Acupuncture Alleviates Anxiety and 22-kHz Ultrasonic Vocalizations in Rats Subjected to Repeated Alcohol Administration by Modulating the Brain-Derived Neurotrophic Factor/Corticotropin-Releasing Hormone Signaling Pathway
Round 1
Reviewer 1 Report
This study by Seo et al. used acupuncture on rats at HT7 Shenmen point to show that it can diminish the deleterious consequences of subchronic alcohol exposure. In a multidisciplinary approach, the authors show that rats exposed to alcohol for 14 days exhibit a loss of weight and an increased plasma corticosterone level. Moreover, simultaneous HT7 stimulation decreases CORT level and reduces rat anxiety. Authors postulate that up-regulation of both BDNF and TrkB in amygdala decreases the expression of CRH within the hypothalamus, thus contributing to a down-regulation of chronic stress, i.e CORT.
The limitation of this project lies in the sub-chronic exposure of rats to alcohol, via intraperitoneal injection. It has been shown in several studies in rodents that when a choice is made between water or water + alcohol, rats prefer at 50-60% water with alcohol (doi: 10.1523/ENEURO.0221-19.2019. ; doi: 10.1016/j.neuropharm.2016.07.031). Why did you choose to perform intraperitoneal injections, which do not at all model the self-administration of alcohol in humans or animals? This point seems to be an important limitation of this study.
In general, there is a second point that is quite problematic with this paper: the statistical analysis of the data shows significant differences, which are not obvious when reading the study. The statistical treatment that has been carried out should be clearly explained and argued in such a way that these differences are justified.
Fig.1a: “after repeated administration for 14 days” What is the frequency of alcohol administration? Once a day?
Fig. 1d: considering the SEM, it is hard to see that there is a statistical difference between Sal and ETOH for CORTplasm. Same applies for Fig. 2b.
Fig. 2a should be completed to show the Sham point used as a negative control
Fig. 2c: please indicate if the difference between ETOH+NonAcu and ETOH+Veh group was non-significant
Figure 7d: I think the arrows of CRH have been inverted. Repeated ETOH is supposed to make CRH going up, and HT7 is supposed to make CRH going down… (L282: ETOH exposure leads to an increase in CRH signaling)
L30-31 : Please do not restrict the impact of alcohol to the US. Present it gobally (see https://www.who.int/news-room/fact-sheets/detail/alcohol)
L57: ”anxiety-like behaviors in rats”. Do you mean anxiety associated to alcohol-induced withdrawal and dysphoria ? Please precise.
L334: “the MAI stimulated the HT7 334 point for 30 s”. Please indicate what was the precise intensity and the frequency used.
Minor
L43: β-endorphin
L50: an essential component of the reward system
L68: and that its stimulation regulates
L69: “Sigma1 R”. Is it “σ1 receptors” ?
L80: addiction, anxiety, and depression
L130: was treated with a mechanical acupuncture instrument (MAI)
L132: In the EPM (Elevated Plus Maze) test
L325-327: please indicate the ETOU dose used for i.p injection.
L329: were used in this study (Figure 2a)
L344-345: an equation should be written as “using the following equation :
AI = 1 - [(open arm entries/total arm entries) + (open arm duration/total 345 duration)]/2”
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Seo et al. describe a mechanism by which acupuncture HT7 (acupuncture point heart 7: HT7) stimulation alleviates the increase in stress hormone levels and mitigates increased 22-kHz vocalizations and anxiety-like behaviors caused by repeated alcohol (ETOH) administration. I found the manuscript very interesting, well-written, and free from obvious typographical errors. The authors with competently done assays showed that MAI stimulation of the HT7 acupoint significantly 1) decreased the plasma CORT level, 2) decreased the anxiety index, 3) decreased aversive 22-kHz USVs, 4) increased the levels of mBDNF and phosphorylated TrkB in the amygdala, and 5) decreased CRH levels in the rat PVN induced by the repeated injection of ETOH for 14 days. Microinjection of the TrkB receptor antagonist ANA-12 into the amygdala prior to HT7 stimulation significantly suppressed the recovery of the CRH level in the PVN. I consider that this manuscript has valuable and relevant information and deserves to be published.
Author Response
Thank you for your comment. Corrected English and minor statistical errors and reattached.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
I think this study is quite interesting, and well presented. The authors responded rather favourably to all the questions addressed. However, can I ask them to:
- Clearly justify in the Materials and Methods section (L332) the use of the intraperitoneal route which, I still believe, constitutes a particular paradigm in exposure to alcohol (like to any other toxic substance), compared to self-administration.
- Return version V1 with all the changes appearing in yellow, so that you can compare V1 to V3
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Round 3
Reviewer 1 Report
The article can published in its present form.
