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. 2014 Jun 10;8:151. doi: 10.3389/fncel.2014.00151

Figure 2.

Figure 2

The AIS shifts distally on chronic depolarization, but GABAergic synapse positions are not affected. (A) As Figure 1A. Neuron stained for ankG and GABAAR-α2 subunit. Soma, AIS and GABAAR cluster positions along the axon are indicated (open arrowhead: cluster before AIS; closed arrowhead: cluster within AIS; arrow: cluster beyond AIS). AIS endpoints are determined from ankG intensity. (A') as (A), for a KCl-treated neuron. (B) AIS start position is greater after KCl treatment (p = 0.007, n = 10 experiments); (B') AIS length is not affected (p > 0.05). (C) Number of GABAAR clusters per axon is unchanged (p > 0.05). (D) Positions of all GABAAR clusters from control and KCl pools. (E) GABAAR cluster position along axons (control, black, n = 1944 clusters; KCl, blue, dashed, n = 2130 clusters, p > 0.05, Mann-Whitney U-test). (F) Cluster ratio (in AIS / before AIS) is lower in KCl-treated neurons, p = 0.004, n = 5 experiments. (G) Example neurons stained for ankG and gephyrin (labeled as in A). Top: control, bottom: KCl. Scale bar = 10 μm. (H) Total number of gephyrin clusters per axon is not significantly different (p > 0.05, n = 5). (I) Cumulative frequency plot of gephyrin cluster position along axons (control, black, n = 1585 clusters; KCl, blue, dashed, n = 1686 clusters). Distributions not significantly different (p > 0.05). (J) Gephyrin cluster ratio (in AIS/before AIS) is reduced in KCl-treated neurons, (control: 1.9 ± 0.1; KCl: 0.8 ± 0.1, p = 0.008, n = 5 preps). Under control conditions, axons contained on average 2.5 ± 0.4 gephyrin clusters before their AIS and 3.8 ± 0.5 clusters within their AIS; in KCl treated neurons, axons contained 3.6 ± 0.6 clusters before their AIS and 3.2 ± 0.5 within their AIS. (K) Example neuron stained for ankG and VGAT (labeled as in A). Top: control, bottom, KCl. Scale bar = 10 μm. (L) Total number of VGAT puncta per axon is not significantly different (p > 0.05, n = 5). (M) Cumulative frequency plot of VGAT puncta position along axon (control, black, n = 2219 clusters; KCl, blue, dashed, n = 1962 clusters). Distributions not significantly different (p > 0.05), suggesting that the tight coupling between pre- and post-inhibitory synapses is not affected by chronic depolarization. (N) Cluster ratio (in AIS/before AIS) is lower in KCl treated neurons, (control: 1.9 ± 0.1; KCl: 0.8 ± 0.1, p = 0.003, n = 5 experiments). Under control conditions, axons contained on average 1.7 ± 0.3 VGAT puncta before their AIS and 3.1 ± 0.5 puncta within their AIS; in KCl treated neurons, axons contained 3.3 ± 0.2 puncta before their AIS and 2.7 ± 0.5 within their AIS.