down syndrome is caused by trisomy 21 and is frequently associated with cognitive impairments . based on the partial triplication of chromosome 16 , the mouse homologue of human chromosome 21 , a mouse model ( ts65dn ) has been developed that shows behavioral abnormalities , including deficient hippocampus - dependent learning and memory [ 1 , 2 ] . although most studies regarding the neurobiology of down syndrome have been focused on neurodevelopment , recent evidence suggests that pathophysiological processes in the adult brain significantly contribute to cognitive impairments in this disorder [ 38 ] . in ts65dn mice , enhanced inhibitory synaptic transmission suppresses proper induction of hippocampal synaptic plasticity , an important cellular mechanism for learning and memory formation . strikingly , using a variety of different gabaa receptor antagonists to suppress the abnormally increased level of inhibition in adult ts65dn mice fully restored their learning and memory impairments without affecting wild - type controls . these beneficial effects were not evident , however , with only acute administration of gabaa receptor antagonists but instead became clear only with a more prolonged ( 2 - 3 weeks ) treatment . this prolonged treatment was then sufficient to cause improvements in cognitive function lasting well beyond the actual treatment period , suggesting that treatment triggered lasting neurobiological changes . the neurobiological basis of these nonacute gabaa receptor antagonist treatment - induced behavioral modifications is currently unknown . key insights into potential mechanistic aspects , however , may be provided by a brief review of brain development . during postnatal brain development , inhibition plays an important role in regulating the temporal extent of critical periods ( i.e. , developmental time windows during which sensory input can substantially shape brain structure and function ) [ 9 , 10 ] . remarkably , several experimental manipulations that lower inhibitory synaptic transmission have been found to reinstate high levels of plasticity in the adult brain resembling those found in critical periods [ 1113 ] . it is possible that high levels of plasticity , characteristic of critical periods , are actively suppressed in adults by mechanisms including increased inhibition . therefore , one strategy to enhance plasticity in the adult brain could be the removal of these constraints by lowering inhibition [ 14 , 15 ] . one pharmacological manipulation particularly interesting from a translational point of view reported that ocular dominance plasticity is reinstated in the mature rat visual cortex by chronic treatment with the widely prescribed antidepressant fluoxetine , presumably also via a decrease in inhibitory synaptic transmission . used brain in vivo microdialysis to show reduced levels of extracellular gaba in the visual cortex of fluoxetine treated animals . white matter ltp , a form of synaptic plasticity that is normally absent in the adult brain due to matured intracortical inhibition , was present in fluoxetine - treated animals . bdnf expression was increased as a consequence of fluoxetine treatment , and intracortical bdnf administration was sufficient to cause an ocular dominance shift in response to monocular deprivation . to test if reduced inhibition underlies the effects of fluoxetine on ocular dominance plasticity , the gabaa receptor agonist diazepam was administered intracortically in fluoxetine - treated mice , which fully occluded the effect of fluoxetine on ocular dominance plasticity . translation of the preclinical gabaa receptor antagonist findings in ts65dn mice ( see above ) [ 4 , 8 ] to clinical populations is hampered by the fact that none of the employed gabaa receptor antagonists is currently in clinical use . gabaa receptor antagonists have narrow therapeutic windows and harsh side effect profiles and therefore have limited potential for translational applications , warranting the search for novel treatment approaches . we note that other strategies safer than gabaa receptor antagonists have been proposed for the treatment of down 's syndrome - related cognitive impairments , including gabaa receptor 5-selective inverse agonists and other compounds [ 1619 ] . here , we followed up on the finding that chronic fluoxetine treatment