GABA signaling promotes synapse elimination and axon pruning in developing cortical inhibitory interneurons

Abstract

Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses.

Figure 1.

Blockade of GABA release induces basket cell axons and bouton overgrowth. a–d, Comparison of axon arbors in basket cells with different GABA levels at EP24. Basket cells were transfected at EP18. a, A control (Ctrl, Gad67^+/+;Gad65^+/+) cell with a complex axon arbor. b, A Gad67^+/+;Gad65^−/− cell showed an axon arbor comparable to control. c, A Gad67^−/−;Gad65^+/− cell showed a simple axon arbor. d, A Gad67^−/−: Gad65^−/− cell showed an over exuberant axon arbor. Scale bar, 100 μm. e, Quantification of local axon density in basket cells with different GABA levels. Compared with Ctrl cells, there was no difference in Gad67^+/+;Gad65^−/− cells (p > 0.05, Mann–Whitney test), a significant decrease in Gad67^−/−:Gad65^+/+ cells (p < 0.05, Mann–Whitney test), and a highly significant increase in Gad67^−/−:Gad65^−/− cells (p < 0.001, one-way ANOVA, post hoc Dunn's test; n = 5 cells for each group). f1, A control basket cell (green) at EP24 with an extensive axon arbor that innervates hundreds of nearby pyramidal cells. f2, Axon branches carry prominent presynaptic boutons. f3, Terminal branches innervate pyramidal cell soma with numerous clustered boutons (arrowheads; red, NeuN immunostaining). Scale bars: f1, 100 μm; f2, 10 μm; f3, 2 μm. g, A Gad^−/− basket cell shows overall similar arbor size (g1) but increased axon and bouton density (g2) around pyramidal cell soma (g3). h, vGAT^−/− basket cells display a similar phenotype as Gad^−/− cells. i, Axon density is significantly increased in Gad^−/− and vGAT^−/− cells compared with controls (n = 10 basket cells for each group; one-way ANOVA, post hoc Dunn's test, p < 0.001). j, Interbouton distances are significantly reduced in Gad^−/− and vGAT^−/− cells compared with controls (n = 10 basket cells for each group; one-way ANOVA, post hoc Dunn's test, p < 0.001). Asterisks indicate p < 0.05.

Figure 2.

GABA depletion does not alter intrinsic biophysical properties of basket cells. a, A basket cell was identified by GFP expression for patch-clamp recording. b, Protocol for analysis of input resistance. R_a current and R_input current are measured as shown and Rm = R_input − R_a. c, Protocol for analysis of action potential (AP). The half-width of the first AP is measured as shown. d, Analysis of fast-spiking adaptation index. Adaptation = last interval/fourth interval. e, Control and Gad^−/− basket cells are not significantly different in intrinsic property parameters: Rm, action potential half-width, and fast-spiking adaptation (FS) (n = 6 cells for each group; numbers present mean ± SD; t test, p > 0.05).

Figure 3.

Blockade of GABA release in developing basket interneurons results in smaller and more homogenously sized boutons. a, In organotypic cultures, perisomatic bouton size is significantly reduced in Gad^−/− and vGAT^−/− cells compared with controls (10 cells for each group and 100 boutons from each cell; one-way ANOVA, post hoc Dunn‘s test, p < 0.001). Bar represents mean. Error bars represent SEM. b, The cumulative distribution of bouton size in mutant cells is shifted toward the smaller size (Kolmogorov–Smirnov test, p < 0.001). c, The variance of bouton size is significantly smaller in mutant cells (one-way ANOVA, post hoc Dunn's test, p < 0.001). d, In visual cortex, Gad67^flx/flx;Gad65^−/− mice and AAV-GFP-ires-Cre-infected basket interneurons (arrowheads; blue, Pv immunostaining) show no GABA immunostaining (red) 7 d after injection compared with neighboring untransfected basket cells (arrows). Scale bar, 20 μm. e, Gad^−/− and vGAT^−/− cells (in vGAT^flx/flx mice) in the visual cortex have smaller perisomatic boutons (arrows) compared with control cells. Pyramidal cell somata are labeled by NeuN (red). Note that the interval between boutons appears reduced in these mutant cells. Scale bar, 2 μm. f, Bouton size is significantly reduced in infected Gad^−/− and vGAT^−/− cells compared with controls (3 animals for each group, 50 boutons from each animal; one-way ANOVA, post hoc Dunn's test, p < 0.001). Bars represent the mean. Error bars represent SEM. g, The cumulative distribution of bouton size in mutant cells is shifted toward the smaller size (Kolmogorov–Smirnov test, p < 0.001). Asterisks indicate p < 0.05.

Figure 4.

