CLC-3 chloride channels moderate long-term potentiation at Schaffer collateral-CA1 synapses

Abstract

The chloride channel CLC-3 is expressed in the brain on synaptic vesicles and postsynaptic membranes. Although CLC-3 is broadly expressed throughout the brain, the CLC-3 knockout mouse shows complete, selective postnatal neurodegeneration of the hippocampus, suggesting a crucial role for the channel in maintaining normal brain function. CLC-3 channels are functionally linked to NMDA receptors in the hippocampus; NMDA receptor-dependent Ca(2+) entry, activation of Ca(2+)/calmodulin kinase II and subsequent gating of CLC-3 link the channels via a Ca(2+)-mediated feedback loop. We demonstrate that loss of CLC-3 at mature synapses increases long-term potentiation from 135 ± 4% in the wild-type slice preparation to 154 ± 7% above baseline (P < 0.001) in the knockout; therefore, the contribution of CLC-3 is to reduce synaptic potentiation by ∼40%. Using a decoy peptide representing the Ca(2+)/calmodulin kinase II phosphorylation site on CLC-3, we show that phosphorylation of CLC-3 is required for its regulatory function in long-term potentiation. CLC-3 is also expressed on synaptic vesicles; however, our data suggest functionally separable pre- and postsynaptic roles. Thus, CLC-3 confers Cl(-) sensitivity to excitatory synapses, controls the magnitude of long-term potentiation and may provide a protective limit on Ca(2+) influx.

Figure 1. CLC-3 colocalizes with postsynaptic density 95 (PSD-95) and NMDA receptors (NMDARs) at postsynaptic sites

Cryosections were made from the hippocampus of postnatal day 42 wild-type (WT) and Clcn3^−/− (KO) mice, stained for desired proteins, and imaged at ×63 magnification with ×4 zoom on a confocal microscope. A, CLC-3 is expressed throughout the CA1 region of the hippocampus in WT but not Clcn3^−/− sections. B, CLC-3 immunostaining overlaps extensively with staining for the postsynaptic proteins NR1 and PSD-95 in the WT stratum radiatum. Manders’ coefficients quantifying the degree of overlap are 0.82 for the fraction of NR1 overlapping CLC-3 and 0.70 for CLC-3 overlapping NR1, with a correlation coefficient of 0.84. The fraction of PSD-95 overlapping CLC-3 is 0.67 and CLC-3 overlapping PSD-95 is 0.80, with a correlation coefficient of 0.81. Qualitative analysis of colocalization was done using ImageJ plug-in JACoP (Fabrice et al. 2006).

Figure 2. Long-term potentiation (LTP) is increased in Clcn3^−/− slices

A, stimulation intensity (left panel) for WT (74 ± 13 μA, n= 10) and Clcn3^−/− slices (124 ± 22 μA, n= 8, P= 0.06) required to produce a field excitatory postsynaptic potential (fEPSP) that is 30% of the maximal response (right panel; WT, 1.86 ± 0.18 mV, n= 10; and KO, 1.58 ± 0.13 mV, n= 8; P > 0.05). Box plots depict the mean (line across box), ± SEM (top and bottom of box) and range of the data. B, mean normalized fEPSP amplitude in response to theta burst stimulation at t= 0. Both WT (1.35 ± 4, n= 8) and Clcn3^−/− slices (1.54±7, n= 10) show significant LTP at 50 min (P < 0.001, Student's paired t test). The Clcn3^−/− (red traces and symbols) and WT slices (black traces and symbols) display significantly different magnitudes of LTP (P < 0.001, Student's unpaired t test). Each point is the mean ± SEM. Insets are averages of 10 fEPSPs (normalized to 1.0) for WT and Clcn3^−/− slices before and after induction of LTP. Scale bar represents 0.5 and 10 ms.

Figure 3. Paired-pulse facilitation (PPF) is increased in Clcn3^−/− slices

A, field response in CA1 stratum radiatum to paired-pulse stimulation of the Schaffer collaterals. Stimulation intensity was adjusted to 30% of the maximal evoked response. Traces shown are at a 50 ms interstimulus interval (ISI) in WT (black traces) and Clcn3^−/− slices (red traces). Top traces, average fEPSPs from six consecutive sweeps. Bottom traces, average fEPSPs normalized to the peak of the first fEPSP. B, paired-pulse ratios for WT and Clcn3^−/− slices, calculated as the amplitude of second/first peak (at 50 ms, WT, 1.60 ± 0.06, n= 14; and KO, 2.16 ± 0.09, n= 8). The Clcn3^−/− slices display significantly stronger facilitation at all intervals between 30 and 200 ms (*P < 0.01, **P < 0.001, Student's unpaired t test). C, paired-pulse stimulation before and following 50 min of stable LTP expression in WT (n= 7) and Clcn3^−/− slices (n= 4). At short ISIs, WT slices displayed a slight decrease in PPF (ISI = 30–100 ms, P < 0.05, Student's paired t test), but no significant difference appeared at longer intervals (ISI = 150–200 ms, P > 0.05, Student's paired t test). The Clcn3^−/− slices showed small, inconsistent changes in PPF across ISIs, but these were significant at only one interval (ISI = 60 ms, P < 0.05; all other ISIs, P > 0.05; Student's paired t test), suggesting postsynaptic expression of LTP. Each point is the mean ± SEM.

