Solanki et al. · Behavioural Brain Research · 2020

Administration of a putative pro-dopamine regulator, a neuronutrient, mitigates alcohol intake in alcohol-preferring rats

Solanki Naimesh, Abijo Tomilowo, Galvao Carine, Darius Philippe, Blum Kenneth, Gondré-Lewis Marjorie C.

The study

What was asked, and what was found

Solanki et al., Behavioural Brain Research, 2020 asked whether a nutrient formulation designed to raise dopamine availability would reduce binge drinking in rats bred to prefer alcohol, and if so, whether it worked by raising expression of the dopamine D2 receptor. Rats were dosed for four consecutive days by mouth, intraperitoneally or subcutaneously, then tested on an operant binge drinking protocol against saline or water controls. The compound cut lever pressing and ethanol consumption in both sexes, quickly by the injected routes and after about 3 days by mouth. Subcutaneous dosing, the only route tested against sucrose, left sucrose drinking unchanged.

The expression question was answered with in situ hybridization. Digoxigenin labelled probes against DRD2 messenger RNA, supplied by AUM BioTech, were hybridized to fresh frozen sections through the nucleus accumbens of alcohol naive rats at 500 ng per mL, and the resulting spots were counted per cell under unbiased stereological rules. The probe gave abundant signal over medium spiny neurons and none over the anterior commissure beside the region, which is the internal check that the signal is regionally specific. The paper reports no probe-omission and no sense-probe control, so that is the check it carries.

The result was negative for the proposed mechanism and positive for the anatomy. Four days of subcutaneous dosing did not change DRD2 messenger RNA per cell in either the core or the shell, so the drinking effect does not run through increased D2 transcription at that dose. Independent of treatment, the shell carried significantly more DRD2 messenger RNA per cell than the core. Both of those results rest on the six female rats used for the stereological analysis, three treated and three controls. Our own material in this study was a labelled detection probe, used to find the transcript in fixed brain sections. Nothing here was silenced.

Key findings

Our own material in this study was a digoxigenin labelled probe against DRD2 messenger RNA, used to read expression in rat brain sections. Nothing in this study was silenced or knocked down.(Materials and methods, In Situ Hybridization)
The probe gave punctate signal over neuronal cell bodies that could be counted per cell under unbiased stereological rules, and gave none over the anterior commissure beside the region of interest.(Results, In Situ Hybridization for DRD2 mRNA in Nucleus Accumbens)
The measurement did not support the mechanism the authors had proposed: the compound cut binge drinking, but at the minimum recommended dose it did not raise DRD2 messenger RNA in the nucleus accumbens.(Discussion)

Product: AUMsilence
Targets: DRD2 · rat · mRNA, detected by a labelled probe rather than knocked down
Models: genetically alcohol-preferring rats, male and female, from the Indiana University Alcohol Research Center · in vivofresh frozen rat brain, nucleus accumbens core and shell, from alcohol naive animals · tissue sections
Delivery: Not applicable: a labelled probe applied to fixed brain sections in hybridization buffer, rather than a reagent delivered into living cells
Readouts: in situ hybridization, unbiased stereological counting, operant lever pressing and ethanol consumption, locomotor activity, elevated zero maze
Measured: probe concentration in the hybridization solution: 500 ng per mL (overnight at 72 degrees C on 10 micron fresh frozen sections)difference in DRD2 messenger RNA per cell between the two subregions detected by the probe: F(1, 8) = 108.423, p below 0.001, shell higher than core (nucleus accumbens shell against core, regardless of treatment, in the six female rats used for the stereological analysis, three treated and three controls)effect of the test compound on DRD2 messenger RNA per cell: no change in either subregion (4 days of subcutaneous dosing, in the six female rats used for the stereological analysis, three treated and three controls)
Figures: Fig. 5 In situ hybridization for DRD2 messenger RNA in the nucleus accumbens, with the probe signal counted per cell in core and shell.
Citation: Solanki et al. Administration of a putative pro-dopamine regulator, a neuronutrient, mitigates alcohol intake in alcohol-preferring rats. Behavioural Brain Research 2020;385:112563. doi:10.1016/j.bbr.2020.112563

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