α-Conotoxin TxIB and Alcohol Withdrawal in Zebrafish

Background and Purpose

Alcohol use disorder (AUD) is a significant public health concern.
Previous research suggests a connection between nicotinic acetylcholine receptors (nAChR) and alcohol addiction.
The specific role of α6β2* nAChR in alcohol addiction is not well understood.
The study aims to investigate α6β2* nAChR as a potential drug target for alcohol withdrawal.

Methods

Zebrafish (Danio rerio) were exposed to 0.2% alcohol for 14 days, followed by 7 days of repeated withdrawal.
α-conotoxin TxIB (a selective α6β2* nAChR antagonist) was injected retro-orbitally.
Open Field Test was used to assess zebrafish behavior.
ELISA and quantitative real-time PCR (RT-PCR) were used to measure monoamine neurotransmitter levels and mRNA expression in the zebrafish brain.
RNA-sequencing (RNA-seq) and bioinformatics analysis were conducted to explore the potential regulatory network of TxIB after alcohol withdrawal.

Results

The withdrawal group showed a significantly higher max speed in the center area of the Open Field Test compared to the control group.
TxIB injection corrected this behavioral abnormality.
No significant changes were observed in the amount and mRNA expression of monoamine neurotransmitters after alcohol withdrawal and TxIB administration.
RNA sequencing revealed 657 genes with aberrant expression in the zebrafish brain after alcohol withdrawal.
TxIB injection reversed the expression of 225 of these genes.
These reversed genes were significantly enriched in the calcium ion binding pathway.
The gene expression profile was further validated by RT-PCR.

Conclusion

α-conotoxin TxIB improved behavioral abnormality induced by alcohol withdrawal in zebrafish.
TxIB altered gene expression, primarily in the calcium signaling pathway.
α-conotoxin TxIB shows promise as a potential therapeutic agent for alcohol withdrawal.

Keywords

Alcohol withdrawal
α6β2p nAChR
α-conotoxin TxIB
Zebrafish
Transcriptome analysis
RT-PCR
ELISA
Monoamine neurotransmitters

Introduction

Alcohol abuse poses a significant risk to human health and creates an economic burden.
According to the WHO, 3 million deaths annually are attributed to alcohol misuse, accounting for 5.3% of global deaths.
Alcohol is a predisposing factor for approximately 5.1% of diseases.
Alcohol-related deaths are notably high in the 20-39 age range, accounting for 13.5% of deaths.
Alcohol can damage multiple organs, especially the cardiovascular system, increasing the risk of atrial fibrillation, myocardial infarction, and congestive heart failure (Whitman et al., 2017).
Four medications are FDA-approved for AUD treatment: disulfiram, naltrexone (oral and injectable), and acamprosate, each with different mechanisms and limitations.
The neural basis of AUD is not well understood.
nAChRs in the mesolimbic dopamine system regulate the rewarding effects of alcohol (Powers et al., 2013).
Mecamylamine, a non-selective nAChR antagonist, prevents alcohol-induced dopamine level increases in the pre-VTA, suggesting nAChRs in the VTA regulate alcohol reward (Touchette et al., 2018).
High expression of α6 subunit-containing nAChRs in midbrain dopamine neurons suggests involvement in reward-related behaviors (Yang et al., 2009).
Mice with high α6 nAChR expression consumed more alcohol and formed conditioned place preferences at low alcohol concentrations (Powers et al., 2013).
Patch-clamp recordings show α6* nAChRs are selectively activated by low-dose alcohol (0.1–5 mM) (Gao et al., 2019).
Alcohol increases the transient frequency and amplitude of dopamine neurons in the nucleus accumbens, blocked by the α6β2* nAChR antagonist α-conotoxin MII (Gao et al., 2019).
Selective α6β2* nAChR antagonists are expected to play a significant role in studying α6β2* nAChR's role in alcohol abuse and may suggest a new therapeutic strategy.
nAChRs are involved in the withdrawal process.
Mecamylamine attenuates ethanol withdrawal-induced signs (Bhutada et al., 2010).
Chronic mecamylamine administration attenuates ethanol withdrawal signs, supporting nAChR involvement in ethanol dependence.
Acute nicotine exposure prevents abstinence symptoms in animals undergoing ethanol withdrawal, while acute alcohol injection prevents symptoms in mice undergoing nicotine withdrawal (Perez et al., 2015).
MII[H9A:l15A], a selective α6* nAChR antagonist, blocks nicotine withdrawal-associated conditioned place aversion and anxiety-related behavior (Jackson et al., 2009).
Blocking α6β2 nAChR in the medial habenula (MHb) alleviates anxiety in mice undergoing nicotine withdrawal (Pang et al., 2016).
Conotoxins are polypeptide toxins from marine mollusks that block ion channels, including nAChRs (Lebbe et al., 2014).
Several conotoxins targeting α6β2* nAChR have been discovered, such as MII and ArIB (Everhart et al., 2004; Whiteaker et al., 2007).
α-conotoxin MII reduced alcohol-induced locomotor stimulation and dopamine overflow in the ventral striatum and reduced voluntary alcohol intake in rats and mice (Larsson et al., 2004).
Conotoxin TxIB is the most specific α6β2* subtype nAChR antagonist with high affinity (Luo et al., 2013).
TxIB inhibits nicotine-induced conditioned place preference (CPP) and reduces monoamine neurotransmitters in the nucleus accumbens, hippocampus, and prefrontal cortex (You et al., 2019).
Zebrafish (Danio rerio) is a prominent model system in behavioral and neuroscience research (Stewart et al., 2014).
Zebrafish are used to study alcohol-induced behavior change (Gerlai et al., 2008, 2009) and the impact of alcohol exposure on prey-predator relationships and stress axis activation (Oliveira et al., 2013).
nAChR function and mechanism are conserved across species (Zirger et al., 2003).
nAChR is expressed shortly after fertilization.
nAChR agonists and antagonists have similar impacts on learning and memory in zebrafish as in rodents or humans (Braida et al., 2014).
α6 nAChR in zebrafish is expressed mainly in the central nervous system, especially in catecholaminergic neurons in the midbrain, consistent with rodents and humans (Ackerman et al., 2009).
The current study aims to identify behavior changes induced by alcohol withdrawal and TxIB administration in zebrafish and correlate behavior alterations with gene expression profiles.
Behavior recording, ELISA, RT-PCR, RNA-seq, and bioinformatics analysis were used to identify regulated genes and pathways.