Release blocked GABAergic boutons represent bona fide synapses. a, Control basket cell presynaptic boutons (green, arrowheads) around a pyramidal cell soma (red) colocalize with the presynaptic marker vGAT (blue). Scale bar, 5 μm. b, Perisomatic boutons (green) from Gad^−/− basket cells remain colocalized with vGAT (blue). c, Control presynaptic boutons (circled) are juxtaposed to the postsynaptic marker Gephyrin (red). Scale bar, 1 μm. d, vGAT^−/− presynaptic boutons (circled), although much smaller in size, remain juxtaposed to gephyrin. e, Control boutons (green) are juxtaposed (white arrowheads) to postsynaptic GABA_A R β2 (red). Scale bar, 1 μm. f, vGAT^−/− boutons (green), although much smaller in size, remain juxtaposed to postsynaptic GABA_A R β2. g, Expression pattern of neurexin1β-SEP in control axon together with TdTomato. Neurexin1β-SEP puncta localized to subregions within boutons (arrowheads), with substantial variations in puncta size independent of bouton size revealed by tdTomato signals. Scale bar, 1 μm. h, In a Gad^−/− axon, neurexin1β-SEP puncta remain localized to boutons (arrowheads) but appear homogenous and tiny in size. i, Syn-GFP puncta are colocalized to presynaptic marker vGAT. Scale bar, 2 μm. j, k, Syn-GFP puncta localize to axonal boutons in a control basket cell (j) (labeled by tdTomato) and in a Gad^−/− basket cell (k). Scale bar, 5 μm. Arrowheads point to examples of Syn-GFP puncta that are colocalized with tdTomato-labeled boutons. l, Local reconstruction of control and Gad^−/− basket cell axons (red lines) with Syn-GFP puncta (green dots). The average interpuncta distances of Syn-GFP puncta in Gad^−/− and vGAT^−/− cells are significantly decreased compared with controls (n = 5 cells for each group; p < 0.05, Mann–Whitney test). Scale bar, 5 μm. Asterisks indicate p < 0.05. m, n, Serial EM sections showing a control (m) and Gad^−/− (n) basket cell bouton, which forms a symmetric synapse with a postsynaptic cell soma in P26 visual cortex. GFP labeling was conferred by AAV-GFPires-Cre injection into Gad67^flx/flx;Gad65^−/− mouse cortex from P18 to P26. Both unlabeled (Ctrl) and labeled (Gad^−/−, arrow, labeled by dark precipitate from immunogold against GFP) boutons contain misshaped vesicles, typical of GABAergic synapses, which cluster around the active zone (arrow). The synaptic cleft appears typically narrower than those of asymmetric synapses (arrowheads) that are found on nearby dendrites. Scale bar, 500 nm. 3D reconstructions from serial EM images of the same WT and Gad^−/− axonal boutons are shown at the bottom, respectively.

Figure 5.

Increased stability of Syn-GFP puncta after blockade of GABA release. a, Dynamics of Syn-GFP puncta along a stretch of control basket cell axon at EP19. Note that puncta appear (green arrows) and disappear (red arrows) over a 60 min period. The profile plot shows that the intensity of signals fluctuates (areas shaded in red), reflecting puncta dynamics. b, Dynamics of Syn-GFP puncta along a stretch of vGAT^−/− axon at EP19. Puncta are more stable, and a new one appeared (t40, green arrows). The profile plot along the axon shows that the intensities are relatively stable (shaded in blue), reflecting the more static nature of vGAT^−/− puncta. c, The mIPDs in Ctrl, Gad^−/−, and vGAT^−/− axons are not significantly different (one-way ANOVA, post hoc Dunn's test, p > 0.05). d, The average interpuncta distances are smaller in Gad^−/− and vGAT^−/− axons than in Ctrl axons at both t0 and t60 (one-way ANOVA, post hoc Dunn's test, p < 0.05). Note that, at EP19, most synGFP puncta are small regardless of genotypes, compared with those of EP24 control cells (Fig. 1f); in fact, synGFP puncta in E19 Gad^−/− cells appear larger than in control cells, likely because these puncta are more stable in Gad^−/− cells than those that are more mobile in control cells. Asterisks indicate p < 0.05.

Figure 6.

Blockade of GABA release reduces elimination of Syn-GFP puncta in developing basket cell axons. a, Dynamics of Syn-GFP puncta in a Ctrl cell over a 3 h period at EP24. Note that punctum d is lost at t120. Scale bar, 1 μm. b, Dynamics of a Syn-puncta in vGAT^−/− cell at EP24. Note that three new puncta appeared over the 3 h period. Puncta 10 and 11 appeared and disappeared (the puncta 10 and 11 intensity dropped below threshold,) and then appeared again. c, d, Plot of puncta intensity fluctuation over time for puncta annotated in the Ctrl (c) and vGAT^−/− (d) example. Note that newly formed puncta (j and k, for example) have much lower intensity.

