Endokannabinoidide süsteemi ja Wfs1 roll emotsionaalse käitumise regulatsioonis: käitumuslikud, farmakoloogilised ja geneetilised uuringud

Väitekirja elektroonilisest versioonist puuduvad publikatsioonid.Anxiety disorders are the most prevalent psychiatric disorders. The molecular mechanisms of the development of anxiety disorders are largely unknown, therefore possible new therapeutic targets in the brain were investigated. In the present study we used cat odour and the elevated plus-maze exposure to elicit anxiety-like response in Wistar rats or Wfs1 gene-deficient mice. For gene expression analysis of the endocannabinoid system (ECS), Wfs1, Gabra1 and Gabra2 genes quantitative real-time-PCR was performed. We found that only strong stress – cat odour exposure – increased some ECS genes in the mesolimbic area, but decreased their expression in the amygdala and in periaqueductal gray matter. This is probably related with different activation of brain structures in response to anxiety. High exploratory activity rats had reduced expression of Wfs1 mRNA in the mesolimbic area and hippocampus, whereas low exploratory activity rats displayed inhibited expression of the Wfs1 gene in the temporal lobe compared to control group animals. Increased anxiety after cat odour exposure in C57Bl/6 mice was accompanied by decreased level of the Wfs1 gene in the mesolimbic area and temporal lobe. Female Wfs1-deficient mice displayed increased anxiety and sensitivity to the anxiolytic action of diazepam and ethanol. This is related to the reduced level of Gabra1 and Gabra2 mRNA expression in the frontal cortex and in temporal lobe. In the present study we found that ECS and Wfs1 protein are playing a major role in the adaptation of rodents to a novel and aversive environment.Ärevushäired on maailmas enim levinud psühhiaatrilised häired. Ärevushäirete molekulaarsed tekkemehhanismid on siiani ebaselged, seetõttu püüdsime käesolevas uuringus leida ajus uusi võimalikke sihtmärke uute ravimite väljatöötamiseks. Käesolevas doktoritöös kasutasime kassilõhna ja tõstetud pluss-puuri katset ärevuse esilekutsumiseks isastel Wistar liini rottidel ja Wfs1 geeni puudulikkusega hiirtel. Endokannabinoidi süsteemi (ECS), Wfs1, Gabra1 ja Gabra2 geenide ekspressiooni muutuste välja selgitamiseks kasutasime kvantitatiivset reaalaja-PCR-i. Uuringute tulemused näitasid, et ainult tugev stress – kassilõhn – vallandab rottidel muutused ECS-i geenide ekspressioonis. Mesolimbilises struktuuris täheldati osade ECS-i geenide mRNA ekspressiooni tõusu, mandelkehas ja periakveduktaalses hallaines seevastu oli samade geenide ekspressioon vastusena kassilõhnale langenud. See võib olla seotud erinevate ajuosade erineva aktivatsiooniga vastuseks ärevusele. Wfs1 geeni ekspressioon oli vähenenud madala uudistamisaktiivsusega rottide oimusagaras, samasugust muutust täheldasime kõrge uudistamisaktiivsusega loomade mesolimbilistes struktuurides ja hipokampuses võrreldes kontrollgrupiga. Lisaks täheldadi Wfs1 geeni ekspressiooni langust C57Bl/6 liini hiirte aju mesolimbilistes struktuurides ja oimusagaras vastusena kassilõhnast indutseeritud ärevusele. Emased Wfs1 geeni puudulikkusega hiired olid ärevamad tõstetud pluss-puuri katses võrreldes metsiktüüpi loomadega, samuti oli neil tõusnud tundlikkus diasepaamile ja etanoolile. Need muutused võivad olla seotud Gabra1 ja Gabra2 mRNA ekspressiooni vähenemisega oimusagaras ja frontaalkoores. Käesolevas uurimistöös saadud tulemused näitavad ECS-i ja Wfs1 valgu olulist tähtsust katseloomade kohanemisel uudse ja ebameeldiva keskkonnaga

Effect of Chronic Valproic Acid Treatment on Hepatic Gene Expression Profile in Wfs1 Knockout Mouse

Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype

Long-term spatial memory and reversal learning ability in the Morris water maze is not altered in <i>Trib3</i>-deficient mice.