Materials and Methods

Adult zebrafish (wildtype, AB strain, 1:1 gender ratio) were used.
Fish were housed in a 60 L tank with filtered water at 25 ± 0.5°C under 14 h light/10 h dark cycles and fed granular tropical fish food twice daily.
Zebrafish were acclimated for at least 2 weeks before the experiment and were drug-naive, used in a single experiment only.
The animal study was approved by the Animal Ethics Committee of Hainan University and was in accordance with ethical standards.
Zebrafish were treated with escalating alcohol concentrations, starting at 0.1% and increasing to 0.2% every other day, maintaining 0.2% for 14 days (Tran et al., 2015).
From the third week, zebrafish were withdrawn from alcohol for 3 hours per day.
On the 21st day, zebrafish were withdrawn from 0.2% alcohol for the last time before drug injection and the behavior test.
Crude linear TxIB was synthesized by GL Biochem (Shanghai, China) with cysteine residues protected.
The linear TxIB was oxidized to form two disulfide bonds (Luo et al., 2013).
The molecular weight of the folded TxIB was determined using Electrospray Ionisation Mass Spectrometry, with purity >95%.
The in-vitro bioactivity of TxIB was similar on rat and human α6/α3β2β3 nAChR (Zhangsun et al., 2017), confirmed using Voltage-clamp Recording (Luo et al., 2013).
Lyophilized TxIB was dissolved in normal saline solution (0.9%) and injected at 10 μL per fish for a final dose of 1 mg/kg using the retro-orbital injection method (Pugach et al., 2009).
The control and withdrawal groups were injected with 10 μL of normal saline solution per fish.
Behavior and other tests were conducted five hours after injection.
Drug treatments were divided into three groups: control, withdrawal, and withdrawal + TxIB.
Behavior tests were performed during the daytime from 10:00 a.m. to 5:00 p.m.
Fish were moved to the behavior testing apparatus followed by video recording.
The open field apparatus consisted of a transparent cylinder (diameter: 20 cm) filled with water up to 15 cm, divided into center and periphery zones (Kyzar et al., 2012).
After a 2-min adaption period, individual fish were recorded for 5 min.
Time spent in and the number of entries into the center area were monitored as indexes of anxiety-like behavior.
The total distance traveled and the velocity of the movement were recorded as parameters of locomotion.
Video files were analyzed using automated video-tracking software: Smart 3.0 (Panlab Harvard Apparatus, Spain).
Following five hours after drug injection, zebrafish were anesthetized and decapitated for whole-brain tissue sampling.
Brain tissue was suspended with 15 µL/mg ice-cold PBS buffer, homogenized, and centrifuged.
The supernatant was used for ELISA assay to quantify dopamine, serotonin, noradrenaline, and gamma-aminobutyric acid, using commercial ELISA kits (X-Y Biotechnology, Shanghai).
Total protein content was measured using the BCA protein quantification kit (X-Y Biotechnology, Shanghai).
Following treatment, zebrafish were anesthetized and decapitated, and the brain was dissected and rinsed in cold PBS solution.
Ten brains were pooled together for one biological replicate, and total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech co., Ltd.).
mRNA was enriched, fragmented, and used to synthesize the first cDNA strand with random hexamers.
dNTPs, RNase H, and DNA polymerase I were added to synthesize the second cDNA strand.
