Necessary, but not sufficient: insights into the mechanisms of mGluR mediated long-term depression from a rat model of early life seizures

Abstract

Using the rat model of early life seizures (ELS), which has exaggerated mGluR mediated long-term depression of synaptic strength (mGluR-LTD) in adulthood, we probed the signaling cascades underlying mGluR-LTD induction. Several inhibitors completely blocked mGluR-LTD in control but not in ELS rats: the proteasome, the mammalian target of rapamycin (mTOR), S6 kinase (S6K), or L-type voltage-gated calcium channels (L-type VGCC). Inhibition of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) resulted in a near complete block of mGluR-LTD in control rats and a slight reduction of mGluR-LTD in ELS rats. "Autonomous" CaMKII was found to be upregulated in ELS rats, while elevated S6K activity, which is stimulated by mTOR, was described previously. Thus, modulation of each of these factors was necessary for mGluR-LTD induction in control rats, but even their combined, permanent activation in the ELS rats was not sufficient to individually support mGluR-LTD induction following ELS. This implies that while these factors may act sequentially in controls to mediate mGluR-LTD, this is no longer the case after ELS. In contrast, activated ERK was found to be significantly down-regulated in ELS rats. Inhibition of MEK/ERK activation in control rats elevated mGluR-LTD to the exaggerated levels seen in ELS rats. Together, these results elucidate both the mechanisms that persistently enhance mGluR-LTD after ELS and the mechanisms underlying normal mGluR-LTD by providing evidence for multiple, convergent pathways that mediate mGluR-LTD induction. With our prior work, this ties these signaling cascades to the ELS behavioral phenotype that includes abnormal working memory, fear conditioning and socialization.

Keywords: Autism; CaMKII; Early life seizure; Long-term depression; Metabotropic glutamate receptor; Voltage-gated calcium channels.

Figure 1. Increased phosphorylation of CaMKII following ELS

A, ELS caused no changes in expression of total CaMKIIα. B, ELS caused significant increases in phosphorylation of site T286 on CaMKIIα. C, ELS caused no changes in phosphorylation of the CaMKII site T305/6. D, ELS did not change total expression of CaMKIIβ. E, ELS caused significant increases in phosphorylation of site T287 on CaMKIIβ. F, example western blots for CaMKIIα T286. Semi-quantitative western-blot technique (Cornejo et al., 2007) was used to determine immunoreactivity/mg loaded protein, which were normalized to controls (see Methods).

Figure 2. Increased phosphorylation of synaptic CAMKII

A, CaMKIIa P-T286 was significantly elevated in the P2 fraction (control P2: 100.00 ± 18.00%, n = 7, ELS P2: 177.40 ± 27.20%, n = 9, P < 0.05, Student’s t-test), but not in the S2 fraction (control S2: 100.00 ± 16.40%, n = 11, ELS S2: 138.70 ± 35.50%, n = 12, P = 0.474, Student’s t-test). B, mimicking this trend, CaMKIIβ P-T287 was significantly elevated in the P2 fraction (control P2: 100.00 ± 11.60%, n = 7, ELS P2: 180.76 ± 23.88%, n = 7, P < 0.02, Student’s t-test), but not in the S2 fraction (control S2: 100.00 ± 18.40%, n = 7, ELS S2: 103.75 ± 28.69%, n = 9, P = 0.908, Mann-Whitey Rank Sum). Semi-quantitative western-blot technique (Cornejo et al., 2007) was used to determine immunoreactivity/mg loaded protein, which were normalized to controls (see Methods).

Figure 3. Enhanced mGluR-LTD following ELS in adult rats and the altered role of CaMKII in mGluR-LTD induction

A, Interleaved (vehicle, no inhibitor) mGluR-LTD experiments (control: 80.32 ± 1.56%, n = 18, ELS: 53.50 ± 3.99%, n = 18, P < 0.001, Student’s t-test). B, Inhibition of CaMKII by tatCN21 (2 µM) completely blocked mGluR-LTD in control rats yet mGluR-LTD was still present in ELS rats (control: 93.72 ± 1.31%, n = 11, ELS: 68.39 ± 1.98%, n = 10, P < 0.001, Student’s t-test). Insets show averages of 4 fEPSPs near the numerically indicated time points. Scale bar 0.5 mV × 15 ms. Dashed line indicates baseline; Dashed/dotted line indicates average control mGluR-LTD (vehicle, no inhibitors from (A)) used in subsequent Figures; Dotted line indicates average ELS mGluR-LTD (vehicle, no inhibitors from (A)) used in subsequent Figures.

