Chronic ethanol induces synaptic but not extrasynaptic targeting of NMDA receptors

Abstract

The development of ethanol tolerance and dependence reflects neuroadaptive changes in response to continuous depression in synaptic activity. The present study used confocal imaging and electrophysiology procedures to assess the effects of prolonged ethanol exposure on NMDA receptor trafficking in cultures of hippocampal neurons. Neurons exposed to 50 mm ethanol for 4 d showed an increase in the colocalization of NMDA receptor type 1 (NR1) clusters with the presynaptic marker protein synapsin. This was accompanied by significant increases in the size and density of these synapsin-associated clusters with no change observed in nonsynapsin-associated NR1 clusters. Similar effects were observed with NR2B clustering after chronic ethanol exposure. The increase in synaptic NMDA receptor clustering was prevented by addition of a protein kinase A inhibitor or by coexposure to a low concentration of NMDA and was reversed when ethanol was removed from the cultures. No changes were observed in the synaptic content, cluster size, or density of AMPA receptors after ethanol exposure. Electrophysiological measurements on ethanol-treated neurons revealed a similar enhancement in synaptic NMDA currents with no change in AMPA-mediated events. After isolation of extrasynaptic NMDA receptors by MK801 (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (/) trapping, whole-cell responses to NMDA were not different between control and ethanol-treated neurons These observations demonstrate that neuroadaptive changes in NMDA receptors in response to prolonged ethanol exposure occur through activity-dependent processes that regulate their synaptic targeting and localization.

Figure 1.

Chronic ethanol exposure enhances the dendritic clustering of NMDA receptors. Immunohistochemistry of NR1 and confocal imaging was used to examine the effects of ethanol exposure on NMDA receptor clustering in hippocampal cultures. A-C, Confocal imaging of NR1 revealed punctate clustering of NMDA receptors in control cultures (A). A 4 d exposure of neuronal cultures to 25 mm ethanol (B) did not alter the cluster pattern, whereas 50 mm ethanol (C) exposure resulted in a striking increase in clustering. Scale bar, 40 μm. D, E, Quantification of the effects of ethanol on clustering revealed a significant increase in both NR1 cluster size (D) and density (E). The asterisk indicates a significant difference from control (p < 0.01, Student's t test; n = 90).

Figure 2.

Chronic ethanol induces the synaptic targeting of NMDA receptors. A-F, Dual immunohistochemistry of NR1 (green) and synapsin (syn; red) revealed that ∼50% of the NR1 clusters colocalize with synapsin (A, D) under control conditions. Ethanol exposure (50 mm; 4 d) resulted in a significant increase in NR1/synapsin colocalization to ∼80% (B, D). The ethanol-induced increase in NR1 cluster size (E) and density (F) was limited to the synaptic compartment with no changes occurring in extrasynaptic NR1 clustering. The addition of NMDA (2.5 μm) completely prevented the ethanol-induced increases in NR1 synaptic colocalization (C, D) and increases in synaptic cluster size (E) and density (F). ^*, Significant difference from control; #, significant difference from ethanol treated (p < 0.01; ANOVA with Student Newman-Keuls; n = 90). Scale bar, 5 μm. G, H, Quantification of synapsin clusters revealed that ethanol also significantly increased cluster size (G) and density (H). The asterisk indicates significant difference from control (p < 0.01, Student's t test; n = 90). I, J, Dual immunohistochemistry of synapsin (red) and synaptophysin (sypt; green) revealed that ethanol (50 mm; 4 d) increased clustering of synaptophys in similar to that observed with synapsin. Scale bar, 5 μm. K, Actin staining with fluorescently labeled phallodin revealed a population of NR1 clusters that are localized to the heads of spines (filled arrow) and a population of NR1 clusters along the main dendrite that are not associated with spines (arrowhead). Some immature spines were without NR1 clusters (open arrow). Scale bar, 5 μm.

Figure 3.

Chronic ethanol enhances the synaptic clustering of NR2B. A, B, Immunohistochemistry and confocal imaging of NR2B subunits revealed numerous small punctate clusters along dendrites similar to that observed with NR1. This clustering was greatly increased after exposure to ethanol (50 mm; 4 d) (compare A and B). Scale bar, 40 μm. C, D, Quantification of clustering indicated a significant increase in both cluster size (C) and density (D). The asterisk indicates a significant difference from control (p < 0.01, Student's t test; n = 90). E, G, Dual immunohistochemistry of NR2B (green) and synapsin (syn; red) revealed ∼65% colocalization of NR2B and synapsin clusters under control conditions (E, G). After ethanol exposure (50 mm; 4 d), this colocalization increased to ∼80% (F, G). The asterisk indicates significant difference from control (p < 0.01, Student's t test; n = 90). Scale bar, 5 μm. H, I, Analysis of clustering in the synaptic and extrasynaptic compartments revealed that ethanol treatment greatly increased the size (H) and density (I) of synaptic clusters, with only minor increases in extrasynaptic clusters. The asterisk indicates a significant difference from control (p < 0.01, Student's t test; n = 90).

