Differential alterations of the DA system in BTBRand Fmr1-KOmice
To investigate whether and how the DA pathways are modified inthe ASD models, we labelled dopaminergic neurons with an anti-THantibody in brain sections taken from age-, sex- and backgroundmatched WT, BTBR and Fmr1-KO mice. TH is a rate-limitingenzyme that converts tyrosine to DA precursor L-DOPA. We firstexamined the origins of the DA system, i.e. SNc and VTA inthe midbrain, and their major projections to the dorsal striatum(dSTR) and NAc via the respective nigrostriatal andmesocorticolimbic pathways [4].Diagrams in Fig. 1A depictthe distribution of the soma and axons of dopaminergic neuronsidentified by TH labelling in the four nuclei. Higher-magnificationimages illustrate the specific locations of these neurons withinSNc and VTA (Fig. 1B). Bymeasuring the intensities of anti-TH staining and normalizing themto the average values of the WT group, we found that TH expressionin the BTBR brains was significantly reduced in SNc (U = 6,Z = 2.146, p = 0.032), VTA (U = 7, Z = 2.003,p = 0.045), and dSTR (U = 6, Z = 2.143,p = 0.032; Mann-Whitney U test) (Fig. 1C). In contrast, no differences in THexpression were observed in the brain areas betweenFmr1-KO and WT mice (p > 0.05; Mann-Whitney Utest).
As the STR (mostly dSTR) is a key substrate of the DA outputsand crucially involved in ASD [11], we subsequently focused on thedopaminergic innervations in this region. We noticed a differentpattern of TH-positive axons in Fmr1-KO mice as comparedto WT and BTBR animals (Fig. 2A). Quantitative assessments of fractaldimensions [29] unraveledsignificant “group” effects in Db (F2,20=6.887,p = 0.005) and lacunarity (F2,20=14.4,p < 0.001). The Fmr1-KO group had higher Db(t14=-2.304, p = 0.037; increased “complexity”)and lower lacunarity (t14 = 5.126,p < 0.001; decreased “texture”), while BTBR micedisplayed no such differences from the WT cohort(p > 0.05) (Fig. 2B).
Beside the dopaminergic afferents, the STR receive glutamatergicand GABAergic inputs, and their interactions are essential for thefunctionality of the basal ganglia. The main excitatory projectionscome from the cortex and thalamus, which end with terminal boutonsthat are immunoreactive to VGLUT1 and VGLUT2, respectively[41, 42]. The two populations of nerve terminalsare comparable in the amount and in a similar spatial relation withdopaminergic axons [43]. Theinhibitory synapses largely arise from different types ofinterneurons in the STR and medium spiny neurons in the “direct”(striatonigral) and “indirect” (striatopallidal) pathways[44]. Staining GABAergic neuronswith an anti-GAD67 antibody showed no difference in the intensityof GAD67 labelling among the three groups (WT: 1.00 ± 0.15, BTBR: 0.97 ± 0.09, Fmr1-KO: 1.31 ± 0.20; p > 0.05). Likewise, usingVGLUT1 as a marker for glutamatergic synapses, we did not detectany significant variance in the overall expression of VGLUT1 (WT:1.00 ± 0.11, BTBR: 1.15 ± 0.12, Fmr1-KO: 1.35 ± 0.24; p > 0.05). However, in theanalyses of co-labelled VGLUT1 and TH signals, we uncovered anincreased number of VGLUT1-possitive boutons co-localized with theTH-positive axons/synapses in the BTBR (t16=-3.094,p = 0.007) and Fmr1-KO (t16=-4.309,p = 0.001) STR, compared with the WT brain(Fig. 2C & 2D). The size of these boutons did not differ(p > 0.05).
Next, we performed Western blotting from striatal homogenates toestimate the quantities of TH, VGLUT1 and GAD67 proteins. Inagreement with the immunohistochemical findings(Fig. 1), the total amount ofTH in the STR of BTBR mice was substantially lower than that in theWT brains (U = 3, Z=-2.406, p = 0.016), while the synapticproteins, VGLUT1 and GAD67, were unaltered (Fig. 3A & 3B). Itis worth noting that the level of striatal TH in Fmr1-KOanimals appeared higher than that in the WT ones, although thedifference did not reach statistical significance. Furthermore, wemeasured DAT, an essential presynaptic protein that modulates DAhomeostasis by the reuptake of DA. De novo mutation of DATgene is a risk factor for ASD [8].Interestingly, the DAT levels dropped in both BTBR (U = 1,Z=-2.727, p = 0.006) and Fmr1-KO (U = 0,Z=-2.887, p = 0.004) mice, as compared to the WTcontrols.
