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

Abstract

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long-lasting AIE-induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long-lasting changes in neuroimmune/trophic factor balance and epigenetic-microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE-induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE-induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE-induced neuropathology and AUDs.

Keywords: Acetylcholine; Adolescence; Behavior; Binge Drinking; Development; Epigenetic; Neuroimmune.

Figure 1

Molecular mechanisms of persistent changes in proinflammatory and trophic gene expression induced by adolescent intermittent ethanol (AIE) exposure. Top: Neurobiology of Adolescent Drinking in Adulthood (NADIA) findings support an overall hypothesis that complex mechanisms involving epigenetic and noncoding RNA, particularly microRNA (miRNA), contribute to a persistent increase in proinflammatory gene expression and a loss of trophic factor expression due to signaling across neurons and glia, which contribute to persistent changes in adulthood. AIE exposure changes multiple levels of molecular signaling in adulthood, including alterations in microRNA (miRNA) and epigenetic programing that involves DNA and nuclear histone methylation and acetylation processes involved in silencing or enhancing gene expression (HDAC: histone deacetylase; DNMT: DNA methyltransferase). Bottom: Adolescent development involves signaling across neurons, microglia, and astrocytes that regulate synaptic maturation and neurocircuitry. AIE exposure increases expression of proinflammatory genes and proteins (left), including nuclear factor kappa B (NFκB), high mobility group box 1 protein (HMGB1), Toll‐like receptors (TLRs), cytokines and chemokines, and many other genes. Epigenetic markers of gene silencing such as HDAC2 and H3K9me2 are also increased after AIE. Other proteins, growth factors, and epigenetic markers are persistently increased or decreased after AIE exposure (right). Levels of the epigenetic marker H3K9ac, associated with increasing gene transcription, are reduced in brain by AIE in association with decreases in transcription of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF). AIE exposure reduces adult expression of CREB, CREB‐binding protein (CBP), and the immediate early gene Arc in amygdala. AIE persistently reduces lysine‐specific histone demethylase 1A (LSD1, also known as lysine‐specific demethylase 1A or KDM1A) that demethylates histone lysines to epigenetically regulate gene expression. These signaling mechanisms control gene expression in neurons, astrocytes, and microglia and can thereby alter synapses that change circuits. By targeting these signaling mechanisms, AIE exposure can produce long‐lasting impacts on neurocircuitry and neurobiology.

Figure 2

Glial and neuronal signals across neurons, astrocytes, and microglia lead to long‐lasting changes in synapses. Adolescent brain development occurs in part through maturation of neurocircuits and synapses. Synaptic development is regulated through astroglia and microglia. 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., 2015b). Other studies find AIE alters multiple adult synaptic proteins, particularly the adult hippocampal excitatory synaptic NMDA receptor subtype GluN2B (NR2B) proteome (Swartzwelder et al., 2016) 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). AIE also lowers adult hippocampal CA1 synaptic long‐term potentiation threshold, that is, increased potentiation, suggesting increases in excitatory synapses and/or NMDA (NR2B) receptors within excitatory synapses through association with the postsynaptic density 95 (PSD95) protein, a marker of excitatory synapses (Risher et al., 2015a). AIE increases the amplitude of NMDAR‐mediated currents in CA1 pyramidal cells and the proportion of that current driven by the NMDA excitatory receptor subunit containing GluN2B receptors (NR2B) (Swartzwelder et al., 2017). These findings suggest greater adult hippocampal network excitability after AIE, which alters plasticity mechanisms, like long‐term potentiation (LTP), and renders hippocampal circuits more vulnerable neurotoxic cell loss (Risher et al., 2015b). Similarly, AIE has been found to alter microglia, inducing a hyperramified state. Microglia regulate synapses in part through complement proteins, particularly C1q: complement component 1q, a protein complex that interacts with complement receptor 3 (CR3) (Walter and Crews, 2017). AIE increases adult microglial sensitivity to stimulation. Additional studies are needed to better understand how AIE‐induced persistent sensitization of microglia impacts synaptic regulation. Figure definitions: α2δ‐1: neuronal calcium channel subunit and binding site for thrombospondins; C1q: complement component 1q, a protein complex involved in the complement system of the innate immune system; CR3: phagocytotic complement receptor 3 of the innate immune system; NR2B: subunit of the NMDA receptor; PSD: postsynaptic density; TGFβ: transforming growth factor‐beta; TSP: thrombospondins