The cDNA was purified, terminally repaired, A-tailed, and ligated to sequencing junctions, followed by fragment size selection using AMPure XP beads.
The cDNA library was enriched by PCR.
High-throughput RNA sequencing was performed on an Illumina NovaSeq 6000 sequencer at Biomarker Biotechnology Co., Ltd. (Bei Jing).
Clean Reads were aligned with the designated reference genome (GRCz11) and EdgeR package was used for differential expression analysis (fold change ≥2 and p-value < 0.05).
The RNA-seq raw data has been deposited in the Gene Expression Omnibus - NCBI (https://www.ncbi.nlm.nih.gov/geo/) with the accession number: GSE186926.
Total RNA of the whole zebrafish brain was extracted using FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., China).
Reverse transcription into cDNA was performed using HiScript® II Q Select RT SuperMix (Vazyme Biotech Co., Ltd., China).
RT-PCR analysis was then performed in the qTOWER 3 Real-Time PCR Thermal Cycler (Jena Analytical Instruments GmbH, Germany) using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China).
The mRNA expression level was calculated using Double Delta Ct Analysis and presented as relative fold-change of the control group after normalization to the rpl13a mRNA levels.
Data were visualized and analyzed using Graphpad Prism (version 8.3).
One-way ANOVA followed by a Tukey test was performed to determine the statistical significance between groups.
Heatmap (Figure 4B) was generated using TBtools, Gene Ontology enrichment was conducted using DAVID, and the figure (Figure 4C) was generated using ggplot2 (R package).
Venn diagram (Figure 4A) and the figure of KEGG pathway enrichment (Figure 4D) were generated using BMKCloud (www.biocloud.net).
KEGG map (Figure 8) was generated using KEGG Mapper (https://www.genome.jp/kegg/mapper/).

Results

Behavior Test

The max speed in the center zone of the TxIB group ( $23.67 pm 2.01$ cm/s, p < 0.05) was significantly higher than the withdrawal group ( $16.81 pm 1.82$ cm/s, p < 0.05) and close to the control group ( $23.50 pm 1.52$ cm/s).
The withdrawal group showed significantly lower global activity in the center zone ( $27.03 pm 5.66$ cm^2/s, p < 0.01) than the control group ( $79.45 pm 16.53$ cm^2/s, p < 0.01).
The global activity in the center zone of the TxIB group was slightly higher ( $49.58 pm 10.16$ cm^2/s, $p = 0.52$ ).

Monoamine Neurotransmitter Amounts in the Zebrafish Brain

Monoamine neurotransmitter amounts (dopamine, serotonin, noradrenaline, and GABA) were measured using ELISA assay.
The neurotransmitter levels of the TxIB group were slightly higher than the control and withdrawal groups, but the difference did not reach statistical difference (p > 0.05).
Dopamine levels: Normal group ( $24.50 pm 4.22$ pg/mg protein), withdrawal group ( $33.74 pm 4.94$ pg/mg protein), TxIB group ( $40.03 pm 7.41$ pg/mg protein).
Serotonin levels were similar to dopamine, with marginally higher levels in the withdrawal and TxIB groups compared to the normal group ( $33.88 pm 7.70$ pg/mg protein).
Noradrenaline levels: Normal group ( $19.18 pm 2.33$ pg/mg protein), withdrawal group( $22.17 pm 2.31$ pg/mg protein), TxIB group ( $28.77 pm 4.40$ pg/mg protein).
GABA content: Normal group ( $6.94 pm 1.16$ nmol/mg protein), withdrawal group ( $7.84 pm 0.92$ nmol/mg protein), TxIB group ( $14.43 pm 3.74$ nmol/mg protein).