Taken together, BTBR mice exhibited a global reduction of THexpression in the cell body and axon projections of dopaminergicneurons in multiple nuclei, suggesting severe detriments along theDA pathways. Fmr1-KO animals did not have such alterationsyet showed abnormal morphology of TH-positive axons in the STR.Both strains evidenced more VGLUT1 in close proximity to the THsignals, indicating an altered regulation of the excitatory inputsby DA. Lastly, the decreased amount of striatal DAT impliesdeficient DA reuptakes in the two models.
Effects of intranasal DA on striatal protein expressionin BTBR and Fmr1-KO mice
Knowing the DA system was dysregulated in the ASD models(Fig. 1–3), we hypothesized that application of DAmight rectify their phenotypes. Because DA cannot pass theblood-brain-barrier due to its polar properties, we administratedDA via the nasal passage [45].BTBR and Fmr1-KO mice were randomly assigned to vehicleand DA treatments, separately. They were sacrificed 15 minafter intranasal administration of either reagent. We quantifiedthe proteins that were altered in their STR with immunoblotting.Compared to the vehicle groups, DA application increased theexpression of TH in the BTBR STR (t13=-2.299,p = 0.039) (Fig. 4A), but decreased it in the Fmr1-KOSTR (t13 = 2.42, p = 0.031)(Fig. 4B). Given the basallevel of TH was lower in BTBR yet was slightly higher inFmr1-KO mice than that in the WT controls(Fig. 3), these resultsimplicate that intranasal DA may help normalize their distinctphenotypes in the striatal circuit. Although DAT was reduced inboth of the ASD strains (Fig. 3), no significant changes were found after DAadministration (p > 0.05, Fig. 4). The susceptibility of TH protein tointranasal application of DA rationalizes the utility of DA forbehavioral rescues.
Intranasal delivery of DA alleviates the deficits innon-selective attention, object-based attention and sociability ofBTBR mice
We performed behavioral assays following intranasal delivery ofvehicle or DA to BTBR mice. In the open field test, we quantifiedthe parameters in three intervals by taking into accountconfounding factors (anxiety/habituation) that could influencerodent locomotor activity [46].Analyses of distance travelled with a repeated two-way ANOVArevealed a significant effect of “interval” (F2,12=27.657, p < 0.001), but not of “treatment” or“treatment x interval” (p > 0.05;Fig. 5A). Since the“interval” effect was present, one-way ANOVAs with thewithin-subject factor “treatment” were applied separately for theperiods of 0–5, 5–10, 10–15 min. No “treatment” differences inthe travelling distance were detected at any time intervals(p > 0.05). Similarly, a significant “interval” effect(F2, 12=22.405, p < 0.001), but not“treatment” and “treatment x interval” effects(p > 0.05), was found in the analyses of thigmotaxis.Subsequent analyses for each time bin did not reveal any“treatment” difference either (p > 0.05). As to otherassessments on grooming behavior, center entries and time spent inthe center, no effects of “treatment”, “interval” or theirinteraction were observed (p > 0.05). By contrast,while calculating the average duration of rearing, an indicator fornon-selective attention [35], wefound a significant effect of “treatment x interval” (F2,12=8.736, p = 0.005), but not of “treatment” or“interval” (p > 0.05). Essentially, the animals treatedwith DA spent more time on rearing than those receiving the vehiclein the first 5 min (F1, 6=8.102,p = 0.029; Fig. 5A)but not in other intervals (p > 0.05). As BTBR micehave a non-selective attention deficit [16], we suggest that intranasal administrationof DA improves their non-selective attentional processing withoutaffecting general locomotion or exploratory behaviors.
In the object-based attention test, there was no difference inthe total time of object exploration between the DA andvehicle-treated groups in either the learning or the test session(p > 0.05; Fig. 5B & Table 1). Yet, analyses of the test trial unfolded asignificant effect of “treatment x object” (F1, 7=7.127,p = 0.032), but not of “treatment” or “object”(p > 0.05). Paired t-tests were then used tocompare the exploration time for the old versus the novel objectwithin each treatment. The vehicle-treated BTBR mice explored bothobjects indiscriminately (p > 0.05), consistent withour previous report on their attention/memory deficiency[16]. In contrast, DA-treatedanimals preferred the novel to the old object(t7=-2.847, p = 0.025), giving a highercognitive index (F1,7=11.103, p = 0.013;Fig. 5B). The results implythat DA enhances object-based attention and/or short-term memory ofthe BTBR model.