induced reduced levels of inhibition and enhanced plasticity , suggesting that chronic fluoxetine administration may have beneficial effects on cognitive dysfunction in ts65dn mice , similar to gabaa receptor antagonists . treatments with selective serotonin reuptake inhibitors ( ssris ) , such as fluoxetine , influence the serotonin system and also have complex effects on gabaergic neurotransmission [ 2023 ] , including pre- and postsynaptic effects , which may include an inhibition of evoked inhibitory postsynaptic potentials due to elevated extracellular serotonin levels . in the hippocampus , the serotonergic system has been proposed to be involved in shifting inhibition from dendritic to perisomal areas , which should allow for greater dendritic excitation and synaptic plasticity . we tested if an oral fluoxetine treatment regime has therapeutic effects on cognitive impairments ( spatial learning in the morris water maze ) in the ts65dn mouse model of down syndrome . there were no beneficial effects of fluoxetine treatment on behavioral impairments in ts65dn mice but , instead , unexpected side effects including seizures and death due to treatment that appeared to be genotype specific . experimental animals were generated by crossing c57bl / ejeij  c3sn.blia-pde6b + f1 hybrid wild - type males with b6eic3sn.blia-ts(17)65dn/dnj females ( breeders were purchased from the jackson laboratories ) . a 1 - 1 mating scheme was used , and males were left in the mating cage with the female and her litter . mice received water and food ad libitum . experiments were carried out during the light period of the cycle . male and female animals used for experiments were between 149 and 227 days of age at completion of the study ( sex and age were approximately balanced across groups ) . fluoxetine hydrochloride ( sigma ) was administered to the animals through the drinking water at a concentration of 0.2  mg / ml as previously described . animals were treated for 4 weeks before behavioral assessment commenced and were sacrificed after 6 weeks of treatment . initially , mice were handled for 7 days ( for approximately 2  min / animal / day ) to habituate the animals to investigator contact and procedural elements associated with the task . following handling , mice were trained on the hidden version of the water maze . during water maze training , the escape platform was hidden underneath the water surface in a constant location of the pool . the pool ( med associates ) had a diameter of 1.2  m and was filled with opaque water ( temperature : 2224c ) . behavior of the animals was recorded using an automated tracking system ( ethovision xt , noldus ) . during training trials , accordingly , intertrial intervals were approximately 1  min between trials 1 and 2 , as well as trials 3 and 4 , and were approximately 90  min between trials 2 and 3 . training trials were completed when mice climbed on the escape platform or when 1  min had elapsed , whichever came first . animals were given 15  s posttrial interval on the escape platform after completion of training trials . to evaluate the accuracy with which the animals had learned the position of the escape platform , we performed a probe trial once training was completed . during the probe trial , we removed the escape platform from the pool , and animals were released into the pool from the starting position within the opposite quadrant ( oq ) . we determined the time that mice spent searching in the target quadrant ( which previously contained the escape platform ) or the other quadrants during the probe trial . additionally , we analyzed the number of crossings of the exact target location ( i.e. , where the platform was during training ) and compared it to crossings of analogous positions in the other quadrants . as an additional probe trial measure , we determined the average distance ( proximity ) to the target location and compared it to the average distance to corresponding locations in the other quadrants . to assess if the probe trial measures differed across genotypes and/or treatment groups , we performed a three - way anova with genotype and treatment as between - subjects factors and quadrant as a within - subject factor . additionally , to assess for spatial selectivity of searching during the probe trial , we performed t - tests to compare