Figure 4.

Chronic ethanol does not alter synaptic clustering of GluR1. Dual immunohistochemistry and confocal imaging of GluR1 and synapsin show almost complete colocalization of clusters in control cultures. Ethanol (50 mm; 4 d) had no effect on GluR1 clustering or colocalization with synapsin (n = 10). Scale bar, 20 μm.

Figure 5.

Ethanol-induced increase in synaptic clustering of NR1 is reversed after removal of ethanol. A, B, Culture dishes were removed from the ethanol vapor chambers, and ethanol was allowed to slowly evaporate from the culture media. NR1 (green) and synapsin (red) colocalization returned to control levels after 8 d. Scale bar, 20 μm.

Figure 6.

The ethanol-induced increase in synaptic NR1 clustering is PKA dependent. Dual immunoblotting and confocal imaging were used to examine the effects of a PKA inhibitor on NR1 (green) and synapsin (red) colocalization. A, B, In control cultures, a 24 hr exposure to KT-5720 (KT; 1 μm) reduced NR1/synapsin colocalization. The addition of KT-5720 on the final day of ethanol exposure (50 mm; 4 d) completely prevented the ethanol-induced increase in clustering. ^*, Significant difference from control (p < 0.01); @, significant difference from control and ethanol alone (0.05); #, significant difference from ethanol alone (p < 0.01; ANOVA with Student Newman-Keuls). C, D, Examination of the effects of KT-5720 on NR1 clustering in the synaptic and extrasynaptic compartments revealed that the effect of KT-5720 on NR1 cluster size (C) and density (D) were specific for the synaptic receptor compartment. ^*, Significant difference from control (p < 0.01); @, significant difference from control and ethanol alone (p < 0.01); #, significant difference from ethanol alone (p < 0.01; ANOVA with Student Newman-Keuls). B-D, n = 60 for control, KT, and EtOH plus KT groups; n = 30 for EtOH group.

Figure 7.

Chronic ethanol exposure enhances synaptic but not extrasynaptic NMDA currents and does not affect synaptic AMPA currents. A, In the presence of APV (10 μm) to block NMDA receptors, ethanol (50 mm; 4 d) had no effect on either the amplitude or frequency of mEPSCs_AMPA (Student's t test; n = 7). B, Ethanol treatment (50 mm; 4 d) increased the mEPSC NMDA/AMPA amplitude ratio. When culture dishes were removed from the ethanol vapor chambers and ethanol was allowed to evaporate from the culture media, the NMDA/AMPA current ratio returned to control levels. The asterisk indicates a significant difference from control (p < 0.05, Student's t test; n = 7). C, Ethanol (50 mm; 4 d) did not alter either the amplitude or frequency of mIPSCs_GABAA (Student's t test; n = 3-4). D, Activation of synaptic NMDA receptors by the addition of the NMDA receptor coagonist glycine resulted in enhanced NMDA current amplitude in ethanol-treated cultures compared with untreated controls. The asterisk indicates a significant difference from control (p < 0.05, Student's t test; n = 5-8). E, F, Extrasynaptic NMDA receptor currents were isolated from synaptic currents by selectively blocking synaptic NMDA receptors using the MK-801 trapping technique. D, Stimulation of extrasynaptic NMDA receptors by application of NMDA revealed extrasynaptic currents constituted approximately two-thirds of the total current (compare the size of the before and after MK-801 trace). E, Ethanol treatment (50 mm; 4 d) did not alter the NMDA concentration-response curve for activation of extrasynaptic NMDA receptors (n = 5).

Figure 8.

Chronic ethanol exposure enhances spontaneous EPSCs and network activity. A, B, In the absence of extracellular Mg²⁺, the addition of APV to isolate EPSCs_AMPA (A) or NBQX to isolate EPSCs_NMDA (B) revealed a significant increase in the amplitude and frequency of both AMPA and NMDA components of the EPSC when measured immediately after the removal of the ethanol-containing culture media. Symbols indicate significant difference from control (^*p < 0.01; @ p < 0.05; Student's t test; n = 3). C, In the presence of extracellular Mg²⁺, chronic ethanol treatment resulted in an increase in network driven spontaneous spike activity. The asterisk indicates a significant difference from control (p < 0.05, Student's t test; n = 5-6).