Figure 4. The role of S6K and the proteasome in mGluR-LTD induction was altered after ELS

A, The S6K inhibitor PF-4708671 (25 µM) completely blocked mGluR-LTD in controls, yet mGluR-LTD was still present in ELS rats (control: 106.14. ± 9.71%, n = 6, ELS: 54.96±8.75%, n = 10, P < 0.01, Student’s t-test). B, The proteasomal inhibitor MG-132 (10 µM) completely blocked mGluR-LTD in controls, yet mGluR-LTD was still present in ELS rats (control: 99.93 ± 6.05%, n = 8, ELS: 62.93 ± 7.83%, n = 6, P = 0.0025, Student’s t-test). Insets show averages of 4 fEPSPs near the numerically indicated time points. Scale bar 0.5 mV × 15 ms. Dashed line indicates baseline; dashed/dotted line indicates average control mGluR-LTD (vehicle, no inhibitors); dotted line indicates average ELS mGluR-LTD (vehicle, no inhibitors).

Figure 5. The role of L-type VGCCs and mTOR in mGluR-LTD induction was altered after ELS

A, mGluR-LTD was completely blocked in control rats in the presence of the L-type voltage-gated calcium channel antagonist isradipine (10 µm), yet mGluR-LTD was still present in ELS rats (control with isradipine: 100.44 ± 4.70%, n = 5, ELS with isradipine: 75.67 ± 5.26%, n = 9, P < 0.01, Student’s t-test). B, mGluR-LTD was completely blocked in control rats in the presence of the mTOR antagonist rapamycin (20 nM), yet mGluR-LTD was still present in ELS rats(control: 96.71 ± 2.45%, n = 7, ELS: 58.09 ± 7.20%, n = 5, P < 0.001, Student’s t-test). Insets show averages of 4 fEPSPs near the numerically indicated time points. Scale bar 0.5 mV × 15 ms. Dashed line indicates baseline; dashed/dotted line indicates average control mGluR-LTD (vehicle, no inhibitors); dotted line indicates average ELS mGluR-LTD (vehicle, no inhibitors).

Figure 6. Reduced phosphorylation of ERK and role of ERK inhibition following ELS

A, ELS caused no changes in expression of total ERK (control: 100.00 ± 29.05%, n = 11, ELS: 113.97 ± 22.50%, n = 11, P = NS, Student’s t-test). B, ELS caused significantly reduced phosphorylation of ERK at Thr202/Tyr204 (control: 100.00 ± 34.12%, n = 7, ELS: 28.88 ± 8.52%, n = 8, P < 0.05, Student’s t-test). Semi-quantitative western-blot technique (Cornejo et al., 2007) was used to determine immunoreactivity/mg loaded protein, which were normalized to controls (see Methods). C, The MEK/ERK 1/2 inhibitor U0126 (5 µM) did not significantly alter mGluR-LTD between ELS and controls (control: 46.97 ± 9.56%, n = 7, ELS: 68.77 ± 6.06%, n = 7, P < 0.08, Student’s t-test). Scale bar 0.5 mV × 15 ms. Dashed line indicates baseline; dashed/dotted line indicates average control mGluR-LTD (vehicle, no inhibitors); dotted line indicates average ELS mGluR-LTD (vehicle, no inhibitors).

Figure 7. Summary of mGluR-LTD experiments

* - indicates significantly different than similarly treated control; # - indicates significantly different than naïve control mGluR-LTD (vehicle, no inhibitors); $ - indicates significantly different from naïve ELS mGluR-LTD (vehicle, no inhibitors). Dashed line indicates baseline; dashed/dotted line indicates average control mGluR-LTD (vehicle, no inhibitors); dotted line indicates average ELS mGluR-LTD (vehicle, no inhibitors).