staining (red) indicates that coupling initiated by the recording from an astrocyte does not involve neurons. The inset demonstrates that the yellow spots observed in B are not caused by colocalization of the two markers, but rather by the superposition of the two distinct stainings resulting from the projection of several confocal plans. C, D, Dye coupling studied with biocytin (red) and combined with NG2 staining (green) indicates that coupling initiated by the recording of an astrocyte does not involve NG2-positive cells. E, F, Dye coupling with biocytin (red) and combined with S100 (green) staining indicates that coupling initiated by the recording of an astrocyte mainly involves S100-positive cells. Note that as expected from previous reports, this antibody also stains blood vessels. The inset illustrates the colocalization of the two markers. Similar observations were performed in four to eight independent experiments. Scale bar, 50 μm.

Figure 3.

Developmental changes in electrophysiological phenotypes of astrocytes recorded in the barrel cortex. A₁–C₂, Whole-cell current profiles recorded from morphologically identified astrocytes in layer IV of the somatosensory cortex. Voltage steps for the current induction were 150 ms pulses applied at a holding potential of −80 mV and ranging from −180 to +40 mV with 10 mV increments. A₁, A₂, CGs were characterized by a high input resistance and by the activation of inward and outward currents in response to depolarizing steps resulting in a strong outward rectification as illustrated by the plotting of the I–V relationship. B₁, B₂, IGs showed lower input resistance and weaker voltage-dependent currents compared with CGs with a weak change in the slope of the I–V curve. C₁, C₂, PAs exhibited a very low input resistance and a typical linear relationship between current and voltage. D, Developmental pattern of astrocytic electrophysiological phenotypes. Note that CGs and IGs were only observed at P5 and P10 in rather low proportion. This diagram was made from 33, 131, 35, and 18 cells recorded at P5, P10, P15, and P20, respectively.

Figure 4.

Distribution of Cx43 and Cx30 in layer IV of the barrel cortex. A–C, Double staining of coronal sections from barrel cortex, performed with NeuN (green) and Cx43 (red) antibodies, showing that at P6, Cx43 expression was already enriched in the barrels. In the merge (C), NeuN staining allowed delineation of the barrel walls. The inset in B illustrates at a higher magnification the pattern of Cx43 staining. At this age, this immunoreactivity was characterized by a diffuse stellate pattern and the presence of few immunoreactive puncta. D, E, Single staining of Cx43 in coronal (D) and tangential (E) sections from P10 mouse barrel cortex. F, G, Single staining of Cx43 in coronal sections in (F) and out (G) of the barrel field (IB and OB, respectively) from P20 mouse. H, Single staining of Cx30 in tangential sections from P20 mouse barrel cortex. Note that the compartmentalization of Cx expression observed in the barrel cortex is not observed in layer IV of the visual cortex. I–K, Single staining of Cx43 (I, coronal section; K, tangential section) and Cx30 (J), indicating that the enrichment of their expression in the barrels is maintained at adult stage. In C, D, and G, dotted lines delineate the location of the indicated cortical layers. Similar observations were made in three independent experiments performed at the different ages. Scale bar: (in K) A–D, F, G, I, J, 100 μm; E, H, K, 140 μm.

Figure 5.

Shaping of astrocytic networks in the barrel cortex. A, B, Comparison of the shape of biocytin intercellular diffusion after injection performed at P20 in an astrocyte located in layer IV outside (OB) (A) and within (IB) (B) the barrel field. Dashed lines with arrowheads indicate the two axes (x and y) taken for the measurement of dye coupling. C, Summary diagram showing the comparison of dye coupling performed outside or in the barrel field at two developmental ages. This analysis of the shape of the coupling area was undertaken by considering two axes, x and y, drawn in A and B. The x/y ratios were calculated from 3–10 independent experiments. **p < 0.01 between the OB and IB at both ages using unpaired t test. Scale bar, 100 μm.

Figure 6.