<p>Data are means ± SEM from littermate <i>Trib3</i>^−/− (n = 16) and <i>Trib3</i>^+/+ (n = 10) mice. (A) Escape latencies during four days of hidden-platform training performed at four trials per day. (B) Pool quadrant occupancy in a probe trial performed 24 h after the completion of training. The submerged platform was removed from the pool and the swim trajectory of mice was monitored for 1 min. The pool quadrant that previously contained the platform is designated the target quadrant, and the time spent in each quadrant of the pool is presented as percent of the total search time. (C) Reversal training escape latencies during two days of training performed at four trials per day. For reversal training, the hidden platform was repositioned to the pool quadrant opposite of the initial platform location. (D) Results of a probe trial performed 24 h after reversal training. The probe trial was carried out as in B. The reversal target denotes the pool quadrant that contained the platform during the reversal trainings trials. (E) Swimming speed at different stages of the experiment. The mean speed ± SEM from 60-second swimming sessions with no platform are shown. The habituation session was performed one day before the start of training. For <i>Trib3</i>^+/+, n = 5 per sex, and for <i>Trib3</i>^−/−, n = 8 per sex.</p

Rejection of amino acid-imbalanced diet in mice is not influenced by the deletion of <i>Trib3</i>.

<p>(<b>A</b>) Consumption of a diet lacking the essential amino acid leucine (Leu−), compared to the consumption of a corresponding nutritionally complete control diet (Leu+). Adult <i>Trib3</i>^+/+ and <i>Trib3</i>^−/− (n = 7 per genotype) mice were trained with food deprivation during the dark phase, and, during the light phase, food intake was measured for each animal at the indicated time-points by weighing the remaining food. For each animal, the intake of Leu− diet was compared to that of the Leu+ diet, and the average difference in the consumption of the Leu− diet relative to the Leu+ diet is expressed in percent ± SEM for each genotype. (<b>B</b>) Body weight and (<b>C</b>) body weight loss due to overnight food deprivation do not differ between <i>Trib3</i>-deficient and wild type adult mice. For B and C, four-month-old group-housed <i>Trib3</i>^+/+ (n = 5 for both males and females) and <i>Trib3</i>^−/− (n = 8 for both males and females) mice were maintained on the Leu+ diet <i>ad libitum</i> for two weeks to determine their diet-habituated body weight, followed by a single iteration of overnight fasting. The data in B and C are presented as the group means ± SEM.</p

<i>Trib3</i> is upregulated in the mouse anterior piriform cortex in response to leucine-deficient diet.

<p>Adult wild type mice consumed either a diet lacking leucine (Leu−; n = 5) or a corresponding control diet containing leucine (Leu+; n = 5), and, after 6 h of feeding, <i>Trib3</i> expression in the indicated brain regions was quantified by RT-qPCR. The results are presented as the mean ± SEM, and expressed relative to the level of <i>Trib3</i> mRNA in the anterior piriform cortex of the control diet (Leu+) group. *<i>P</i><0.05 comparing leucine-starved and control diet-fed groups.</p

<i>Trib3</i> is developmentally regulated in the mouse brain.

<p>(<b>A</b>) RT-qPCR quantification of <i>Trib3</i> expression in wild type C57BL/6J mouse brains at embryonic day (E) 14, 15, 17 and 18, and at postnatal day (P) 0, 2 and 4. The mean <i>Trib3</i> expression level ± SEM at the indicated age is presented relative to the level of <i>Trib3</i> expression at E14 (n = 7 for E17, E18 and P0, n = 6 for E15, n = 5 for E14 and P2, and n = 3 for P4). Means marked with the same letter are not significantly different at the 5% significance level. (<b>B</b>) Lack of <i>Trib3</i> does not lead to altered expression of other <i>Tribbles</i> family genes in the P3 mouse brain. RT-qPCR was used to determine the level of <i>Trib1</i> and <i>Trib2</i> mRNA expression in littermate <i>Trib3</i>^+/+, <i>Trib3</i>^+/− and <i>Trib3</i>^−/− mice (n = 5 per genotype). For both genes, the mean ± SEM is presented relative to the level of expression in <i>Trib3</i>^+/+ mice.</p