target quadrant measures to the corresponding average values of the other quadrants . escape latencies during training were analyzed by two - way anova with genotype and treatment as between - subjects factors . swim speed during training and probe trial was analyzed by two - way anova with genotype and treatment as between - subjects factors . also , distance travelled during the probe trial was analyzed by two - way anova with genotype and treatment as between - subjects factors . mice were placed for 10  min in a square open field made of acrylic ( footprint 27.5  cm  27.5  cm ) ; activity was recorded and analyzed using an automated system ( ethovision xt , noldus ) . light levels were set to 100 lux in the center of the open field . mice were anesthetized and perfused transcardially with 0.9% saline and 4% paraformaldehyde in cold 0.1  m phosphate buffer ( ph 7.4 ) . brains were extracted , postfixed in 4% paraformaldehyde over night , and subsequently transferred into 30% sucrose . forty micrometer coronal section series were then created using a sliding microtome ( leica ) . sections were stored in cryoprotectant solution ( 25% ethylene glycol , 25% glycerine , and 0.05  m phosphate buffer ) at 20c . every sixth section of the coronal section series mentioned above ( i.e. , sections 240  m apart ) was used for free - floating immunohistochemistry . sections were rinsed in tbs , incubated in a solution containing 0.6% h2o2 in tbs to inhibit residual endogenous peroxidase , and blocked for 30  min in tbs with 3% donkey serum and 0.1% triton x-100 . sections were then incubated for 48  h at 4c in a primary antibody solution ( rabbit anti - choline acetyltransferase ( anti - chat ) , millipore , 1  :  100 ) containing tbs , 3% donkey serum , and 0.1% triton x-100 . subsequently , sections were rinsed in tbs , blocked in tbs with 3% donkey serum and 0.1% triton x-100 , and incubated for 1  h at room temperature in secondary antibody ( biotinylated donkey anti - rabbit igg , dianova , 1  :  500 ) . next , sections were washed with tbs and subjected to 1  h incubation in avitin - biotin peroxidase complex in tbs ( abc elite , vector ) . sections were mounted , air - dried , dehydrated with a graded series of ethanol , and mounted with permount . stereological analyses of cell number and cell size of choline acetyltransferase - immunostained cells were performed on section series stained against choline acetyltransferase with sections 240  m apart , covering the entire fronto - occipital extension of the hemisphere . analyses were carried out using a nikon eclipse 90i microscope equipped with stereo investigator ( microbrightfield ) . quantitative analysis of the total number of chat - positive neurons in the basal forebrain including ventral diagonal band nuclei ( vdb ) and medial septum nuclei ( msn ) was performed in an unbiased fashion using the optical fractionator method as described previously [ 24 , 25 ] . this method allows for a systematic random sampling of the region of interest ( roi ) . the landmarks outlining the roi ( msn and vdb ) were taken from the mouse brain atlas . rois were manually marked , and pictures from every roi were taken using the stereo investigator software . with a sampling grid of 90  m  90  m systematically moving through the outlined roi , we counted chat - positive neurons that were either within the dissector counting frame ( 60  m  60  m ) or that were touching its right / upper edge . cell size of chat - immunoreactive neurons was determined on the same section series using the nucleator probe within the stereo investigator software . six rays extending from the nucleus were used to mark the boundaries of the cells , allowing an estimation of cell surface area . to test if adult - onset fluoxetine treatment has a beneficial effect on behavioral features associated with the ts65dn mouse model of down syndrome , we initiated fluoxetine or vehicle control treatment in ts65dn mice and 2n ( diploid ) littermate controls ( 2n / vehicle :  n = 9  mice ; 2n / fluoxetine :  n = 11  mice ; ts65dn / vehicle :  n = 7  mice ; ts65dn / fluoxetine :  n = 9  mice ) . body weight measurements were taken after 1 month of treatment with fluoxetine or vehicle control ( figure 1 ; 2n , vehicle :  n = 8  mice ; 2n , fluoxetine :  n = 11  mice ; ts65dn , vehicle :  n = 7  