The mRNA Expression Level of Monoamine Neurotransmitter Synthetase in the Zebrafish Brain

The mRNA expression level of rate-limiting enzymes in monoamine neurotransmitter (dopamine, serotonin, noradrenaline) synthesis was assessed.
Six genes were chosen and analyzed via RT-PCR: Th1, Th2, Dbh, Tph1a, Tph1b, Tph2.
The expression level of these genes was not altered significantly by alcohol withdrawal or TxIB treatment in the whole zebrafish brain.

TxIB Regulated Calcium Signaling Pathway in Zebrafish Brain

RNA sequencing was used to identify genes and signaling pathways impacted by alcohol withdrawal and TxIB administration.
Between the control and withdrawal groups, 657 genes were detected as differentially expressed.
344 genes differed in expression between the withdrawal and TxIB groups.
225 genes appeared in both groups, indicating their expression pattern was regulated by alcohol withdrawal and reversed by TxIB.
The heatmap of these 225 genes showed that they were mainly up-regulated by alcohol withdrawal treatment and down-regulated by TxIB.
KEGG and GO enrichment analysis revealed that genes in calcium ion binding and calcium signaling pathways were significantly enriched.
12 genes enriched in the calcium signaling pathway were identified from RNA-seq results.
These genes were mainly upregulated by alcohol withdrawal and downregulated by TxIB. $erbb2$ , $atp2a1$ , $cacna1sb$ , $mylk4b$ , $ptk2bb$ were significantly down-regulated by TxIB, and $adcy2a$ was substantially up-regulated by TxIB administration.

Protein-Protein Interaction (PPI) Analysis, Module and Hub Gene Screening

The global protein-protein interaction (PPI) network of the DEGs was identified using the STRING database.
176 nodes were identified together with 621 edges (regulations). The average node degree is 7.06 and the PPI enrichment p-value is < $1.0 e^{-16}$ .
The densest area of interactions appeared around ttna, ttnb, and myl1.
MCODE (molecular complex detection) plug-in of Cytoscape software was employed to identify densely connected regions in the PPI network.
Top 3 modules calculated by MCODE were identified: Cluster 1 (35 nodes and 472 edges with an overall score of 27.76 and its core is tpma), Cluster 2 (11 nodes and 49 edges with the core of cyt1 and the overall score of 9.8), and Cluster 3 (5 nodes and 9 edges were found with mdh1aa as the core and the overall score is 4.5).
The top 10 Hub genes were identified using Cytohubba plug-in in Cytoscape software.
The full name and description of these hub genes were searched in The Zebrafish Information Network (ZFIN) (https://zfin.org/) and listed in Supplementary Table S1.
As shown in Supplementary Table S1, the 10 hub genes were mainly involved in the regulation of muscle contraction. As to the Human ortholog(s) of these genes, they were primarily indicated in intrinsic cardiomyopathy and myopathy.

RT-PCR Validation

Ten genes enriched in the calcium signaling pathway and cardiac muscle contraction pathway ( $tpma$ , $trdn$ , $cacna1sb$ , $atc1b$ , $atp1a1a.4$ , $myl4$ , $cox6a2$ , $cacng1a$ , $atp2a1$ , $atp2a1l$ ) were validated through RT-PCR.
The results were generally consistent with RNA sequencing data: withdrawal from alcohol increased the expression of these genes, while TxIB administration suppressed the expression.
$Myl4$ expression was elevated up to 16 fold by alcohol withdrawal and reduced to normal status in the TxIB group.
$Atp2a1$ was upregulated 9 fold in the withdrawal group, and the TxIB group was reduced to 5 fold compared to the control group.
The rest of the genes showed an upregulation trend by the withdrawal treatment whereas downregulated by TxIB administration.

