A. V. Kulikov

Evidence for the involvement of central serotonin in mechanism of domestication of silver foxes

Silver foxes selected for more than 30 years for tame behavior and displaying no defensive reaction to human contact were shown to have a higher serotonin level in midbrain and hypothalamus, and a higher 5-hydroxyindole acetic acid (5-HIAA) content in midbrain, hypothalamus and hippocampus in comparison to nonselected wild silver foxes bred in captivity over the same time span. Tryptophan hydroxylase (TPH) activity in midbrain and hypothalamus in domesticated foxes was increased as compared with their aggressive/defensive counterparts. Monoamine oxidase type A (MAO A) activity was was decreased with an increased Km and unchanged Vmax in domesticated foxes. No changes in specific [3H]ketanserin or [3H]8-OH-DPAT binding in frontal cortex was revealed. A reduced density (Bmax) of 5HT1A receptors in hypothalamic membranes in domesticated foxes was shown. It is suggested that the brain serotonergic system is involved in the mechanism of domestication converting wild aggressive/defensive animals into tame ones.

Association between Tph2 gene polymorphism, brain tryptophan hydroxylase activity and aggressiveness in mouse strains

The brain neurotransmitter serotonin is involved in the regulation of aggressive behavior. The main factor determining the brain serotonin level is the activity of the rate‐limiting enzyme in the biosynthesis of the neurotransmitter – tryptophan hydroxylase isoform (TPH) 2 encoded by the Tph2 gene. Recently the C1473G single‐nucleotide polymorphism in the Tph2 gene was reported. Here we study the C1473G polymorphism in 10 inbred mouse strains (C57BL/6J, AKR/J, DD/He, C3H/HeJ, YT/Y, BALB/cJLac, CC57BR/Mv and A/He) and demonstrate the association of the polymorphism with brain TPH activity and intermale aggressiveness. TPH activity in the midbrain of mice homozygous for the 1473C allele was higher than that in mice carrying 1473G alleles. A close association of the 1473C allele with increased number of attacks towards another male was found. The results support a link between the C1473G polymorphism in Tph2 gene, trypthophan hydroxylase activity and intensity of intermale aggression.

Reduction in 5-HT1A receptor density, 5-HT1A mRNA expression, and functional correlates for 5-HT1A receptors in genetically defined aggressive rats

The present experiments tested the hypothesis that one of the critical mechanisms underlying genetically defined aggressiveness involves brain serotonin 5‐HT1A receptors. 5‐HT1A receptor density, the receptor mRNA expression in brain structures, and functional correlates for 5‐HT1A receptors identified as 8‐OH‐DPAT‐induced hypothermia and lower lip retraction (LLR) were studied in Norway rats bred for 59 generations for the lack of aggressiveness and for high affective aggressiveness with respect to man. Considerable differences between the highly aggressive and the nonaggressive rats were shown in all three traits. A significant decrease in Bmax of specific receptor binding of [3H]8‐OH‐DPAT in the frontal cortex, hypothalamus, and amygdala and a reduction in 5‐HT1A receptor mRNA expression in the midbrain of aggressive rats were found. 5‐HT1A receptor agonist 8‐OH‐DPAT (0.5 mg/kg, i.p.) produced a distinct hypothermic reaction in nonaggressive rats and did not affect significantly the body temperature in aggressive rats. Similar differences were revealed in 8‐OH‐DPAT‐induced LLR: LLR was expressed much more in nonaggressive than in aggressive animals. Additionally, 8‐OH‐DPAT (0.5 mg/kg i.p.) treatment significantly attenuated the aggressive response to man. The results demonstrated an association of aggressiveness with reduced 5‐HT1A receptor expression and function, thereby providing support for the view favoring the idea that brain HT1A receptor contributes to the genetically defined individual differences in aggressiveness.

Quantitative RT-PCR assay of 5-HT1A and 5-HT2A serotonin receptor mRNAs using genomic DNA as an external standard.

Brain serotonin 5-HT(1A) and 5-HT(2A) receptors have been implicated in both normal and pathological behavior, and in the action of anxiolytic and antidepressant drugs. In this study, detailed description and verification of a new RT-PCR technique to quantify the number of copies of 5-HT(1A) and 5-HT(2A) receptor mRNAs in the brain is presented. The number of copies of beta-actin and 5-HT(1A) or 5-HT(2A) receptor mRNAs in rat brain samples was evaluated with respect to the genomic DNA solution as the external exogenous standard. The expression of 5-HT receptors was calculated as the number of receptor mRNA copies per 100 copies of corresponding beta-actin mRNA. This presented technique is reliable, simple and can be easily set up in any neurobiological laboratory.

Automated measurement of spatial preference in the open field test with transmitted lighting.

New modification of the open field was designed to improve automation of the test. The main innovations were: (1) transmitted lighting and (2) estimation of probability to find pixels associated with an animal in the selected region of arena as an objective index of spatial preference. Transmitted (inverted) lighting significantly ameliorated the contrast between an animal and arena and allowed to track white animals with similar efficacy as colored ones. Probability as a measure of preference of selected region was mathematically proved and experimentally verified. A good correlation between probability and classic indices of spatial preference (number of region entries and time spent therein) was shown. The algorithm of calculation of probability to find pixels associated with an animal in the selected region was implemented in the EthoStudio software. Significant interstrain differences in locomotion and the central zone preference (index of anxiety) were shown using the inverted lighting and the EthoStudio software in mice of six inbred strains. The effects of arena shape (circle or square) and a novel object presence in the center of arena on the open field behavior in mice were studied.