mice ; ts65dn , fluoxetine :  n = 7  mice ) . two - way anova with genotype and treatment as between - subjects factors revealed significant main effects of genotype ( anova genotype f(1,29 ) = 7.37 ,  p = 0.01 ) and treatment ( anova treatment f(1,29 ) = 5.42 ,  p = 0.03 ) , while there was no significant interaction between the factors ( f(1,29 ) = 0.85 ,  p = 0.77 ) . fluoxetine treatment reduced body weights in ts65dn mice relative to ts65dn vehicle controls ( fisher 's plsd ts65dn / fluoxetine versus ts65dn / vehicle ,  p = 0.04 ) . fluoxetine had no significant effect on body weight within 2n littermate controls ( fisher 's plsd 2n / vehicle versus 2n / fluoxetine ,  p = 0.19 ) . unexpectedly , during the approximate 6-week treatment period , 4 out of 9 ts65dn mice treated with fluoxetine died ( table 1 ) . there were no deaths among the vehicle - treated ts65dn mice ( 0 out of 7 mice ) and the fluoxetine - treated wild - type animals ( 0 out of 11 mice ) . in the vehicle - treated wild - type group , 1 out of 8 mice was found dead during the treatment period ( fisher 's exact test , ts65dn / fluoxetine group versus collapsed data from the other groups ,  p = 0.01 ) . we started our behavioral assessment of fluoxetine - treated and vehicle - treated ts65dn mice and their respective wild - type control groups by testing general exploratory activity in an open field assay ( figure 2 ; 2n , vehicle :  n = 8  mice ; 2n , fluoxetine :  n = 11  mice ; ts65dn , vehicle :  n = 7  mice ; ts65dn , fluoxetine :  n = 7  mice ) . there was no effect of genotype on total distance travelled in the open field ( two - way anova with genotype and treatment as between - subjects factors : f(1,29 ) = 0.73 ,  p = 0.4 ) . statistical analysis showed a significant effect of drug treatment on activity levels ( two - way anova with genotype and treatment as between - subjects factors : f(1,29 ) = 5.24 ,  p = 0.03 ) and suggested a possible genotype  treatment interaction ( two - way anova with genotype and treatment as between - subjects factors : f(1,29 ) = 3.35 ,  p = 0.08 ) . activity levels were significantly reduced in fluoxetine - treated ts65dn mice relative to vehicle - treated trisomic mice ( fisher 's plsd ,  p = 0.04 ) . corresponding post hoc analysis did not show a significant effect of treatment in wild - type animals ( fisher 's plsd ,  p = 0.5 ) . to determine if fluoxetine treatment restores spatial learning and memory deficits in ts65dn mice , we tested ts65dn mice and wild - type littermate controls , either treated with fluoxetine or vehicle , in the hidden platform version of the morris water maze ( figure 3 ; 2n , vehicle :  n = 8  mice ; 2n , fluoxetine :  n = 11  mice ; ts65dn , vehicle :  n = 7  mice ; ts65dn , fluoxetine :  n = 7  mice ) . escape latencies decreased during training in all groups ( figure 3(a ) ) , although to a more limited extent in ts65dn mice ( both vehicle and fluoxetine treated ) . statistical analysis revealed a significant main effect of genotype with higher escape latencies in ts65dn mice compared to 2n controls ( two - way anova with genotype and treatment as between - subjects factors : f(1,29 ) = 20.11 ,  p = 0.0001 ) . there was no significant main effect of treatment ( two - way anova : f(1,29 ) = 0.07 ,  p = 0.79 ) and no genotype  treatment interaction ( two - way - anova : f(1,29 ) = 2.07 ,  p = 0.16 ) . we analyzed swim speed during all training sessions and the probe trial using repeated - measures two - way anova with genotype and treatment as between - subjects factors ( figure 3(b ) ) . swim speed was significantly decreased in ts65dn mice relative to 2n controls ( repeated - measures two - way anova : f(1,29 ) = 9.15 ,  p = 0.005 ) . there was no significant effect of treatment on swim speed ( repeated - measures two - way anova : f(1,29 ) = 2.258 ,  p = 0.144 ) and no significant genotype  treatment interaction ( repeated - measures two - way anova : f(1,29 ) = 0.529 ,  p = 0.473 ) .   