Figure 4. CLC-3 is phosphorylated by Ca²⁺/calmodulin kinase II (CaMKII)

tsA cells were stably transfected with CLC-3 (tsA-CLC-3) or mock transfected with selection vector only (tsA-mock). A, tsA cells were fixed, permeablized, and labelled with an antibody to CLC-3. CLC-3 is expressed strongly in stably transfected tsA cells (bottom panels), while mock-transfected cells (top panels) have low endogenous expression. This is confirmed in a Western blot (right); tsA-mock cells do not show appreciable CLC-3 protein compared with tsA-CLC-3 cells. B, α-CLC-3 was incubated with protein A-conjugated magnetic Dynabeads and used to immunoprecipitate (IP) CLC-3 from tsA cells. We were able to immunoprecipitate CLC-3 from tsA-CLC-3 lysate but not from tsA-mock cells. C, CLC-3 is phosphorylated by CaMKII in vitro. Immunoprecipitated CLC-3 was incubated with activated CaMKII for 30 min, run out with SDS-PAGE, and probed for phosphorylation with an α-phosphoserine antibody. In the absence of kinase, there is negligible phosphorylation of the precipitated protein.

Figure 5. Application of the Tat-peptide Tat-CLC-3_107−116 to a WT slice mimics increased LTP in adult Clcn3^−/− slices

A, uptake of fluorescein-labelled Tat-CLC-3_scr into cultured neurons. Cells displayed strong uptake within 15 min. B, in vitro phosphorylation of Tat-peptides by CaMKII using ³²P-labelled ATP. When kinase or peptides are not included in the reaction mixture, there is no difference in baseline counts per minute (80 ± 4 c.p.m., n= 3; 85 ± 13 c.p.m., n= 3; P > 0.05, Student's unpaired t test), measured by a scintillation counter. Addition of Tat-CLC-3_107−116 results in a 12-fold increase in ³²P counts (1077 ± 51 c.p.m., n= 3; ***P < 0.001) compared with the no-peptide condition. Scrambled peptide also increases ³²P counts from baseline (350 ± 33 c.p.m., n= 3; **P < 0.01) but significantly less than Tat-CLC-3_107−116 (***P < 0.001). All bars are the average of three experiments ± SEM. C, stimulation intensity (left) for WT slices with 10 μm Tat-CLC-3_107−116 (118 ± 18 μA, n= 6) and WT slices with 10 μm Tat-CLC-3_scr (105 ± 9 μA, n= 13; P > 0.05) required to produce an fEPSP that is 30% of the maximal response (right; Tat-CLC-3_107−116, 2.19 ± 0.14 mV, n= 6; and Tat-CLC-3_scr, 2.57 ± 0.16 mV, n= 13; P > 0.05). Two different scrambled peptides were tested with the same result, so these data are combined. Box plots depict the mean (line across box), ± SEM (top and bottom of box) and range of the data. Time course (D) and average magnitude of LTP (E) in WT slices with 0, 5 and 10 μm Tat-CLC-3_107−116 compared with Clcn3^−/− slices (from left to right: 1.33 ± 0.04, n= 10; 1.43 ± 0.06, n= 4; 1.55 ± 0.06, n= 6; and 1.58 ± 0.07, n= 8; **P < 0.01, ***P < 0.001, Student's unpaired t test). At 10 μm, Tat-CLC-3_107−116 produces LTP that is not significantly different from that in Clcn3^−/− slices (P > 0.05).

Figure 6. Tat-CLC-3_107−116 specifically blocks the postsynaptic interaction of CLC-3 and CaMKII

A, application of 10 μm Tat-CLC-3_scr to a WT slice results in LTP that is not significantly different from that in WT slices alone (1.35 ± 0.05, n= 6; P > 0.05, Student's unpaired t test). B, 10 μm Tat-CLC-3_107−116 does not alter Clcn3^−/− LTP expression (1.54 ± 0.10, n= 5; P > 0.05), suggesting that it does not interfere with CaMKII phosphorylation of other substrates important for LTP. C, summary of LTP magnitudes in A and B, measured during the last 10 min of recording. D, presynaptic release is not significantly changed compared with WT slices alone (1.60 ± 0.06, n= 14) for WT slices with application of Tat-CLC-3_107−116 (1.57 ± 0.04, n= 6; P > 0.05) or Tat-CLC-3_scr (1.58 ± 0.06, n= 6; P > 0.05). In addition, facilitation measured in Clcn3^−/− slices treated with Tat-CLC-3_107−116 (2.05 ± 0.15, n= 5) is not significantly different from Clcn3^−/− slices alone (2.16 ± 0.09, n= 8; P > 0.05), suggesting a purely postsynaptic action of Tat-CLC-3_107−116 on LTP. Comparisons (Student's unpaired t test) were carried out for all ISIs, but reported for only ISI = 50 ms for brevity.

Figure 7. Model of CLC-3 regulation of LTP

Left side, at a WT synapse in a mature animal, Ca²⁺ influx through the NMDAR activates CaMKII, which subsequently phosphorylates/gates CLC-3. The Cl⁻ flux through CLC-3 results in shunting of membrane potential, reduces depolarization and promotes Mg²⁺ block of the NMDAR, thereby exerting negative feedback on the Ca²⁺ transient at the synapse. This moderates the amount of potentiation that the synapse undergoes. Right side, at a Clcn3^−/− synapse, there is no regulation of Ca²⁺ influx through NMDARs by CLC-3. This leads to addition of excessive AMPA receptors (or changes in AMPA receptor conductance or distribution) and thus greater LTP for an equivalent stimulus.