C1473G polymorphism in mouse tph2 gene is linked to tryptophan hydroxylase-2 activity in the brain, intermale aggression, and depressive-like behavior in the forced swim test

Tryptophan hydroxylase‐2 (TPH2) is the rate‐limiting enzyme of brain serotonin synthesis. The C1473G polymorphism in the mouse tryptophan hydroxylase‐2 gene affects the enzymes activity. In the present study, we investigated the linkage between the C1473G polymorphism, enzyme activity in the brain, and behavior in the forced swim, intermale aggression, and open field tests using mice of the C57BL/6 (C/C) and CC57BR/Mv (G/G) strains and the B6‐1473C (C/C) and B6‐1473G (G/G) lines created by three successive backcrossings on C57BL/6. Mice of the CC57BR/Mv strain had decreased brain enzyme activity, aggression intensity, and immobility in the forced swim test, but increased locomotor activity and time spent in the central part of the open field arena compared with animals of the C57BL/6 strain. Mice of the B6‐1473G line homozygous for the 1473G allele had lower TPH2 activity in the brain, aggression intensity, and immobility time in the forced swim test compared with animals of the B6‐1473C line homozygous for the 1473C allele. No differences were found between the B6‐1473G and B6‐1473C mice in locomotor activity and time spent in the central part of the arena in the open field test. Thus, the C1473G polymorphism is involved in the determination of TPH2 activity and is linked to aggression intensity and forced‐swim immobility in mice. At the same time, the polymorphism does not affect locomotion and anxiety‐related behavior in the open field test. The B6‐1473C and B6‐1473G mice represent a valuable experimental model for investigating molecular mechanisms of serotonin‐related behavior.

Inheritance of predisposition to catalepsy in mice

Abstract“Pinch-induced” catalepsy (excessive freezing) was studied in mice of nine inbred strains. The CBA/Icg strain had the highest predisposition to catalepsy, with 56% males and 73% females exhibiting 120-s immobility after four tests. A Mendelian analysis of predisposition to catalepsy was performed on CBA/Icg and AKR/J mice contrasting in this trait. Reciprocal F1 hybrids did not display freezing after 10 tests. The number of cataleptic and noncataleptic animals among the F2s and in the families of the CBA×F2 backcrosses agreed with the hypothesis that the pronounced predisposition to catalepsy in the CBA/Icg mice was determined by a single autosomal locus in homozygous recessive condition.

Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain.

In this study, we applied for real-time PCR the two-standard system that we had worked out previously for PCR with gel-detection of products. Genomic DNA of a known concentration was used as external standard and mRNA of the DNA-dependent RNA-polymerase II was used as internal standard. It was shown that PCR with gel-detection of products and real-time PCR provide similar results and demonstrate almost identical accuracy and repeatability when the two-standard system is used. With the help of the both methods and using the two-standard system we have confirmed the link between the genetically determined freezing reaction in mice and reduced 5-HT1A receptor mRNA level in the midbrain. We have also found that the genetically determined freezing reaction in mice is not connected with changes in Tph2 gene expression.

Quantitative assay of 5-HT1A receptor gene expression in the brain

Abstract5-HT1A receptors are involved in the regulation of various behaviors and the mechanism of action of anxiolytics and antidepressants. It is rather difficult to study the expression of the 5-HT1A receptor gene in the brain because of the low concentration of its mRNA. A method developed for quantitating the level of 5-HT1A receptor gene expression in brain structures involves estimation of the copy number for contaminant genomic DNA, the cDNA of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (a housekeeping gene), and the 5-HT1A receptor gene cDNA in a cDNA preparation. To estimate the GAPDH and 5-HT1A receptor cDNA copy numbers, the fluorescent intensity of the corresponding PCR products is calibrated using genomic DNA standards of known concentrations. The expression of the 5-HT1A receptor gene is corrected for the content of contaminant genomic DNA and presented as a 5-HT1A receptor cDNA copy number per 100 copies of the GAPDH cDNA. The method was used to demonstrate for the first time that expression of the 5-HT1A receptor gene is increased in the frontal cortex and the amygdala of mice knocked-out in the monoamine oxidase A gene.

Genetic analysis of different kinds of aggressive behavior

Various kinds of aggressive behavior such as spontaneous intermale aggression, predatory aggression (locust-killing behavior), and irritable (shock-induced) aggression were investigated in inbred strains of mice. Genotype was shown to affect significantly the phenotypic variety of these kinds of aggression. There were, however, no interstrain correlations either between intermale aggression and predatory behavior or between intensity of intermale, shock-induced aggression and locust-killing behavior. Moreover, the intermale aggression level (percentage of fighting mice in each strain) did not correlate with the intensity of fighting. It has been shown by Mendelian analysis on C57BL/6J and BALB/c strains that these indices of intermale aggression are under different genetic control. The selection of Norway rats over 20 generations for reduced fear-induced aggressiveness toward man resulted in a decrease in irritable aggression and loss of an aggressive response to man. No changes in intermale and predatory aggression, however, were found. Hence, different kinds of aggressive behavior—intermale, predatory, and fear-induced aggression—seem to be controlled by different genetic mechanisms.