Nikolay N . Dygalo

1. Functional neurogenomics: brain neurochemical systems in regulation of psychoneuroendocrine functions Nikolay N. Dygalo Institute of Cytology & Genetics, RAS, Novosibirsk International WorkshopEC-Russia: Cooperation in Medicine and Public Health St.-Petersburg 11-12 October, 2007

2. Experimental Model of Neurodegeneration (natural apoptotic cell death in the week-old rat brain) DNA fragmentation Caspase-3 protein Cortex Brainstem Cerebellum Hippocamp. Bannova et al., Developmental Brain Res. 2004 Menshanov, Bannova, Dygalo, Neurochem. Res. 2006

3. Alpha2-adrenergic receptors (α2-ARs) regulate apoptosis in the cortex Antisense knock-down of α2A-ARs increased caspase-3 expression in the week-old rat cortex and α2-AR agonist clonidine decreased it. Data evidence for neuroprotective potency of α2-AR agonists in the cortex.

4. Control Yohimbine Clonidine Clonidine + Yohimbine 180 160 140 120 caspase-3 mRNA,% 100 80 , 60 40 20 0 Stimulation of 2-ARincreases caspase-3 mRNA in the week old rat brainstem caspase-3 mRNA β-actin mRNA Clonidine + Control Yohimbine Yohimbine Clonidine - Antagonist - Agonist Dygalo N.N. et al., Developmental Brain Res. 2004. Menshanov PN, Bannova AV, Dygalo NN. Neurochem Res. 2006.

5. RNA interference: Sequence-specific inhibition of the gene expression in the brain in vivo 5-day-old rat brain Oligonucleotides (ОDN) enter brain cells Site of injection Hematoxylin & Eosin Green fluorescence- ОDN

6. Saline dcRNA siRNA 5-day-old rat brain siRNA inhibited targeted-gene expressionin the brain in vivo Site of injection of siRNAtargetedα2A-adrenergic receptor (α2A-AR) gene α2А-АR mRNA -actin mRNA Substances injected into the brain Shishkina G.T., Kalinina T.S., Dygalo N.N. Neuroscience, 2004.

7. RNA interference(targeting2A-ARmRNA) blocks behavioral response of the animal to the receptor stimulation 600 Saline dsRNA siRNA 480 360 Time to the first paw movement, sec * 240 120 0 0 5 10 Clonidine, mkg F2,55=15.82; p<0.001

8. Up-regulation of tryptophan hydroxylase-2 mRNA in the rat brain by chronic fluoxetine treatment correlates with its antidepressant effect Immobility in the forced-swim test TPH2 mRNA Correlation immobility - TPH2 mRNA Real-time PCR Tissue sample r = -0.485, p<0.05 Shishkina, Kalinina, Dygalo, Neuroscience, 2007

9. Antidepressant Sensitive Catalepsy (ASC) mouse strain with depressive-like features * * Distance run, cm AKR CBA ASC ASC mice have decreased open field locomotion * Catalepsy in ASC mice is sensitive to chronic, but not acute, treatment with classic antidepressant drugs, imipramine and fluoxetine. Immobility time, s AKR CBA ASC ASC mice have increased immobility in the forced swim test ASC – originated from backcrossesbetweenCBA и AKR mouse strains Bazovkina et al., Genetika, 2005 Tikhonova et al., Bull. Exp. Biol. Med., 2006

10. Linkage between the main gene of catalepsy and 5-HT1A serotonin receptor in mice QTL analysis mapped the main gene of catalepsy on the 50-75 cM fragment of mouse chromosome 13 near the 5-HT1A receptor gene Selective breeding of mice for catalepsy removes the AKR-derived marker linked to 5-HT1A receptor and fixed the CBA-like. Kondaurova et al., Genes Brain Behav., 2006 Kulikov et al., Dokl. Acad. Sci. 2003, Genetika, 2003

11. C1473G polymorphism in mouse tph2 gene (tryptophan hydroxylase-2) determines the activity of this key enzyme of serotonin synthesis in the brain and intermale aggressiveness in mice C1473G polymorphism in mouse tph2 gene Tph2 activity Number of attacks Strains: 1-CBA, 2-PT, 3-C57BL/6, 4-C3H/He, 5-AKR, 6-YT, 7-DD, 8-BALB/c, 9-A/He, 10-CC57BR, 11-DBA/2 Kulikov et al., Genes Brain Behav., 2005

12. Sequencing of the 5-HT1A serotonin receptor gene of hibernating ground squirrel revealed insertion of three nucleotides (marked in red) in the third intracellular loop that is lacking in the 5-HT1A receptor gene of nonhibernators Naumenko et al., Genes Brain and Behavior, 2007

13. base level • water loading • water loading + 5-HTP injection Vasopressin deficient Brattboro rats were used to analyze interrelations between serotonin and vasopressin in regulation of renal function Vasopressin deficiency affects brain 5-HT system reactions to hydration and water deprivation. Serotonin has a role in regulation of renal function. Activity of TPH2 (pmol/mg/min) Glomerular filtration rate • control • water deprivation • hydration WISTAR BRATTLBORO Ivanova et al. Neuroendocrinology, 2007 Popova et al. Pflugers Arch., 2002

14. Models: Experimental Model of Neurodegeneration Genetic Models of Psychopathology Genetic Model of Vasopressin Deficiency Natural Model of specific form of behavior (hibernation) Depression-like behavior Methods: Gene silencing: RNA interference, Antisense knockdown Gene expression: Real-Time PCR, RT-PCR, Westernblot Gene sequencing Quantitative trait linkage analysis Neurochemistry: Enzymes activities, Receptors densities, Neurotransmitters concentrations (HPLC) Behavioral tests Measurements of physiological functions Applications: Neurodegeneration Psychopathology Target validation for new drugs Search for new targets of the existing drugs

15. Participants of the work: Institute of Cytology & Genetics, RAS, Novosibirsk Laboratory of Functional Neurogenomics Laboratory of Behavioral Neurogenomics Laboratory of Physiological Genetics Institute of Chemical Biology & Fundamental Medicine, RAS, Novosibirsk

16. Thank you for attention