Purmorphamine Affects Anxiety-Like Behavior and Expressions of Hedgehog Pathway Components in Rat Brain after Withdrawal

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Abstract

The Hedgehog (Hh) pathway underlies fetal alcohol syndrome during prenatal alcohol exposure. The involvement of Hh in the mechanisms of alcohol consumption in adulthood remains obscure. We aimed to investigate a role of Hh cascade in voluntary ethanol drinking, anxiety-like behavior during early abstinence and changes in expressions of Hh cascade components in brain regions. Intermittent access to 20% ethanol in a two-bottle choice procedure has been used to model voluntary alcohol drinking in Wistar male rats. Purmorphamine, an activator of the Hh cascade that exhibits Smoothened (Smo) receptor agonist properties, was administered systemically at a dose of 5 mg/kg before drinking sessions from 16 to 20. Purmorphamine had no effect on the level of ethanol preference, but the group with access to alcohol and receiving purmorphamine showed a change in anxiety-like behavior during early withdrawal period. Alcohol consumption affected Sonic hedgehog (Shh) and Patched (Ptch) mRNA content only in the amygdala. In the group that had access to ethanol and received purmorphamine, Shh mRNA levels in the amygdala were negatively correlated with time spent in the open arms of the elevated plus maze in the test of anxiety-like behavior. Thus, it was demonstrated for the first time that alterations of the Hh cascade by purmorphamine administration does not affect voluntary alcohol drinking, but Hh is possibly involved in the formation of anxiety during early withdrawal through specific changes in Hh cascade components in the amygdala.

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D. I. Peregud

Serbsky National Medical Research Center for Psychiatry and Drug Addiction; Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences

Author for correspondence.
Email: peregud_d@yahoo.com
Russian Federation, 119034, Moscow; 117485, Moscow

N. I. Shirobokova

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences

Email: peregud_d@yahoo.com
Russian Federation, 117485, Moscow

A. A. Kvichansky

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences

Email: peregud_d@yahoo.com
Russian Federation, 117485, Moscow

M. Yu. Stepanichev

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences

Email: peregud_d@yahoo.com
Russian Federation, 117485, Moscow

N. V. Gulyaeva

Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences; Research and Clinical Center for Neuropsychiatry of Moscow Healthcare Department

Email: peregud_d@yahoo.com
Russian Federation, 117485, Moscow; 115419, Moscow

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2. Fig. 1. Scheme of the experiment. The model of intermittent access to 20% ethyl alcohol solution under free choice conditions was used, according to which the alcohol solution was presented together with water for a period of 24 h followed by replacement of alcohol with water for 24 h. The Hh cascade activator with Smo receptor agonist properties, purmorphamine, was administered systemically at a dose of 5 mg/kg before 16-20 sessions of alcohol presentation. Alcohol consumption was recorded for 20 sessions of alcohol presentation, then alcohol was cancelled. Anxiety-like behaviour was assessed in an open field and an elevated cruciform maze one day and two days after alcohol withdrawal, respectively. On the third day after alcohol withdrawal, mRNA levels of Shh, Ptch, Smo, and Gli signalling cascade elements were assessed in frontal cortex, amygdala, hippocampus, and striatum

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3. Fig. 2. Dynamics of ethanol preference for water in a model of intermittent access to 20% ethanol solution under free-choice conditions. Data are presented as arithmetic mean ± error of the mean. The significance of differences from the level of alcohol preference for water after the first session of alcohol presentation (mixed-effects model and Dunnett's criterion for multiple comparisons): * p < 0.05; ** p < 0.005

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4. Fig. 3. Effect of purmorphamine on ethanol solution preference for water in an intermittent access model under free-choice conditions. P - randomisation by preference level for the last 5 sessions before starting a series of purmorphamine injections. P - intraperitoneal injection of purmorphamine at a dose of 5 mg/kg one hour before 16-20 sessions of ethanol presentation. Data are presented as arithmetic mean ± error of the mean

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5. Fig. 4. Effect of purmorphamine on anxiety-like behaviour in the open field after implementation of an intermittent ethanol access model under free-choice conditions. Purmorphamine was administered intraperitoneally at a dose of 5 mg/kg one hour before 16-20 sessions of ethanol presentation. The ‘Open Field’ test was performed one day after alcohol withdrawal. a - Time spent in the central zone of the arena; b - number of visits to the central zone of the arena. Data are presented as median and interquartile range

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6. Fig. 5. Effect of purmorphamine on behaviour in the elevated cruciform maze after implementation of an intermittent ethanol access model under free-choice conditions. Purmorphamine was administered intraperitoneally at a dose of 5 mg/kg one hour before 16-20 sessions of ethanol presentation. The test was performed two days after alcohol withdrawal. Kraskell-Wallis rank criterion and subsequent post hoc analysis of multiple comparisons using Dunn's test. a - Number of hovering; b - Number of pulling; c - Time spent in the open arms of the maze; d - Number of entries into the closed arms of the maze. Data are presented as median and interquartile range

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7. Fig. 6. Effect of purmorphamine on the relative content of Shh mRNA in the amygdala after implementation of the intermittent ethanol access model under free-choice conditions. Purmorphamine was administered intraperitoneally at a dose of 5 mg/kg one hour before 16-20 sessions of ethanol presentation. The mRNA levels were assessed by PCR three days after alcohol withdrawal. Data are presented as arithmetic mean ± error of the mean

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