Abnormal mGlu 5 receptor/endocannabinoid coupling in mice lacking FMRP and BC1 RNA

Abstract

Transcriptional silencing of the gene encoding the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP acts as a translational repressor at central synapses, and molecular and synaptic plasticity studies have shown that the absence of this protein alters metabotropic glutamate 5 receptors (mGlu5Rs)-mediated signaling. In the striatum of mice lacking FMRP, we found enhanced activity of diacylglycerol lipase (DAGL), the enzyme limiting 2-arachidonoylglicerol (2-AG) synthesis, associated with altered sensitivity of GABA synapses to the mobilization of this endocannabinoid by mGlu5R stimulation with DHPG. Mice lacking another repressor of synaptic protein synthesis, BC1 RNA, also showed potentiated mGlu5R-driven 2-AG responses, indicating that both FMRP and BC1 RNA act as physiological constraints of mGlu5R/endocannabinoid coupling at central synapses. The effects of FMRP ablation on DAGL activity and on DHPG-mediated inhibition of GABA synapses were enhanced by simultaneous genetic inactivation of FMRP and BC1 RNA. In double FMRP and BC1 RNA lacking mice, striatal levels of 2-AG were also enhanced compared with control animals and to single mutants. Our data indicate for the first time that mGlu5R-driven endocannabinoid signaling in the striatum is under the control of both FMRP and BC1 RNA. The abnormal mGlu5R/2-AG coupling found in FMRP-KO mice emphasizes the involvement of mGlu5Rs in the synaptic defects of FXS, and identifies the modulation of the endocannabinoid system as a novel target for the treatment of this severe neuropsychiatric disorder.

Figure 1

Effects of DHPG on striatal mIPSCs in FMRP-KO mice. (a) The reduction of mIPSC frequency induced by the group I mGlu receptor agonist DHPG was potentiated in FMRP-KO mice. (b) Preincubation with the CB1 receptor antagonist AM251 or with mGlu5 receptor antagonist MPEP prevented the depressant action of DHPG in WT and FMRP-KO mice. (c) Examples of voltage-clamp recordings before and during the application of DHPG in control and FMRP-KO mice. (d) Cumulative distribution of mIPSC inter-event interval recorded from WT and FMRP-KO mice before and during the application of DHPG.

Figure 2

Striatal mGlu5R expression and function in FMRP-KO mice. (a) The genetic ablation of fmr1 gene markedly increased MPEP binding to mGlu5R, and DAGL activity in the striatum. (b) Preincubation with the DAGL inhibitor orlistat prevented the depressant action of DHPG on mIPSC frequency in WT and FMRP-KO mice. (c) 2-AG-independent action of DHPG on sIPSC frequency was similar in striatal neurons from both WT and FMRP-KO mice. ^**p<0.01; ^***p<0.001.

Figure 3

Effects of the direct and indirect M1-dependent modulation of CB1Rs in FMRP-KO mice. (a) AM251 failed to increase mIPSC frequency in both WT and FMRP-KO neurons. (b) The histograms show that the genetic inactivation of fmr1 remarkably increases MAGL activity in the absence (basal) or in the presence of DHPG (DHPG) without altering 2-AG levels. (c, d) The depressant effects of HU210 on mIPSC frequency (c) and on mEPSC frequency (d) were similar in WT and in FMRP-KO mice. (e) The reduction of mIPSC frequency induced by muscarinic M1 receptors agonist McN-A-343, was similar in WT and in FMRP-KO mice. (f) Preincubation with the CB1 receptor antagonist AM251 prevented the depressant action of McN-A-343 in WT and in FMRP-KO mice. ^**p<0.01 vs WT; ^##p<0.01 vs pre-DHPG values.

Figure 4

mGlu5R/endocannabinoid interaction in BC1-KO mice. (a, b) The reduction of mIPSC frequency after the application of DHPG was potentiated in BC1-KO mice and was prevented (b) by preincubation with AM251 or with MPEP in WT and in BC1-KO mice. The electrophysiological traces on the right are examples of voltage-clamp recordings before and during the application of DHPG in WT and in BC1-KO mice. (c) MPEP binding to mGlu5Rs and DAGL activity were similar in BC1-KO and in WT mice in control condition (basal), and significantly reduced compared with FMRP-KO mice. The application of DHPG markedly increased DAGL activity in BC1-KO mice (DHPG). (d) The activity of MAGL was similar in BC1-KO and in FMRP-KO mice, both in basal condition and after stimulation with DHPG. (e) The graph shows that 2-AG levels were similar in WT and in BC1-KO mice in control condition (basal), whereas application of DHPG only increased 2-AG levels in WT mice (DHPG). (f, g) The depressant effects of HU210 on mIPSC frequency (f) and on mEPSC frequency (g) were similar in WT and in BC1-KO mice. ^**p<0.01; ^***p<0.001.

Figure 5

mGlu5R/endocannabinoid interaction in double FMRP-BC1-KO mice. (a) The reduction of mIPSC frequency after the application of DHPG was potentiated in FMRP-BC1-KO mice. (b) The electrophysiological traces are examples of voltage-clamp recordings before and during the application of DHPG in WT and FMRP-BC1-KO mice. (c) Simultaneous ablation of FMRP and of BC1 RNA markedly increased MPEP binding to mGlu5Rs and DAGL activity in the striatum. (d) The histogram shows that 2-AG levels were remarkably increased in FMRP-BC1-KO mice both in the absence (basal) and in the presence of DHPG stimulation (DHPG). (e) Preincubation with AM251 prevented the depressant action of DHPG in WT and FMRP-BC1-KO mice. (f) AM251 increased per se mIPSC frequency in FMRP-BC1-KO mice. (g) The depressant effects of HU210 on mIPSC frequency were similar in WT and in FMRP-BC1-KO mice. ^**p<0.01.