Shaping of astrocytic networks in the layer IV of the somatosensory cortex from transgenic mice with modified barrel organization. A₁–A₃, Cytochrome c staining revealing the presence or the absence of a barrel field organization in frontal slices from wild-type C3H/HE, MAOA KO, and MAOA/5-HT_1B KO mice studied at P10. The insets show a higher magnification of layer IV in these slices with detectable barrels in the wild-type and the double KO mice and the lack of compartmentalization in the MAOA KO mouse. For the MAOA KO, the location of the somatosensory cortex was defined in reference to the shape and size of the striatum being part of the slice. B, C, Cx43 immunostaining of frontal sections at P10. B, In wild type, Cx43 is compartmentalized in the barrel field. C, In contrast, a uniform distribution of Cx43 immunoreactivity is observed in the somatosensory cortex of MAOA KO mice. D₁–D₃, Shape of the dye-coupling area after biocytin injection of an astrocyte located in the barrel field of the wild-type (D₁), the MAOA (D₂), and the double KO MAOA/5-HT_1B receptor (D₃) mice studied at P10. E, Summary diagram of the x/y ratio calculated from 3–10 independent experiments. ***p < 0.001 between wild type and MAOA KO using ANOVA test analysis; MAOA/5-HT_1B receptor was different at *p < 0.05 from MAOA KO, whereas there is no statistical difference between MAOA/5-HT_1B receptor and wild type. Scale bars: B (for B, C), D, 100 μm.

Figure 7.

Analysis of dye-coupling orientation within a single barrel. A₁, A₂, Direct observation of sulforhodamine B injections performed at P10 in an astrocyte located within a barrel. A₁, Superposition of photomicrographs captured under Nomarski optic and epifluorescence showing the limits of three barrels. Note the spread of the dye after 20 min recording and the location of the recorded astrocyte (white spot). A₂, Detail of the two parameters defining the extent of dye coupling within a barrel. The limits of the three barrels have been drawn from the Nomarski picture illustrated in A₁. d_w and d_i represent the distance between the injected site (white spot) and the nearest barrel wall or the center of the barrel, respectively. B, Double histogram showing, in abscissa, the distance of dye diffusion measured from the injection site toward the nearest barrel wall (d_w, black columns) and toward the center of the barrel (d_c, white columns) versus, in ordinate, the location of the injection site within three arbitrary compartments of a half-barrel, respectively, 0–10%, 10–25%, and 25–50% of the normalized dimension of a barrel. Note that the difference of dye diffusion in the two directions became statistically significant as the injection approached the barrel wall. *p < 0.05 using the unpaired t test. C, Plot of the distance ratio d_i/d_w versus the number of coupled cells indicates that the asymmetry of dye coupling within a barrel is more pronounced when dye coupling is reduced. These data were fitted with an exponential curve. Scale bar: (in A₂) A₁, A₂, 50 μm.

Figure 8.

Differential coupling properties of astrocytes in the barrel cortex. A₁–B₂, Direct observation of sulforhodamine B injections performed at P10 in astrocytes located either in a barrel or in a septum. A₁, Superposition of photomicrographs captured under Nomarski optic and epifluorescence showing the extent of dye spread after 20 min recording and the location of the coupling area within the barrel. A₂, High-magnification fluorescent image showing the shape of the dye coupling and the detail of the cells involved in this process. B₁, Superposition of photomicrographs showing the lack of dye coupling and the location of the astrocyte recorded in the septum. B₂, High-magnification image showing the shape of the recorded cells. Scale bars: A₁, B₁, 150 μm; A₂, B₂, 20 μm. A₁, B₁, Insets, Family of current traces recorded in response to voltage steps of 150 ms pulses ranging from −180 to +40 mV with 10 mV increments, applied at a holding potential of 80 mV. Note that the two recorded astrocytes were characterized by similar passive properties. A₃, B₃, Summary diagrams of dye injections performed at P10 with the two different sites of recording, i.e., in the barrel (white columns) or in the septum (black columns).

Figure 9.

Confocal analysis of the morphology of stained astrocytes located in a barrel or in a septum. A, Low magnification of the dye-coupling area after the recording of an astrocyte located in a barrel with a patch pipette filled with biocytin. The inset delineates a region shown at a higher magnification in B. B, Projection of 10 optical sections (1 μm) showing the morphology of one stained cell typical of protoplasmic astrocytes and its organization in individual domain. C, D, Examples of the morphology of astrocytes loaded with sulforhodamine B located in septa. Note that the feature of these astrocytes, as well as the domains defined by their processes, were similar to those of the astrocyte illustrated in B. Scale bar: (in D) A, 50 μm; B–D, 10 μm.