to evaluate how accurately the mice had learned the escape platform position during training , we performed a single - probe trial ( no platform in the water tank ) after completion of training ( figures 3(c ) , 3(d ) , and 3(e ) ) . we analyzed several probe trial measures that report spatial selectivity of searching and , hence , indicate the extent of spatial learning that occurred . first , we measured the time that the animals spent in the target quadrant ( which previously contained the escape platform ) and the other quadrants during the probe trial ( figure 3(c ) ) . statistical analysis of the quadrant occupancy data showed significant genotype  quadrant and treatment  quadrant interactions ( three - way anova with genotype and treatment as between - subjects factors and quadrant as within - subjects factor : genotype  quadrant interaction , f(3,116 ) = 5.24 ;  p = 0.002 , treatment  quadrant interaction : f(3,116 ) = 5.48 ,  p = 0.002 ) , indicating that genotype and treatment had significant effects on quadrant occupancy . vehicle - treated 2n controls spent significantly more time in the target quadrant than the other quadrants ( t - test , target quadrant occupancy versus average occupancy of the other quadrants :  p = 0.001 ) , indicating memory for the platform location . in contrast , in the other groups , target quadrant occupancy was not significantly different from average occupancy of the other quadrants ( t - test , target quadrant occupancy versus average occupancy of the other quadrants : 2n / fluoxetine ,  p = 0.292 ; ts65dn / vehicle ,  p = 0.267 ; ts65dn / fluoxetine ,  p = 0.241 ) . we also recorded the number of target crossings ( i.e. , crossings of the exact target location , where the platform was located during training ) during the probe trial , which was compared to the number of crossings over corresponding locations in the other quadrants ( figure 3(d ) ) . with respect to target crossings , anova analyses yielded a significant effect of genotype ( three - way anova with genotype and treatment as between - subjects factors and quadrant as within - subjects factor : f(1,116 ) = 17.3 ,  p < 0.0001 ) , reflecting an overall reduced number of crossings in ts65dn mice and , additionally , a possible genotype  quadrant interaction ( three - way anova with genotype and treatment as between - subjects factors and quadrant as within - subjects factor : f(3,116 ) = 2.25 ,  p = 0.09 ) . to further probe for spatial selectivity of searching , we asked whether animals showed significantly more crossings over the target location than over corresponding locations in the other quadrants . fluoxetine - treated 2n mice showed significantly more crossings of the target location than the other locations ( t - test , target crossings versus average crossings of corresponding locations in the other quadrants :  p = 0.010 ) , while this was not the case for the other groups ( t - test , target crossings versus average crossings of corresponding locations in the other quadrants : 2n / vehicle ,  p = 0.100 ; ts65dn / vehicle ,  p = 0.362 ; ts65dn / fluoxetine ,  p = 0.784 ) . as a third measure for spatial selectivity of searching , we looked at proximity to the target ( i.e. , average distance to the target location , where the platform was located during training ) and compared it to the average distance to corresponding locations in the other quadrants ( figure 3(e ) ) . three - way anova with genotype and treatment as between - subjects factors and quadrant as within - subjects factor showed a significant main effect of genotype ( f(1,116 ) = 7.10 ,  p = 0.009 ) , which was reflective of the overall larger distances that ts65dn mice had to the target position and corresponding positions in the other quadrants . this anova also revealed a significant genotype  quadrant interaction ( f(3,116 ) = 4.10 ,  p = 0.008 ) and a significant treatment  quadrant interaction ( f(3,116 ) = 5.25 ,  p = 0.002 ) , showing that the search pattern was influenced by genotype and treatment , such that target - preferential searching was less pronounced or absent in ts65dn mice / fluoxetine - treated mice . vehicle - treated 2n mice showed significantly lower proximity values to the target than average distance to the other positions ( t - test , proximity to target versus average proximity to corresponding locations in the other quadrants :  p = 0.005 ) , again indicating preferential searching in the relative vicinity of the target in this group . this comparison yielded nonsignificant results for the other groups ( t - test , target crossings versus average crossings of corresponding locations in the other quadrants : 2n / fluoxetine ,  p = 0.550 ; ts65dn / vehicle ,  p = 0.442 ; ts65dn / fluoxetine ,  p = 0.077 ) . as expected based on the slower swim speed in ts65dn mice ( see above ) , total distance travelled during the probe trial was significantly reduced in ts65dn mice ( figure 3(f ) ; two - way anova with genotype and treatment as between - subjects factors : genotype effect , f(1,29 ) = 10.98 ,  p = 0.003 ; treatment effect , f(1,29 ) = 3.21 ,  p = 0.084 , genotype  treatment interaction , f(1,29 ) = 0.14 ,  p = 0.706 ) . taken together , the probe trial revealed indications of spatially selective searching and , hence , successful spatial learning and memory in vehicle - treated ( see quadrant occupancy and proximity to target ) and fluoxetine - treated ( see target crossings ) 2n mice , but not in ts65dn mice , irrespective of treatment group . these data show clear behavioral abnormalities of ts65dn mice in the water maze , which are consistent with published data [ 2 , 8 , 27 ] and reflect motor impairments ( see swim speed ) and spatial learning and memory difficulties ( see probe trial measures ) in ts65dn mice . neither motor impairments nor spatial learning deficits in ts65dn mice were improved by fluoxetine treatment . during behavioral experimentation , we incidentally witnessed tonic - clonic seizures triggered by handling in a few animals ( a total of 3 mice ) ( table 2 ) . seizures were not observed in the other groups ( fisher 's exact test , ts65dn / fluoxetine group versus collapsed data from the other groups ,  p = 0.01 ) . one of the neuropathological hallmarks of both down syndrome and alzheimer 's disease is the age - dependent loss of basal forebrain cholinergic neurons ( bfcns ) [ 24 , 28 ] . previous studies pointed to a correlation between bfcn degeneration and abnormalities in memory and learning in aging ts65dn mice . to probe whether fluoxetine is able to ameliorate or prevent early bfcn degeneration in ts65dn mice , we performed unbiased stereological cell counts of choline acetyltransferase ( chat ) antibody - stained sections in fluoxetine - treated animals and vehicle controls . we determined the cholinergic cell number and size ( figure 4 ) in the medial septal nuclei ( msn ) and ventral diagonal band ( vdb ) of each group ( 2n , vehicle :  n = 8 mice ; 2n , fluoxetine :  n = 11 mice ; ts65dn , vehicle :  n = 7 mice ; ts65dn , fluoxetine :  n = 5 mice ) . no clear differences with regards to cholinergic cell number were evident between trisomic and euploid mice ( two - way anova with genotype and treatment as between - subjects factors : f(1,27 ) = 0.04 ,  p = 0.84 ) . treatment also had no obvious effects on the number of chat - positive cells in basal forebrain cholinergic nuclei ( two - way anova with genotype and treatment as between - subjects factors : f(1,27 ) = 0.61 ,  p = 0.44 ) . since neuronal atrophy precedes cell loss , we analyzed cell size of chat - positive cells in bfcn of fluoxetine-/vehicle - treated ts65dn mice / wild - type controls . cholinergic neurons of euploid mice were insignificantly larger than those in trisomic mice ( two - way anova with genotype and treatment as between - subjects factors : f(1,27 ) = 2.2 ,  p = 0.15 ) . fluoxetine treatment had no significant effect on cell size , although there was a trend towards increasing cell size in ts65dn mice ( two - way anova with genotype and treatment as between - subjects factors : treatment effect , f(1,27 ) = 3.08 ,  p = 0.091 ; genotype  treatment interaction , f(1,27 ) = 3.14 ,  p = 0.08 ) . in the present study , we set out to test whether a 4-week , adult - onset fluoxetine treatment is effective against behavioral alterations in the ts65dn mouse model of down syndrome . in particular , we did not observe a treatment effect on the clear ts65dn behavioral phenotype in the water maze , which included slower swim speed and effects on probe trial measures indicative of spatial learning and memory impairments . adverse side effects due to fluoxetine treatment of ts65dn mice , however , appeared to be profound in our cohort . four out of 9 ts65dn mice died while on fluoxetine treatment during which time there were no deaths in the other groups ( with the exception of 1 dead wild - type / vehicle animal ) . these results suggest that fluoxetine may have a genotype - specific adverse effect and warrant further experimentation