Fluoxetine Induced Gene Expression Of △FosB In Primary Cultures Of Astrocytes

△FosB is a member of the Fos family of transcription factors.It is derived from the fosB gene via alternative splicing.△FosB as many other Fos family members is induced rapidly and transiently in response to many types of acute stimulations.△FosB accumulates in a region-specific manner in brain uniquely by many types of chronic perturbations,including repeated administration of drugs of abuse or antidepressant or antipsychotic treatments.Importantly,once induced,△FosB persists in brain for relatively long periods due to its extraordinary stability.In the present work, we found that△FosB was expressed in mouse astrocytes in primary cultures.The expression of△FosB was increased by chronic treatment with pharmacologically relevant concentration of fluoxetine.Astrocytes express 5HT_2Aand 5-HT_2Breceptors, but not 5-HT_2Creceptors or 5-HT transporters.Fluoxetine-induced expression of△FosB could be inhibited by SB204741,an antagonist of the 5-HT_2Breceptor.Wherease, DOI,a 5-HT_2A/2Creceptor agonist did not have effect.It was suggested that the effect of fluoxetine on△fosB expression in astrocytes was mediated by 5-HT_2Breceptor.Objective1.Whether△FosB is expressed in primary cultures of mouse astrocytes.2.Whether fluoxetine has effect on expression of△fosB mRNA and protein.3.Whether 5-HT_2Breceptor is involved.MethodsPrimary cultures of astrocytes were from cerebal hemisphere of new born mice. The neopallia of the cerebral hemispheres were aseptically isolated,vortexed to dissociate the tissue,filtered through nylon meshes with pore sizes of 100μM and subsequently 70μM,diluted in culture medium,and planted in Falcon Primaria culture dishes.The culture medium was a Dulbecco's Medium with 7.5 mM glucose,initially containing 20%horse serum,and the cultures were incubated at 37℃in a humidified atmosphere of CO2/air(5:95%).The culturing medium was exchanged with fresh medium of similar composition on Day 3,and subsequently every 3-4 days.From Day 3,the serum concentration was reduced to 10%,and after the age of 2 weeks,0.25 mM dBcAMP was included in the medium.The cultures were used after at least three weeks of culturing.For determination of△FosB expression,the cells were incubated in corresponding medium without seruna at 37℃for specific time periods(30 min-7 day)in the absence or presence of fluoxetine(5μM-60μM)and 1μM DOI,with or without the 2μM SB204741.△fosB mRNA expression was detected by reverse transcription-polymerase chain reaction(RT-PCR),The PCR products were separated by 1.5%agarose gel electrophoresis,and captured by Fluorchem 5500.△FosB protein expression was detected by western bloting.The results were collected by Fluorchem imaging system.Band density was measured with Window AlphaEaseTM FC 32-bit software.The differences between multiple groups were analyzed by one-way analysis of variance(ANOVA)followed by Fisher's LSD multiple comparison test for unequal replications.The level of significance was set at P＜0.05.Results1.The stimulation of△fosB mRNA expression by fluoxetine was concentrationdependent in astrocytes.There was no significant change at 5μM.At 10μM,a pharmacologically relevant concentration,the level of△fosB mRNA was about 200% of the control value,which was statistically significant(P＜0.05).The stimulation was reached to the maximum at 40μM.The effect of fluoxetine was observed rapidly after 30 min of drug treatment,and there was no significant different between 30 min and 1hr or 2hr.2.The expression of△FosB protein was significantly increased(P＜0.05)after chronic treatment with 10μM fluoxetine for 1,3 and 7 days in astrocytes.Whereas, DOI,an agonist of 5-HT_2A/2C,at 1μM did not have any effect after 3 days of treatment.3.The effect of fluoxetine on△FosB expression was abolished by SB204741,an antagonist of 5-HT_2Breceptor.Conclusion△FosB was expressed in mouse astrocytes in primary cultures.The expression of△fosB mRNA and△FosB protein were increased by chronic treatment with a pharmacologically relevant concentration of fluoxetine.DOI,5-HT_2A/2Creceptor agonist,did not affect the expression of△FosB in astrocytes.SB204741,an antagonist of 5-HT_2Breceptor inhibited the effect of fluoxetine,suggesting that the effect of fluoxetine on△FosB expression was mediated by the activity of 5-HT_2Breceptors.It may be one of the underlying mechanisms of drug action of fluoxetine as an antidepressant.