Discussion

The study elucidated the potential effects of α-conotoxin TxIB on alcohol withdrawal zebrafish at behavioral, molecular, and transcriptomic levels.
Several studies have focused on the role of nAChR in alcohol and nicotine addiction (Feduccia et al., 2012; Rahman et al., 2015).
α6 nAChR is particularly relevant to alcohol addiction.
Transgenic mice of hypersensitive mutant α6 nAChR (α6L9′S mice) consumed significantly more alcohol than the control group in the Drinking in the Dark test (Powers et al., 2013).
A low dose of alcohol (0.5 g/kg) can lead to alcohol-induced place preference in α6L9′S mice, whereas it was ineffective in control mice.
Low concentrations of alcohol (0.1–5 mM) can significantly enhance α6* nAChR-mediated currents, and this modulation is ineffective on other nAChR subtypes (Gao et al., 2019).
The frequency and amplitude in nucleus accumbens slices of mouse brain were also enhanced by 5 mM alcohol and further blocked by α6β2* nAChR antagonist MII.
A novel small molecule compound—bPiDI, which selectively targets and blocks α6β2* nAChR, markedly reduced alcohol self-administration in alcohol-preferring rats (Srisontiyakul et al., 2016).
Compounds that act on α6β2* nAChR might serve as potential therapeutic agents to alleviate and treat alcohol and nicotine use disorders.
In the open field test, the max speed in the center zone was altered by the withdrawal treatment and further reversed by TxIB.
The global activity in the center zone of the withdrawal group was significantly lower than the control group, indicating an increased degree of anxiety after alcohol withdrawal, consistent with several studies (Krook et al., 2019; Mocelin et al., 2019).
However, the global activity in the center zone was not altered significantly by TxIB administration in withdrawal zebrafish.
TxIB injection in control fish leads to reduced general locomotion in the center area of the open field, consistent with Kamens et al., 2017.
TxIB increased the max speed in withdrawal fish but not in control fish, which might suggest α6β2* nAChR is activated in alcohol withdrawal and blocking α6β2* nAChR by TxIB attenuated this effect in withdrawal fish.
In this study, a marked change of monoamine neurotransmitter amount was not observed after alcohol withdrawal in the whole zebrafish brain.
Dopamine displayed a significant fall in the ventral striatum of mice after 6 days of alcohol administration and withdrawal (Rossetti et al., 1992).
The dopamine amount alteration might happen in certain brain regions, whereas in the whole brain it is hard to detect, therefore, the ventral striatum should be focused on in future studies.
The zebrafish brain's small size makes it difficult to distinguish and extract different brain regions.
The intermittent weekly ethanol exposure found dopamine levels increased significantly after 9 days following 3 week’s intermittent weekly ethanol exposure (Alexandre et al., 2019).
The difference from previous researches might be derived from the different protocols of alcohol exposure.
Noradrenaline level in Alexandre’s study showed no marked change after intermittent weekly ethanol exposure, consistent with study results.
Acute alcohol exposure increased dopamine and its metabolite dihydroxy-phenyl acetic acid in the whole brain of zebrafish (Tran et al., 2017).
However, in reference article study, after 14 days of alcohol exposure and 7 days of repeated withdrawal, the expression of tyrosine hydroxylase mRNA in the whole zebrafish brain did not change significantly between the control and alcohol withdrawal groups.
TxIB ameliorated the gene expression abnormality induced by alcohol withdrawal in the cardiac muscle contraction pathway, indicating its potential to regulate the cardiovascular system in the context of alcohol withdrawal.
TxIB administration strongly regulates the calcium signaling pathway, and a total of 10 proteins in the calcium signaling pathway were affected, including upregulated $ADCY$ and downregulated $Cav1$ , $RTK$ , $SERCA$ , $TRDN$ , $CASQ$ , $ANT$ , $Tnc$ , $MLCK$ , and $FAK2$ .
The calcium signaling pathway plays a significant role in the alcohol withdrawal process (Spanagel et al., 2014).
Calcium influx plays an important role in alcohol-withdrawal-induced seizures (Newton et al., 2021).
Targeting the calcium signaling pathway can modulate alcohol withdrawal symptoms (Finn and Crabbe, 1997).
The top 10 genes that were regulated significantly by TxIB administration were identified, and these genes were mainly involved in the calcium-binding process.

Data Availability Statement

The datasets presented in this study can be found in online repositories: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186926.

Conclusion

This study showed that the specific α6β2* nAChR antagonist TxIB can alleviate the behavioral abnormality induced by alcohol withdrawal in zebrafish.
Transcriptome analysis of zebrafish brain tissue indicated that the expression profile of a total of 657 genes was altered by alcohol withdrawal, among which 225 genes were recovered by TxIB injection.
These genes were mainly enriched in calcium signaling pathways.
The results of RNA-seq were further validated by RT-PCR.
From PPI network construction, 10 hub genes were identified, and these genes were primarily myosin and actin coding genes.
Our findings showed α-conotoxin TxIB improved behavioral abnormality induced by alcohol withdrawal and altered gene expression in the calcium signaling pathway in the zebrafish model.
Therefore, TxIB would be a potential drug candidate to treat alcohol withdrawal syndrome.