Vladimir S. Naumenko

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.

5-HT1A receptor as a key player in the brain 5-HT system.

Abstract Among an impressive variety of identified serotonin receptors, 5-HT1A attracts particular attention due to its central role in the regulation of 5-HT-ergic neurotransmission and the data on its involvement in the mechanisms of stress response, aggressive behavior, anxiety, and depression. This review concentrates on the cross-regulation between 5-HT receptors and the implication of the 5-HT1A receptor in the genetic control of 5-HT-related behavior. Specifically, it describes the (1) functional interactions between 5-HT1A, 5-HT2A, 5-HT3, and 5-HT7 receptors; (2) cross-talk between 5-HT1A receptor and genes encoding key members of the brain 5-HT system; (3) implication of the 5-HT1A receptor in natural hibernation and genetic predisposition to different kinds of defensive behavior; and (4) role of 5-HT1A autoreceptors and heteroreceptors in anxiety, depression, and suicide, and in the antidepressant effect of serotonin reuptake inhibitors. This review provides converging lines of evidence that the 5-HT1A receptor contributes to the action of other 5-HT receptors, modulating their effect on behavior, and describes new data on the unique role of the 5-HT1A receptor in the indirect regulation of gene expression and in the autoregulation of the brain 5-HT system.

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.

Interplay between Serotonin 5‐HT1A and 5‐HT7 Receptors in Depressive Disorders

Serotonin (5‐hydroxytryptamine or 5‐HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5‐HT receptors. Besides the important role of 5‐HT receptors in the pathogenesis of depressive disorders and in their clinical medications, underlying mechanisms are far from being completely understood. This review focuses on possible cross talk between two serotonin receptors, 5‐HT1A and the 5‐HT7. Although these receptors are highly co‐expressed in brain regions implicated in depression, and most agonists developed for the 5‐HT1A or 5‐HT7 receptors have cross‐reactivity, their functional interaction has not been yet established. It has been recently shown that 5‐HT1A and 5‐HT7 receptors form homo‐ and heterodimers both in vitro and in vivo. From the functional point of view, heterodimerization has been shown to play an important role in regulation of receptor‐mediated signaling and internalization, suggesting the implication of heterodimerization in the development and maintenance of depression. Interaction between these receptors is also of clinical interest, because both receptors represent an important pharmacological target for the treatment of depression and anxiety.

On the role of brain 5-HT7 receptor in the mechanism of hypothermia: Comparison with hypothermia mediated via 5-HT1A and 5-HT3 receptor

Intracerebroventricular administration of selective agonist of serotonin 5-HT(7) receptor LP44 (4-[2-(methylthio)phenyl]-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1-pyperasinehexanamide hydrochloride; 10.3, 20.5 or 41.0 nmol) produced considerable hypothermic response in CBA/Lac mice. LP44-induced (20.5 nmol) hypothermia was significantly attenuated by the selective 5-HT(7) receptor antagonist SB 269970 (16.1 fmol, i.c.v.) pretreatment. At the same time, intraperitoneal administration of LP44 in a wide range of doses 1.0, 2.0 or 10.0 mg/kg (2.0, 4.0, 20.0 μmol/kg) did not cause considerable hypothermic response. These findings indicate the implication of central, rather than peripheral 5-HT(7) receptors in the regulation of hypothermia. The comparison of LP44-induced (20.5 nmol) hypothermic reaction in eight inbred mouse strains (DBA/2J, CBA/Lac, C57BL/6, BALB/c, ICR, AKR/J, C3H and Asn) was performed and a significant effect of genotype was found. In the same eight mouse strains, functional activity of 5-HT(1A) and 5-HT(3) receptors was studied. The comparison of hypothermic responses produced by 5-HT(7) receptor agonist LP44 (20.5 nmol, i.c.v.) and 5-HT(1A) receptor agonist 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg), 5-HT(3) receptor agonist m-CPBG (40.0 nmol, i.c.v.) did not reveal considerable interstrain correlations between 5-HT(7) and 5-HT(1A) or 5-HT(3) receptor-induced hypothermia. The selective 5-HT(7) receptor antagonist SB 269970 (16.1 fmol, i.c.v.) failed to attenuate the hypothermic effect of 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg) and m-CPBG (40.0 nmol, i.c.v.) indicating that the brain 5-HT(7) receptor is not involved in the hypothermic effects of 8-OH-DPAT or m-CPBG. The obtained results suggest that the central 5-HT(7) receptor plays an essential role in the mediation of thermoregulation independent of 5-HT(1A) and 5-HT(3) receptors.

Involvement of brain serotonin 5-HT1A receptors in genetic predisposition to aggressive behavior.

Experiments were performed on Norwegian rats selected over more than 59 generations for high and low levels of high-affective defensive aggressivity and on highly aggressive (offensive) Tg8 mice with irreversible monoamine oxidase A knockout. There were significant differences in the functional state and expression of 5-HT1A receptors between highly aggressive and non-aggressive animals. Functional activity assessed in terms of hypothermia evoked by a 5-HT1A agonist was significantly greater in non-aggressive rats and mice than in aggressive animals. The high level of functional activity in non-aggressive rats coincided with a greater level of expression of 5-HT1A receptors in the midbrain. The level of 5-HT1A receptor mRNA in aggressive mice was unchanged in the midbrain and hypothalamus and was increased in the frontal cortex and amygdaloid complex. These results led to the conclusion that 5-HT1A receptors play a significant role in the mechanisms of genetic predisposition to aggressive behavior.

Functional Characteristics of Serotonin 5-HT2A and 5-HT2C Receptors in the Brain and the Expression of the 5-HT2A and 5-HT2C Receptor Genes in Aggressive and Non-Aggressive Rats

The functional activity of serotonin 5-HT2A and 5-HT2C receptors and the expression of the genes encoding them were studied in Norway rats bred for 60 generations for the presence and absence of high levels of stress-evoked aggression to humans. There were no significant differences in the levels of 5-HT2A receptor mRNA in the midbrain, frontal cortex, and hippocampus and the extents of head twitching evoked by the 5-HT2A agonist DOI in rats with and without genetically determined high levels of aggression. Administration of the selective 5-HT2C agonist MK-212 weakened reflex startle in response to an acoustic signal (the acoustic startle response) in non-aggressive animals but had no significant effects on the response in aggressive animals. Increases in the level of 5-HT2C receptor mRNA were seen in the frontal cortex and hippocampus in non-aggressive rats as compared with aggressive animals. Increases in the expression of the 5-HT2C receptor gene and the functional state of 5-HT2C receptors were seen in the brains of non-aggressive rats, without any changes in the 5-HT2A receptor mRNA level or receptor sensitivity; this is evidence for the involvement of 5-HT2C receptors in the mechanisms inhibiting fear-evoked aggressive behavior.