Figure 3

Adolescent intermittent ethanol (AIE)‐induced reductions in adult neurogenesis are prevented or reversed by multiple strategies. Doublecortin immunoreactivity‐positive (DCX+IR) cell counts are adapted from Neurobiology of Adolescent Drinking in Adulthood (NADIA) publications across multiple NADIA laboratories studying the impact of AIE on adult neurogenesis. Note different ordinate scales, as pixel densities are influenced by within‐experiment differences in chromagen color, antibodies, and signal‐to‐background ratios. (A) Vetreno and colleagues (2018) exposed rats to AIE (postnatal day [P] 25 to 55, i.g.) and assessed DCX+IR neurogenesis at P80. Voluntary wheel running from P24 to P80, concurrent with and extending beyond AIE exposure, prevented the AIE‐induced loss of DCX+IR as well as other markers of neurogenesis without significantly altering neurogenesis in control rats. Exercise also prevented AIE induction of pNFκB p65 and proinflammatory gene mRNA. (B) Vetreno and colleagues (2018) exposed rats to AIE (P25 to 55, i.g.) and assessed DCX+IR neurogenesis at P56, shortly after AIE. Indomethacin, an antiinflammatory drug, was administered during AIE (4 mg/kg, i.p.) and prevented the AIE‐induced loss of DCX+IR. Indomethacin also prevented AIE induction of pNFκB p65 and cleaved caspase‐3. (C) Sakharkar and colleagues (2016) exposed rats to AIE (P28 to 41, i.p.) and assessed DCX+IR neurogenesis at P94. Three days of trichostatin A (TSA; 1 mg/kg, i.p., P92 to 94) reversed the AIE reduction in DCX+IR. TSA also reversed AIE‐induced increases in histone deacetylase activity and reductions in brain‐derived neurotropic factor (BDNF) mRNA and H3K9ac levels of the BDNF promoter. (D) Swartzwelder et al. (2019) exposed rats to AIE (P30 to 46, i.g.) and assessed DCX+IR neurogenesis at P72. Donepezil, an anticholinesterase with antiinflammatory activity, was administered for 4 days (2.5 mg/kg, i.p., P67 to 72) and reversed AIE reductions in DCX+IR. Donepezil also reversed the AIE‐induced increases of RAGE, phosphorylated (activated) nuclear transcription factor pNFκB p65, and the gene silencing marker dimethylated histone H3K9.

Figure 4

Adolescent intermittent ethanol (AIE)‐induced reductions in choline acetyltransferase (ChAT) are prevented or reversed by multiple strategies. ChAT immunoreactivity‐positive (DCX+IR) cell counts are adapted from Neurobiology of Adolescent Drinking in Adulthood (NADIA) publications studying the impact of AIE on cholinergic phenotype. Note different ordinate scales. (A) Vetreno and Crews (2018) exposed rats to AIE (postnatal day [P] 25 to 55, i.g.) and assessed ChAT+IR at P80. Voluntary wheel running from P24 to P80, concurrent with and extending beyond AIE exposure, prevented the AIE‐induced loss of ChAT+IR without significantly altering levels in control rats. (B) Vetreno and colleagues (2019) exposed rats to AIE (P25 to 55, i.g.) and assessed ChAT+IR at P95. Voluntary wheel running from P56 to P95, after AIE exposure, reversed the AIE‐induced loss of ChAT+IR without significantly altering levels in control rats. (C) Vetreno and Crews (2018) exposed rats to AIE (postnatal day [P] 25 to 55, i.g.) and assessed ChAT+IR at P56, shortly after AIE. Indomethacin, an antiinflammatory drug, was administered during AIE (4 mg/kg, i.p.) and prevented the AIE‐induced loss of ChAT+IR.

Figure 5

Prevention and reversal of adolescent intermittent ethanol (AIE) neuropathology by multiple strategies. Top: Epigenetic mechanisms involving histone and DNA methylation and acetylation are known mechanisms regulating gene expression, that is, silencing or enhancing transcription, and are persistently altered by AIE. Neurobiology of Adolescent Drinking in Adulthood (NADIA) findings support a shift in proinflammatory/trophic balance caused by AIE activation of neuroimmune signaling through increased high mobility group box 1 protein (HMGB1), Toll‐like receptors, and other signaling that increase nuclear factor kappa B (NFκB) transcription and blunt transcription of trophic factors such as brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF). These changes likely lead to the observed AIE‐induced reductions in neurogenesis and neuronal phenotype markers (choline acetyl transferase [ChAT], dopamine [DA], serotonin [5‐HT]), as well as a shift toward synaptic excitability (middle). Together, these physiological changes may manifest as AIE alterations in a variety of neuronal circuit and behavioral effects (bottom), including disrupted sleep, anxiety, risky decisions, alcohol drinking, and cognitive inflexibility. NADIA AIE studies find that a variety of pharmacological and behavioral strategies (shown in red) can prevent or reverse these AIE effects. Exercise and the antiinflammatory drug indomethacin prevent AIE‐induced changes in neurogenesis, ChAT, and cognitive inflexibility (Vetreno et al., 2018). Donepezil, an anticholinesterase that is also antiinflammatory, reverses AIE‐induced changes in epigenetic programing of proinflammatory genes, trophic factors, histones, and synapses ([Link]). The histone deacetylase inhibitor trichostatin A (TSA) reverses AIE‐induced decreases in trophic factor expression, synapses, and neurogenesis as well as increased alcohol drinking and anxiety (Sakharkar et al., 2016). Other pharmacological agents including the positive allosteric modulator of type‐5 metabotropic glutamate receptor CDPPB (Gass et al., 2014), oxytocin and vasopressin receptor ligands (Dannenhoffer et al., 2018), and the anticonvulsant gabapentin (Sanchez‐Alavez et al., 2018b; Swartzwelder et al., 2017) have reversed specific behavioral effects. Although no individual study has integrated all potential mechanisms and level of assessment, overall the prevention and reversal studies together strongly support an AIE‐induced persistent shift in brain proinflammatory/trophic gene expression that crosses glia and neurons, altering synapses and neurotransmitters that contribute to long‐lasting changes in adult neurophysiology, neurocircuitry, and behavior.