in larger animal cohorts that also assess the cause of death in fluoxetine - treated ts65dn mice . we observed handling - induced seizures in a few mice , all of which were fluoxetine - treated ts65dn mice , suggesting interactive effects of ts65dn genotype and fluoxetine treatment on seizure susceptibility . seizures represent a relatively rare side effect of therapeutic fluoxetine regimens in general clinical populations but are a more common feature of fluoxetine intoxications [ 2931 ] . several epilepsy paradigms in animal models also illustrate a proconvulsive effect of fluoxetine ; for instance , fluoxetine pretreatment potentiates the convulsive effects of pentylenetetrazol ( ptz ) and electrically evoked seizures [ 32 , 33 ] . collectively , these reports show that treatments with selective serotonin reuptake inhibitors , including fluoxetine , can lower seizure thresholds in humans and animal models . epilepsy is not uncommon in down syndrome and may affect 113% of all individuals with trisomy 21 [ 34 , 35 ] . although spontaneous seizures are not a feature of the ts65dn model , ts65dn mice are more prone to audiogenic seizures than controls , suggesting reduced seizure thresholds in this model . in sum , the data raise the possibility that behavioral convulsions in ts65dn mice , described above , may result from an interaction between fluoxetine and the lowered seizure thresholds in ts65dn mice . future studies should examine these effects in larger animal cohorts and include electrophysiological assessments of seizure activity . for the present study , we adopted a fluoxetine treatment regime used previously , that is , administration of 0.2  mg / ml fluoxetine via the drinking water . this treatment regime was found to lower intracortical inhibition and to reinstate ocular dominance plasticity in the adult visual cortex . the fluoxetine dose is higher than rodent equivalents of clinically used doses ( i.e. , clinically employed doses converted into equivalent surface area doses for mice / rats ) but is within the range of fluoxetine dosing schemes generally used in mice and rats ( e.g. , [ 20 , 37 , 38 ] ) . in future work , it will be important to establish a dose - response function with respect to the side effects in ts65dn mice . based on our present data , we can not rule out the possibility that lower fluoxetine doses exert beneficial effects in the ts65dn model of down syndrome . it will also be important to determine if drug  genotype interactions are caused by genotype - dependent differences in cns effects of fluoxetine or , possibly , different pharmacokinetic profiles in ts65dn mice and wild - type controls ( e.g. , metabolism of fluoxetine , tissue distribution , etc . ) . the animals used in the present study were partly in the age range in which neurodegenerative processes may slowly begin in ts65dn mice ( i.e. , > 6 months of age ) . we assessed neurodegeneration via stereological quantification of basal forebrain cholinergic neurons and did not observe significant differences between trisomic and euploid animals ( figure 4 ) , showing that neurodegenerative changes were not a prominent feature in our ts65dn cohort ( consistent with other reports showing that first signs of degenerative changes affecting the cholinergic system in ts65dn mice emerge between 6 and 12 months ; [ 28 , 39 ] ) . nevertheless , we can not rule out the possibility that subtle degenerative changes were already present , yet not measureable in our cohort . therefore , it remains possible that adult - onset fluoxetine treatment has beneficial effects in 36 months old ts65dn mice ( i.e. , in the narrow time window during adulthood , wherein ts65dn mice are thought to be free of degenerative changes ) . a prior study had assessed the effect of fluoxetine treatment on hippocampal neurogenesis in 25 months old ts65dn mice . and treatment was shorter ( 15 days ) and involved lower dosing ( 5  mg / kg ) than treatment in the present study . fluoxetine treatment increased neurogenesis in ts65dn mice and wildtype controls , which is in agreement with earlier reports regarding the effects of chronic fluoxetine treatment on adult hippocampal neurogenesis . behavioral results were not reported and , hence , it remains unclear whether there was a behavioral effect associated with fluoxetine treatment of