Sexually dimorphic and asymmetric effects of embryonic ethanol exposure on hypocretin/orexin neurons as related to behavioral changes in zebrafish

Adam D Collier,Avi S Albeg,Samantha Campbell,Nailya Khalizova,Sarah F Leibowitz,Nushrat Yasmin,Milisia Fam,Amanda Onoichenco

doi:10.1038/s41598-021-95707-y

Abstract

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote reward-related behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH). We used here in zebrafish three-dimensional analyses of the entire population of Hcrt neurons to examine how embryonic EtOH exposure at low-moderate concentrations (0.1% or 0.5% v/v) alters these neurons in relation to behavior. We found that EtOH in the water for 2 h (22–24 h post fertilization) increases the number of Hcrt neurons on the left but not right side of the brain through a stimulation of cell proliferation, this is accompanied by a decrease in locomotor activity under novel conditions but not after habituation, and these effects are evident in both larvae and adults indicating they are long lasting. Our analyses in adults revealed sexually dimorphic effects, with females consuming more EtOH-gelatin and exhibiting more freezing behavior along with an asymmetric increase in Hcrt neurons and males exhibiting increased aggression with no change in Hcrt. These findings suggest that a long lasting, asymmetric increase in Hcrt neurons induced by EtOH results from an asymmetric increase in proliferation specific to Hcrt and contributes to behavioral changes in females.

Highlights

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote rewardrelated behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH)
We demonstrate here that embryonic exposure to EtOH, administered at low-moderate concentrations during the period of peak hypothalamic neuronal development, stimulates cell proliferation throughout the entire anterior hypothalamus (AH) but increases asymmetrically the proliferation of Hcrt neurons only on the left side of the AH
Using two-dimensional image analysis, we have previously demonstrated that embryonic EtOH exposure in zebrafish, similar to prenatal exposure in r ats[9], increases the number of differentiated neurons throughout the developing hypothalamus[16] and of neurons positive for the proliferation marker BrdU in the A H11, supporting the idea that low-moderate EtOH concentrations stimulate hypothalamic neurogenesis

Summary

Introduction

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote rewardrelated behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH). This is supported by rodent studies in our laboratory[9] and others[10] showing prenatal alcohol exposure at low-to-moderate doses to increase alcohol consumption It is evident in our studies of zebrafish[11], a species that has high physiological and genetic homology to humans, a comparable CNS that develops early and r apidly[12], and a sophisticated behavioral repertoire including voluntary consumption of EtOH-gelatin that produces pharmacologically relevant blood EtOH c oncentrations[13]. Asymmetric effects of EtOH on Hcrt neurogenesis may be important in light of our earlier finding that the EtOH-induced increase in number of Hcrt neurons is stronger on the left than right side[31], and of evidence in other studies showing asymmetry of neuronal development to be an important factor that contributes to behavioral disturbances and the development of alcohol use d isorder[33]

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Conclusion