Changes in the Adult GluN2B Associated Proteome following Adolescent Intermittent Ethanol Exposure

Abstract

Adolescent alcohol use is the strongest predictor for alcohol use disorders. In rodents, adolescents have distinct responses to acute ethanol, and prolonged alcohol exposure during adolescence can maintain these phenotypes into adulthood. One brain region that is particularly sensitive to the effects of both acute and chronic ethanol exposure is the hippocampus. Adolescent intermittent ethanol exposure (AIE) produces long lasting changes in hippocampal synaptic plasticity and dendritic morphology, as well as in the susceptibility to acute ethanol-induced spatial memory impairment. Given the pattern of changes in hippocampal structure and function, one potential target for these effects is the ethanol sensitive GluN2B subunit of the NMDA receptor, which is known to be involved in synaptic plasticity and dendritic morphology. Thus we sought to determine if there were persistent changes in hippocampal GluN2B signaling cascades following AIE. We employed a previously validated GluN2B-targeted proteomic strategy that was used to identify novel signaling mechanisms altered by chronic ethanol exposure in the adult hippocampus. We collected adult hippocampal tissue (P70) from rats that had been given 2 weeks of AIE from P30-45. Tissue extracts were fractionated into synaptic and non-synaptic pools, immuno-precipitated for GluN2B, and then analyzed using proteomic methods. We detected a large number of proteins associated with GluN2B. AIE produced significant changes in the association of many proteins with GluN2B in both synaptic and non-synaptic fractions. Intriguingly the number of proteins changed in the non-synaptic fraction was double that found in the synaptic fraction. Some of these proteins include those involved in glutamate signaling cytoskeleton rearrangement, calcium signaling, and plasticity. Disruptions in these pathways may contribute to the persistent cellular and behavioral changes found in the adult hippocampus following AIE. Further, the robust change in non-synaptic proteins suggests that AIE may prime this signaling pathway for future ethanol exposures in adulthood.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings

“…Thrombospondins (TSP) are signaling proteins released by astrocytes that promote synaptogenesis and are increased in adult hippocampus following adolescent intermittent ethanol (AIE; Risher et al., ). Other studies find AIE alters multiple adult synaptic proteins, particularly the adult hippocampal excitatory synaptic NMDA receptor subtype GluN2B (NR2B) proteome (Swartzwelder et al., ) consistent with increases in synaptic excitation. Astrocytes stimulate the formation of synapses, and AIE has been found to increase astrocytic volume and area as well as increasing astrocyte‐secreted synaptogenic factors, thrombospondins (TSP), and their neuronal synaptic receptor alpha2delta‐1 (α2δ‐1).…”

“…TSA administration reverses AIE‐increased adult alcohol drinking and anxiety‐like behavior as well as epigenetic and synaptic changes in amygdala (Kyzar et al., ; Pandey et al., ; Sakharkar et al., ). The anticonvulsant gabapentin reverses GluN2B receptor–mediated, AIE‐induced synaptic hyperexcitability (Swartzwelder et al., ; also see Swartzwelder et al., ). Gabapentin also increases slow‐wave sleep power, thereby reversing deficits seen in slow‐wave sleep in AIE‐exposed adults (Sanchez‐Alavez et al., ).…”

Effect of Alcohol on Hippocampal-Dependent Plasticity and Behavior: Role of Glutamatergic Synaptic Transmission

“…The subunit composition of the NMDAR changes during development, and probably this different composition of the NMDAR subunits could explain the way that the hippocampal circuit responds to alcohol at both stages. More recent studies, however, have shown that GluN2B in the extrasynaptic membranes regulates a greater number of proteins than GluN2B in the synaptic membranes in adult rats submitted to intermittent ethanol exposure during adolescence (Swartzwelder et al, 2016). In mice, the chronic intermittent ethanol exposure alters GluN2B interaction with proteins important for a mechanism of LTD dependent on the metabotropic glutamate receptors (Wills et al, 2017), raising a new possible mechanism for cognitive impairment provoked by adolescent alcohol consumption.…”

Changes in Neuroimmune and Neuronal Death Markers after Adolescent Alcohol Exposure in Rats are Reversed by Donepezil

“…Studies have shown evidence of AIE-induced neuronal loss in area CA1 11 and reduced neurogenesis in the granule cell layer of the dentate gyrus 34 in adulthood, well after the termination of AIE exposure. In addition, AIE has been shown to alter long-term potentiation (LTP) 11 , astrocyte activation 35 , N -methy-D-aspartate receptor-mediated current amplitudes 36 , and dendritic spine density 12 in the hippocampal formation in adulthood. An AIE-induced reduction of dendritic spine density was observed in the dentate gyrus, and was reversed by sub-chronic systemic treatment with donepezil in adulthood 12 .…”