In the three-chamber social test, the total exploration time wascomparable between the treatments (p > 0.05,Table 1). In the sociabilitytrial, the DA-treated animals explored the stranger noticeably morethan the empty cup (t7 = 3.845, p = 0.006;paired t-test; Fig. 5C), while the vehicle-treated mice did not(p > 0.05). Accordingly, the DA treatment rendered amore positive sociability index than the vehicle treatment(t7 = 6.902, p < 0.001; one-samplet-test). Considering the characteristics of BTBR mice intheir reduced sociability [15,16], this result indicates abeneficial action of intranasal DA on their social impairments. Asthe BTBR strain has intact social novelty [15, 16], wedid not continue into the social novelty trial to avoid excessiveadministration of DA in the same subjects within a short time.
The elevated plus maze test showed no differences in the totaldistance travelled, entries to and time spent in the center, openand closed arms, and counts of head-dips between the DA and vehicletreatments (p > 0.05; Table 2). This suggests that DA does not amend thehigh non-social anxiety associated with the BTBR animals[16].
Intranasal application of DA to Fmr1-KO mice rectifies their defects inobject-based attention and social novelty preference
We executed the same behavioral testing in Fmr1-KO miceafter vehicle or DA treatments. In the open field test, asignificant effect of “interval” (F2,28=62.865,p < 0.001; mixed two-way ANOVAs), but not of “group” or“group x interval” (p > 0.05), was detected in theanalyses of distance travelled (Fig. 6A). Subsequent one-way ANOVAs showed no groupdifference in the travelling distance at any given time interval(p > 0.05). As for thigmotaxis behavior, there was asignificant effect of “interval” (F2,28=100.306,p < 0.001), but not of “group” or “group x interval”.No group differences were found in the three intervals(p > 0.05). As for the time spent in the center, therewas a significant “interval” effect (F2,28=6.895,p = 0.004), but not of “group” and “group x interval”effects (p > 0.05). No group differences were found atany intervals (p > 0.05). Assessments of rearing,grooming, and center entries indicated no significant effects of“group”, “interval” or their interaction(p > 0.05).
In the object-based attention test, there were no groupdifferences in the total time of object exploration throughout thelearning and test sessions (p > 0.05;Table 1). In the test trial,there was a significant effect of “object” (F1,8=22.516,p = 0.001), but not of “group” or “group x object”(p > 0.05). The DA group explored the novel object morethan the old one (t4=-5.423, p = 0.006),whereas the vehicle group did not (p > 0.05;Fig. 6B). Both cohorts hadpositive object-based attention scores (t4 = 7.378,p = 0.002 for DA; t4 = 3.711,p = 0.021 for vehicle). In light of previous findings onobject-recognition impairment mediated by aberrant DA release inFmr1-KO mice [47], ourresults indicate intranasal application of DA is an effectiveavenue for ameliorating the cognitive deficits in the FXSmodel.
In the three-chamber social test, no group differences werenoticed in general explorative behaviors in any of the sessions(p > 0.05; Table 1). In the sociability trial, there was asignificant effect of “object” (F1,13=73.735,p < 0.001), but not of “group” or their interaction(p > 0.05). Both vehicle and DA groups explored thestranger mouse more than the empty cup (t6 = 5.85,p = 0.001; t7 = 6.493, p < 0.001,respectively) with equally positive sociability indexes(t6 = 7.571, p < 0.001;t7 = 12.666, p < 0.001, respectively;Fig. 6C). In the socialnovelty trial, significant effects of “object”(F1,13=9.375, p = 0.009) and “group x object”(F1,13=11.313, p = 0.005), but not of “group”(p > 0.05), were found. The DA-treated animals exploredthe novel stranger more than the familiar one(t7=-3.756, p = 0.007), but the vehicle-treatedgroup did not (p > 0.05). Thereby, the DA treatmentrobustly elevated the social novelty index as compared to thevehicle (F1,13=10.963, p = 0.006;Fig. 6C). Knowing thatFmr1-KO animals have normal social approaching butatypical social novelty preference [40, 48], wesuggest that intranasal DA particularly alleviates the impairedsocial novelty in the autistic-like Fmr1-KO model.
In the elevated plus maze test, there were no group differencesin the behavioral measurements (p > 0.05;Table 2), indicating aminimal effect of DA on the anxiety level of Fmr1-KOmice.