Figure 7.

Blockade of GABA release results in more filopodia extension in developing basket cell axons. a–c, Filopodia dynamics in control (a), Gad^−/− (b), and vGAT^−/− (c) basket cells at EP19. Yellow arrowheads indicate the sites of filopodium extension over a 3 h period. Top, Axon arbor at t0. Scale bar, 5 μm. Bottom (A1–C3), Filopodia extensions at different time points during the imaging sessions. Scale bar, 2 μm. Red lines are drawn to annotate the filopodia. d, Filopodium density is significantly increased in both Gad^−/− and vGAT^−/− compared wit controls (one-way ANOVA, post hoc Dunn's test, p < 0.05; n = 5 cells for each group). Error bar represents SEM. e, The ratio of boutons bearing filopodia ([number of filopodium]/[number of bouton]) is significantly increased in both Gad^−/− and vGAT^−/− compared with controls (one-way ANOVA, post hoc Dunn's test, p < 0.05; n = 5 cells for each group). f, The percentage of newly acquired boutons is significantly increased in both Gad^−/− and vGAT^−/− compared with controls (one-way ANOVA, post hoc Dunn's test, p < 0.05; n = 5 cells for each group). Asterisks indicate p < 0.05.

Figure 8.

Blockade of GABA release results in reduced branch pruning and increased branch extension. a, From EP19 to EP21, the growth of a basket cell axon arbor involves branch addition (yellow arrows)/extension (red arrows) and loss (green arrows)/retraction (blue arrows). b, vGAT^−/− axon arbors show much more extension events (red arrows) and much less loss/retraction (green arrows) events. Scale bar. 50 μm. c, d, Higher-magnification view of the boxed axon terminals in the control (c) and vGAT^−/− (d) cells. c, Note that in the control cell, a branch extended (red arrow) in day 2 and then retracted in day 3 (green arrow); another branch was added (yellow arrow) in day3. d, In the vGAT^−/− cell, a branch extended (red arrow) in day 2 and then further extended in day 3 (red arrow). Scale bar, 10 μm. e, Histogram showing branch dynamics of Ctrl and vGAT^−/− cells with a lowest smoothed line drawn over the bin heights. Each bin contains 50 data points. Insets, Distributions of skewness and kurtosis of the 1000 bootstrapped values. f, The distribution of each of five branch behaviors in control and vGAT^−/− cell axons are significantly different (χ² test, p < 0.001), with twice as many extended branches (green) and half as many retracted (blue) branches in vGAT^−/− cells. g, In vGAT^−/− cells, the increase in axon length in extended branch group is significantly greater than that in control cells (Student's t test, p < 0.001). However, there is no significance between the reduction in axon length in the retracted branch group compared with that in control cells. Error bars represent SEM. Asterisks indicate p < 0.05.

Figure 9.

Basket cell axon branch growth is accompanied by bouton formation. Two-photon images of the same basket axon terminal branches on 2 consecutive days. From P18 to P19, branch extension (long arrow), branch retraction (short arrow), and bouton formation (arrowheads) are observed. a/b and c/d are from two different basket cells. Note the appearance of numerous distinct boutons along axon branches from c to d. Scale bar, 10 μm.

Figure 10.

A model on the role of GABA transmission in inhibitory synapse and axon development. A developing GABAergic axon (light blue) explores potential synaptic targets (pink rectangles) by making transient synaptic contacts. Dark pink rectangles represent appropriate targets; light pink rectangles represent inappropriate targets. These transient contacts contain release machinery (e.g., synaptic vesicles represented as green circles) and mediate GABA transmission; the strength of transmission might provide a measure of the relative strength among nearby contact sites. GABA transmission may result in an activity-dependent redistribution of synaptic resource, such as removal from weak sites to promote their elimination and supplement to stronger sites to promote their further maturation, thereby promoting competition among these sites. Through GABA transmission (arrow), transient contacts at the inappropriate targets are eliminated, whereas those at the appropriate targets are validated and strengthened. Synapse maturation may result in branch stabilization, new branch extension and growth (WT). At inappropriate contacts, the elimination of transient synapses may lead to the pruning of branches. Reduction of GABA transmission (GABA^+/−) preserves such competition mechanism for synaptic elimination but may limit the capacity to support synapse maturation, therefore resulting in reduced synapse density and strength, and a simple axon arbor. Without GABA transmission (GABA^block), GABAergic axons cannot discriminate between appropriate and inappropriate targets and strong and weak contacts. Thus, appropriate contacts cannot be validated and mature, and inappropriate contacts cannot be eliminated, resulting in increased bouton density and small and more homogenously sized boutons. Such increases in bouton density and stability result in more branch extension and less pruning, leading to massive growth of axon arbor.