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. 2022 Dec:105:9-24.
doi: 10.1016/j.alcohol.2022.08.006. Epub 2022 Aug 30.

Effect of chronic intermittent ethanol vapor exposure on RNA content of brain-derived extracellular vesicles

Annalisa M Baratta  1 , Regina A Mangieri  2 , Heather C Aziz  2 , Marcelo F Lopez  3 , Sean P Farris  4 , Gregg E Homanics  5
Affiliations

Effect of chronic intermittent ethanol vapor exposure on RNA content of brain-derived extracellular vesicles

Annalisa M Baratta et al. Alcohol. 2022 Dec.

Abstract

Extracellular vesicles (EVs) are important players in normal biological function and disease pathogenesis. Of the many biomolecules packaged into EVs, coding and noncoding RNA transcripts are of particular interest for their ability to significantly alter cellular and molecular processes. Here we investigate how chronic ethanol exposure impacts EV RNA cargo and the functional outcomes of these changes. Following chronic intermittent ethanol (CIE) vapor exposure, EVs were isolated from male and female C57BL/6J mouse brain. Total RNA from EVs was analyzed by lncRNA/mRNA microarray to survey changes in RNA cargo following vapor exposure. Differential expression analysis of microarray data revealed a number of lncRNA and mRNA types differentially expressed in CIE compared to control EVs. Weighted gene co-expression network analysis identified multiple male and female specific modules related to neuroinflammation, cell death, demyelination, and synapse organization. To functionally test these changes, whole-cell voltage-clamp recordings were used to assess synaptic transmission. Incubation of nucleus accumbens brain slices with EVs led to a reduction in spontaneous excitatory postsynaptic current amplitude, although no changes in synaptic transmission were observed between control and CIE EV administration. These results indicate that CIE vapor exposure significantly changes the RNA cargo of brain-derived EVs, which have the ability to impact neuronal function.

Keywords: alcohol use disorder; extracellular vesicles; lncRNA; synaptic transmission; transcriptome.

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Conflict of interest statement

Conflicts of interest Authors have no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Extracellular vesicle (EV) characterization following chronic intermittent ethanol (CIE) vapor exposure. (A) Representative nanoparticle tracking analysis (NTA) trace showing particle size distribution for CIE EVs isolated by differential ultracentrifugation. (B) Electron microscopy image of CIE EVs. No significant change in brain-derived EV (C) size or (D) concentration of diluted (1:1000) EVs following CIE vapor exposure compared to air-exposed controls as assessed by NTA. Data are mean ± SEM; N = 6 males per group.
Fig. 2.
Fig. 2.
Differentially expressed probe targets (DEPTs) from extracellular vesicle RNA in response to chronic intermittent ethanol vapor exposure. Blue represents significantly down-regulated DEPTs and red represents significantly up-regulated DEPTs. Volcano plots represent (A) female mRNA (393 down-regulated; 800 up-regulated), (B) female lncRNA (635 down-regulated; 1291 up-regulated), (C) male mRNA (212 down-regulated; 109 up-regulated), and (D) male lncRNA (330 down-regulated; 290 up-regulated).
Fig. 3.
Fig. 3.
Venn diagrams comparing male and female differentially expressed probe targets (DEPTs) of EV RNA following chronic intermittent ethanol vapor exposure. Shown here are results for up-regulated (across the top) and down-regulated (across the bottom) mRNA (left) and lncRNA (right) DEPTs. Males are represented in blue and females in pink. p value represents significant overlap between male and female samples.
Fig. 4.
Fig. 4.
Effects of EVs on synaptic transmission in the NAc. (A) Representative current traces showing spontaneous excitatory postsynaptic currents (sEPSCs) recorded at −55 mV holding potential. (B) Cumulative probability distributions of sEPSC amplitudes in 2.5-pA bins. (C) Mean sEPSC amplitudes. (D) Cumulative probability distributions of sEPSC area (event charge transfer) in 25 pA*msec bins. (E) Mean sEPSC areas (event charge transfer). (F) Cumulative probability distributions of sEPSC decay times (90%–10%) in 1-msec bins. (G) Mean sEPSC decay times (90%–10%). (H) Representative current traces showing spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at +10-mV holding potential. (I) Cumulative probability distributions of sIPSC rise times (10%–90%) in 0.5-msec bins. (J) Mean sIPSC rise times (10%–90%). Inset shows amplitude-normalized, averaged sIPSC traces from one neuron per group overlaid and aligned at baseline. (K) Cumulative probability distributions of sIPSC decay times (90%–10%) in 2-msec bins. (L) Best-fit curves overlaid on each group’s normalized probability distribution (histogram of binned data), with corresponding R2 values for each curve. Inset shows amplitude-normalized, peak-aligned, averaged sIPSC traces for slow- and fast-decaying events from one neuron per group. (M) Mean sIPSC decay times (90%–10%). Bar graphs show individual data for each neuron (circles) overlaid on group means ± SEM (bars). Cumulative probability distributions of binned data show group means ± SEM. For all sEPSC data, group n’s (neurons/mice) = 13/5, PBS control, 12/5, Air, 11/6, CIE. For sIPSC data shown here, n’s (neurons/mice) = 10/5, PBS control, 10/5, Air, 10/6, CIE. When Air and CIE distributions did not differ from each other, they are shown in the panel insets using the same ranges of x- and y-axes as the main panel, and the cumulative distributions of the pooled data from these two groups are shown by the “EVs” plots in the main panels. Dashed lines are used to indicate significance for two-way ANOVAs comparing all three groups; solid lines are used to indicate significance between two groups. *, ****, p < 0.05, 0.0001, effect of group in t test, Tukey's multiple comparisons, F test, or two-way ANOVA. ##, ###, ####, p < 0.01, 0.001, 0.0001, bin × group interaction in two-way ANOVA. ‡, p < 0.05, CIE vs. PBS for the indicated bin, Tukey's multiple comparisons.
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