ts65dn mice in this study . another more recent study reported beneficial effects of fluoxetine , administered early postnatally , in the ts65dn mouse model . in this study , fluoxetine was administered from p3 to p15 ( s.c . injections of 5  mg / kg from p3 to p7 and 10  mg / kg from p8 to p15 ) followed by a brdu injection at p15 to label proliferating cells . fluoxetine increased cell proliferation and generation of new neurons in the dentate gyrus and subventricular zone of ts65dn mice and wild - type controls . fluoxetine treatment increased the number of dentate gyrus granule cells at p45 in ts65dn mice to wild - type control levels , while granule cell numbers were reduced in vehicle ts65dn mice . limited behavioral assessment was performed in the context of this study in juveniles ( at p43 ) . context fear - conditioning impairments in juvenile ts65dn mice were improved by early postnatal fluoxetine treatment , while fluoxetine had no effects on wild - type controls . in the present study we did not find beneficial effects of fluoxetine treatment on behavioral impairments of adult ts65dn mice in the morris water maze . it is possible that early postnatal and adult - onset fluoxetine treatments have different effects on behavioral impairments in ts65dn mice . the protocols in our study , however , differ also in a number of other respects from the published paper ( e.g. , fluoxetine dose , route of administration , and duration of treatment ; behavioral paradigm : here , we assessed learning in the water maze ; bianchi et al . assessed fear conditioning ) , which may also account for the different findings . in future studies , it will therefore be important to further explore this parameter space , that is , to establish dose - response relationships for fluoxetine treatment in ts65dn mice at different ages and to conduct comprehensive behavioral assessments that cover impairments of ts65dn mice in a range of behavioral tasks . here , we determined the impact of an adult - onset , chronic treatment with the antidepressant fluoxetine on behavioral alterations in the ts65dn mouse model of down syndrome . we did not find a beneficial effect of fluoxetine treatment on ts65dn behavioral phenotypes , but instead our findings suggest the presence of genotype - dependent fluoxetine side effects ; we observed seizures and mortality in treated ts65dn mice , but not wild - type controls . future studies should reevaluate these findings in larger animal cohorts , determine what the nature of the possible drug  genotype interaction is ( e.g. , genotype - dependent differences in drug metabolism , tissue distribution , or truly differential effects of equivalent drug tissue concentrations on cellular / tissue functions ) , and establish dose - response relationships for these possible side effects .
down syndrome is caused by triplication of chromosome 21 and is associated with neurocognitive phenotypes ranging from severe intellectual disability to various patterns of more selective neuropsychological deficits , including memory impairments . in the ts65dn mouse model of down syndrome 
 , excessive gabaergic neurotransmission results in local over - inhibition of hippocampal circuits , which dampens hippocampal synaptic plasticity and contributes to cognitive impairments . 
 treatments with several gabaa receptor antagonists result in increased plasticity and improved memory deficits in ts65dn mice . 
 these gabaa receptor antagonists are , however , not suitable for clinical applications . 
 the selective serotonin reuptake inhibitor fluoxetine , in contrast , is a widely prescribed antidepressant that can also enhance plasticity in the adult rodent brain by lowering gabaergic inhibition . for these reasons 
 , we wondered if an adult - onset 4-week oral fluoxetine treatment restores spatial learning and memory impairments in ts65dn mice . 
 fluoxetine did not measurably improve behavioral impairments of ts65dn mice . on the contrary , we observed seizures and mortality in fluoxetine - treated ts65dn mice , raising the possibility of a drug  genotype interaction with respect to these adverse treatment outcomes . 
 future studies should re - address this in larger animal cohorts and determine if fluoxetine treatment is associated with adverse treatment effects in individuals with down syndrome .