Multiple studies suggest that interactions between the higher brain structures, such as cortex and hippocampus, and so called defensive circuitry, which include amygdala, hypothalamus and periaqueductal grey, modulate fear/defensive behaviors. Moreover, changes in such interaction may lead to mental illness.? We have previously found that mice with the conditional knockout (KO) of Brain Derived Neurotrophic Factor (BDNF) restricted to the hippocampal area CA3 are more aggressive than their wild type (WT) counterparts. During the past fiscal year we were searching for neuronal and molecular mechanism responsible for this elevated aggressiveness. Our search was driven by the monoamine hypothesis, which postulates that aggression in BDNF KO mice was caused by changes in serotonergic signaling.? Our aims were ? 1) to examine whether levels of serotonin were altered in BDNF KO mice;? 2) to identify changes in regulation of hippocampal synaptic transmission by serotonin;? 3) to determine if any such changes could be the cause of the highly aggressive behaviors in mice.? The address the first aim we employed microdialysis/HPLC system for measuring levels of extracellular serotonin in the brain and discovered that BDNF KO mice had lower concentration of serotonin in the hippocampus than their wild type counterparts did. In contrast, levels of serotonin in the prefrontal cortex were not affected by the mutation. These findings indicate that the decrease in the local levels of BDNF in the hippocampus resulted in the reduction of serotonin concentration, and that this reduction represents local rather than global change in serotonergic transmission.? To address the second aim we began to test if there were any differences in how serotonin regulated synaptic transmission inside the hippocampus in BDNF KO versus WT mice. One of the main effects of serotonin is augmentation of inhibitory synaptic transmission. Therefore, we measured potentiation of inhibitory postsynaptic currents (IPSC) by serotonin using whole cell recording from CA3- and CA1-pyramidal neurons of hippocampal slices. We found that such potentiation was significantly higher in slices from KO mice. It indicated that functions of serotonin receptors might be altered in the mutant animals. ? The serotonin receptors 5-HTr2 and 5-HTr3 have been previously implicated in modulating IPSC, so we examined effects of the specific 5-HTr2- and 5-HTr3-specific agonists and antagonists on amplitudes and frequencies of IPSCs recorded from KO and WT mice. While activation or suppression of 5-HTr2 had same effects between KO and WT mice, the suppression of 5-HTr3 eliminated the between genotype differences in IPSC responses to serotonin. Moreover, while agonist of 5-HTr3 had no detectable effect on IPSCs in the slices from WT animals, it increased IPSC amplitudes and frequencies in the slices from KO mice. These results indicate that the function of 5-HTr3 in the hippocampus is enhanced in KO mice. Testing expression of serotonin receptors by real time PCR revealed higher levels of mRNA for 5-HTr3a, and 5-HTr2c in KO mice, than their WT counterparts, but there was not difference in expression of 5-HTr1a and 5-HTr2a.? We next asked whether the KO-induced changes in the animals behavior and in serotonergic transmission were the consequence of chronic decrease in the level of serotonin. To this end we increased levels of extracellular serotonin in KO mice by chronic administration of selective serotonin reuptake inhibitor fluoxetine. This treatment dramatically decreased KO mice aggressiveness, reduced levels of 5-HTr3 mRNA in their hippocampus and reduced the augmentation of IPSC by serotonin. These findings suggest that the effects of BDNF knockout on expression and function of serotonin receptor 5-HTr3 are mediated by chronic decrease in the level of extracellular serotonin.? The address the third aim, we examined whether local modulation of specific serotonin receptors in the hippocampus could alter aggression. We chronically implanted mice with double cannulas targeting ventral hippocampus and examined how acute infusion of agonists and antagonists to serotonin receptors 1A, 1B, 2A, 2C and 3 modulated aggressive behaviors in the intruder test. The agonist of 5-HTr3 enhanced aggression in WT mice, and its antagonist had an inhibitory effect. In KO animals, the agonist also increased aggression, however, the antagonist failed to inhibit it. The agonist of 5-HTr1A suppressed aggression regardless of genotype, while modulation of receptors 1B and 2 did not have any effect on aggression. These findings yield two conclusions: 1) serotonin in the hippocampus can inhibit aggression by activating receptor 5-HTr1A, and enhance aggression by activating receptor 5-HTr3; 2) elevated aggression in KO mice is a consequence of higher expression and activity of 5-HTr3.? Our future goals are to understand how elimination of hippocampal BDNF reduces levels of extracellular serotonin, and why activity of 5-HTr3 inside hippocampus can affect aggression.
