Neural activity and neurotransmission regulate the maturation of the innervation field of cortical GABAergic interneurons in an age-dependent manner

Abstract

Neural activity guides the patterning of neuron synaptic territory in the developing nervous system. Evidence supporting this hypothesis comes from numerous studies on projection neurons in neuromuscular and visual systems. It is unknown whether the innervation field of GABAergic interneurons, which forms local dense innervations, follows similar rules. Cortical basket cells innervate hundreds of pyramidal cell somata and proximal dendrites. Thanks to this connectivity pattern, they can tightly control neural excitability and synchronization. Here we show that reducing excitation, and thus neurotransmitter release, in mouse cortical single basket cells in slice cultures decreases the number of innervated cells without changing the pattern of perisomatic innervation, both at the peak and after the proliferation phase of perisomatic synapse formation. Conversely, suppressing neurotransmitter release in single basket cells can have completely opposite effects depending on the developmental stage. Our results reveal a remarkably specific and age-dependent role of neural activity and neurotransmission levels in the establishment of the synaptic territory of cortical GABAergic cells.

Figure 1.

Activation of AlstR reduces activity-dependent increase of pCREB expression. A, Schematics of experimental procedure. Cortical organotypic cultures were biolistically transfected with P_G67-GFP/P_G67-AlstR and P_G67-tdTomato to colabel allatostatin-expressing cells in green and control basket cells in red in the same organotypic slice. B, C, P_G67-GFP/P_G67-AlstR transfected basket cells treated with allatostatin (C) show decreased nuclear pCREB immunoreactivity (C2, arrow) compared with untransfected neighboring cells, whereas pCREB levels in untreated, AlstR-transfected basket cells (B) do not differ from neighboring cells (B2, arrow). Scale bar, 10 μm.

Figure 2.

Reducing basket cell excitability and suppressing neurotransmitter release have opposite effects during the peak of perisomatic synapse maturation. A, Control basket cell (Ctrl, red) at EP24 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons (A2), and terminal branches with prominent and clustered boutons (A3, arrowheads) around pyramidal cell somata (NeuN immunostaining, blue). Stars indicate pyramidal cell somata that are not innervated in the projected confocal stack. B, AlstR-expressing basket cell (green) treated with allatostatin from EP17–24 shows overall similar axon size and morphology (B1), however, it is easier to find noninnervated targets in the axonal arbor territory (B2, stars). Terminal branching, bouton size, and density (B3) around innervated pyramidal cell somata appear similar to those from control cells. C, TeNT-Lc-expressing basket cell (green) shows overall similar axon size (C1). The fraction of innervated targets does not appear affected (C2), however, perisomatic innervations are characterized by more numerous, smaller-looking boutons (C3, arrowheads) compared with Ctrl cells. A3, B3, and C3 are from regions in A2, B2, and C2. Scale bars (in A1) A1–C1, 50 μm; (in A2) A2–C2, 10 μm; (in A3) A3–C3, 5 μm. D, GFP or RFP-positive boutons colocalize with GAD65 (blue) in controls, AlstR or TeNT-Lc basket cells, suggesting that even the smaller boutons in TeNT-Lc-transfected cells express GABAergic synaptic markers. Scale bar, 5 μm. E, F, AlstR-expressing cells show no differences in bouton density (E) and terminal branching (F) compared with aged-matched controls (p > 0.05, n.s.), whereas TeNT-Lc-expressing cells show a statistically significant increase of both parameters (*p < 0.05); n = 67 perisomatic innervations from 6 control basket cells for controls, 58 innervations from 6 AlstR basket cells, and 60 perisomatic innervations from 6 TeNT-Lc basket cells. G, At EP24, the fraction of potentially innervated targeted neurons is not affected for TeNT-Lc-expressing cells, but it is significantly reduced for AlstR-transfected cells (*p < 0.05); n = 6 basket cells for all experimental groups. H, Bouton size is significantly reduced in TeNT-Lc-transfected cells compared with controls and AlstR-expressing cells (Kolmogorov-Smirnov (K-S) test, p < 0.001); n = 150 boutons from 6 basket cells for each group. Values in D–F represent mean ± SEM.

Figure 3.

Basket cell axon analysis suggests the occurrence of pruning after the peak of perisomatic synapse proliferation. A, Examples of fully reconstructed basket cells. Scale bar, 150 μm. B, Full axon reconstruction of control basket cells at EP24 and EP32 show that the percentage of potentially innervated cells is significantly smaller at EP32 compared with EP24 (*p < 0.05, n = 5 for both ages). In addition, AlstR activation from EP17–24 (n = 3) and from EP26–32 (n = 3) significantly reduces the fraction of innervated cells (*p < 0.05), consistent with what was shown by quantifying the innervation percentage in confocal stacks acquired in the first 150 μm from the basket cell soma (Fig. 2G).

Figure 4.

Suppressing neurotransmitter release after the peak of perisomatic synapse maturation induces a more severe loss of perisomatic innervations than reducing neural excitability. A–C, Activation of AlstR from EP26–32 does not affect terminal branching or perisomatic boutons formed by AlstR-expressing cells (B3, arrowheads) compared with controls (A3), but it diminishes the number of potentially innervated postsynaptic cells (A2, B2; stars represent noninnervated pyramidal somata in the projected confocal stack). On the other hand, TeNT-Lc-expressing cells show a reduction both in percentage of innervated cells (C2) and perisomatic innervation (C3). Boutons appear more irregular with some large and many smaller ones (arrowheads). A3, B3, and C3 are from regions in A2, B2, and C2. Scale bars: (in A1) A1–C1, 50 μm; (in A2) A2–C2, 10 μm; (in A3) A3–C3, 5 μm. D, E, Quantification shows that terminal branching and perisomatic bouton density is significantly affected only in TeNT-Lc-transfected cells, but not by AlstR activation (*p < 0.001); n = 60 perisomatic innervations from 6 basket cells for each experimental group. F, At EP32, both AlstR- and TeNT-Lc-expressing cells show reduced innervation of pyramidal somata compared with control (*p < 0.001); n = 6 basket cells for each group. G, AlstR-expressing cells have normal-sized boutons compared with controls, but TeNT-Lc-expressing cells show a significant reduction in bouton size (K-S test, p < 0.001); n = 6 basket cells and 150 boutons. Values in D–F represent mean ± SEM.