Alcohol inhibits NR2B-containing NMDA receptors in the ventral bed nucleus of the stria terminalis

Abstract

Components of the mesolimbic dopamine system, in particular dopaminergic cells in the ventral tegmental area (VTA), have been implicated in the acute reinforcing actions of ethanol. The ventral bed nucleus of the stria terminalis (vBNST) potently regulates dopaminergic cell firing in the VTA, and has been implicated in the behavioral actions of ethanol. The N-methyl-D-asparate receptor (NMDAR) is a major molecular target of ethanol, however, current evidence suggests that ethanol regulation of NMDAR function is widely variable and likely depends on a number of factors. Thus, it is critical to investigate ethanol regulation of NMDAR function at synapses relevant to ethanol-regulated behaviors, such as in the vBNST. Here we show, using multiple techniques, that ethanol inhibits NMDAR function in vBNST neurons in a postsynaptic fashion. Further, we demonstrate the functional presence of both NR2A and NR2B-containing NMDARs in the vBNST. While genetic removal of NR2A did not alter the magnitude of ethanol inhibition, pharmacological blockade of NR2B rendered synaptically activated NMDARs insensitive to ethanol inhibition. Finally, we demonstrate that ethanol inhibits NMDARs in cells in the vBNST that project to the VTA, providing a direct means by which ethanol in the vBNST can modulate the dopaminergic system.

Figure 1

Ethanol inhibits NMDA currents in vBNST in a concentration-dependent and reversible fashion via a postsynaptic mechanism. (a) Ethanol inhibited NMDA-EPSCs in a concentration-dependent fashion. (Inset) Representative traces from an experiment demonstrating the effect of 50 mM ethanol on NMDA-EPSCs and reversal during washout (25 mM, n = 5; 50 mM, n = 5; 100 mM, n = 5). (b) A total of 50 mM of ethanol did not alter the kinetics of the NMDA-EPSC, as shown in representative normalized traces. (c) Average decay time, shown here as the weighted τ, demonstrates the lack of effect of 50 mM ethanol on the decay kinetics of the evoked NMDA-EPSC (n = 5). (d) Application of 50 mM ethanol had no effect on electrically evoked AMPA receptor-mediated EPSCs (n = 6). (e) Representative traces demonstrating the lack of an effect of 50 mM ethanol on the paired pulse ratio of AMPA EPSCs. (f) Pooled data demonstrating the lack of an effect of 50 mM ethanol on the paired pulse ratio of AMPA-EPSCs. (g) A total of 50 mM ethanol inhibited currents evoked by exogenous application of NMDA (n = 5). (inset) Representative traces demonstrating the inhibitory effect of 50 mM ethanol on exogenously applied NMDA.

Figure 2

Synaptic NMDARs in the vBNST contain the NR2B subunit. (a) Bath application of the NR2B-selective antagonists, ifenprodil (n = 5), and Ro 25–6981 (n = 5) (b) inhibited evoked NMDA currents in the vBNST. (c) Ro 25–6981 did not alter the kinetics of the NMDA-EPSC, as demonstrated in the representative normalized traces. (d) Average decay time, shown here as weighted τ, demonstrates the lack of effect of Ro-25–6981 on the decay kinetics of the evoked NMDA-EPSC (n = 5).

Figure 3

Synaptic NMDARs in the vBNST contain the NR2A subunit. (a) Representative amplitude-normalized NMDA-EPSC from NR2A knockout and wild-type demonstrating alterations in the kinetic profile. (b) There was a significant increase in both the decay and (c) rise time when compared to NMDA-EPSCs from wild-type animals (NR2A knockout, n = 12; wild type, n = 10). (d) Representative traces demonstrating the robust inhibition of NMDA-EPSCs by the NR2B-selective antagonist, Ro 25–6981, in the NR2A knockout animal. *p < 0.05 using Student’s t-test.

Figure 4

Ethanol inhibits NMDA-EPSCs in an NR2B-dependent fashion. (a) The ability of 100 mM ethanol to inhibit NMDA-EPSCs was intact in NR2A knockout mice, n = 5. (b) A representative experiment showing the reduced effect of 100 mM ethanol following application of 2 µM Ro 25–6981. (c) Pooled data demonstrating the impaired ability of 100 mM ethanol to inhibit NMDA-EPSCs following application of Ro 25–6981, n = 5. (d) A representative experiment showing the effect of 100 mM ethanol following application of 10 µM dl-APV, n = 4. (e) Pooled data demonstrating the ability of 100 mM ethanol to inhibit NMDA-EPSCs in the presence of 10 µM dl-APV. (f) The inhibitory effect of 100 mM ethanol is altered following treatment of Ro 25–6981, but not following treatment of dl-APV or in the NR2A knockout when compared to wild-type animals.

Figure 5

Ethanol inhibits NMDA-EPSCs in vBNST projection neurons. (a) Diagram adapted from mouse brain atlas showing coronal section at AP level of the VTA. (b) Bright field image of a typical VTA microsphere injection site at the same level as A. (c) Merged fluorescent and IR-DIC images of vBNST cells demonstrating the presence of labeled fluorescent microspheres following injection of microspheres into the VTA. The tracer-labeled cell in the center was patched and tested for effects of ethanol. Fluorescent channel of the tracer-filled cell (inset). (d) A total of 50 mM ethanol inhibited NMDA-EPSCs in vBNST cells that project to the VTA (n = 7).

Figure 6

Model illustrating potential effects of NR2B antagonists on diheteromeric and triheteromeric NMDARs. (Aa) Schematic representation of a synapse containing a combination of diheteromeric NR2A and NR2B NMDARs. The individual kinetic profiles are shown color-coded below the receptor diagrams. The total NMDA-EPSC profile is in black. The NMDA-EPSC from the NR2A diheteromers are shown with a rapid decay, as has been noted previously. The NMDA-EPSC from the NR2B diheteromers are shown with a slower decay, as was seen in the NR2A knockout. (Ab) A schematic representation of the effects of an NR2B antagonist (denoted simply by the black X) on a mixed population of diheteromeric NMDARs. The resulting synaptic trace (in black) has a faster decay compared to the nodrug condition, as has been shown in the hippocampus in young rat hippocampus. (Ba) Schematic representation of a synapse containing triheteromeric NMDARs containing NR2A and NR2B subunits. (Bb) A schematic representation of the effects of an NR2B antagonist on triheteromeric receptors. Recent evidence (Hatton and Paoletti, 2005) demonstrated that NR2B antagonists can inhibit triheteromeric receptors, albeit with reduced efficacy, as demonstrated with the reduced amplitude of the traces as compared to those in b1. However, it is important to note the lack of alteration in the decay kinetics, reflecting the results obtained in the vBNST. Our data are most consistent with the hypothesis that the majority of NMDARs in the vBNST neurons are of this type. It is important to note that the traces shown in this figure are illustrations.