Analysis of the Melanocortin Brain System of a Krushinsky–Molodkina Rats with Genetic Predisposition to Audiogenic Seizures

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Abstract

The study was conducted on 4-month-old male rats of the Krushinsky–Molodkina (KM) line, genetically predisposed to audiogenic seizures, and Wistar rats, which are not sensitive to the effects of sound. In KM rats, real-time PCR revealed an increase in the level of AgRP mRNA (4-fold, p<0.05) and melanocortin receptors MC4R (2.4-fold, p<0.05) in the hypothalamus vs. Wistar rats. No differences in the level of proopiomelanocortin mRNA were detected. The results of immunohistochemical analysis indicate an increased (p<0.05) level of optical density of AgRP, MC3R and MC4R in the hypothalamic structures of KM rats vs. Wistar rats. In the dorsal hippocampus a statistically significant increase in the level of MC3R (by Western blotting) and MC4R (by immunohistochemistry) was also detected in KM rats vs. Wistar rats. The obtained results are discussed in connection with the revealed blocking dose-dependent effect of SHU9119, a non-selective MC3R/MC4R inhibitor, on seizure activity in KM rats.

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About the authors

I. V. Romanova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Author for correspondence.
Email: irinaromanova@mail.ru
Russian Federation, Saint Petersburg

A. L. Mikhrina

Hebrew University

Email: irinaromanova@mail.ru
Israel, Jerusalem

S. I. Vataev

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: irinaromanova@mail.ru
Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Expression analysis of genes encoding POMC, AgRP, MC3R and MC4R in Wistar rat hypothalamus (n = 8-9) and CM (n = 10), data are presented in conventional units. Denotations: * - statistically significant differences from the Wistar group (p < 0.05), Student's t-test

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3. Fig. 2. Analysis of the level of POMC, AgRP, MC3R and MC4R in the arcuate nucleus of the hypothalamus of the rat Wistar (n = 5) and CM (n = 5). * - Significance of differences between the KM group and the corresponding Wistar group (p < 0.05) (Mann-Whitney U-test). Results are presented as median with interquartile ranges in conventional units

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4. Fig. 3. Immunohistochemical reaction to AgRP in the arcuate nucleus (ARC) of Wistar rat hypothalamus (a) and CM (b). Scale of 100 μm

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5. Fig. 4. Immunohistochemical reaction to MS3R in the arcuate nucleus (ARC) of Wistar rat hypothalamus (a) and CM (b). Arrows indicate the bodies of immunopositive neurons, 3 v indicates the third ventricle of the brain. Scale 100 µm

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6. Fig. 5. Western blotting results demonstrating: a - MC3R protein level and control protein GAPDH in the dorsal hippocampus of Wistar rat (n = 6) and CM (n = 6), b - statistical analysis of data, which are presented as median with interquartile ranges in conventional units, * - significance of differences (p < 0.05), Mann-Whitney U-test

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7. Fig. 6. Immunohistochemical reaction to MS4R in the CA3 field of the hippocampus of the Wistar rat (a), CM (b) and the result of statistical analysis (c) of the optical density of MS4R in neurons of the CA1 and CA3 fields of the hippocampus. In micrographs (a, b), arrows indicate the perikaryon of immunopositive neurons, scale 100 μm. In graph (c) data are presented as median (M) with interquartile ranges in conventional units, * - significant difference (p < 0.05), between the respective groups, n = 5 in each group, Mann-Whitney U-test

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8. Fig. 7. Results of administration of CM SHU9119, a non-selective MC3R/MC4R blocker, to rats. a - number of rats in each group (numbers on the column), in which seizures were detected after the drug administration (in % relative to the baseline 100%); b - change in seizure intensity (in points) in these rats, reliability of the difference from the baseline (Wilcoxon's T-criterion with Bonferroni correction): * - p < 0.05, *** - p < 0.0001; c - change in latent period (in seconds) in these rats

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