Differential alterations of the DA system in BTBR and Fmr1-KO mice
To investigate whether and how the DA pathways are modified in the ASD models, we labelled dopaminergic neurons with an anti-TH antibody in brain sections taken from age-, sex- and background matched WT, BTBR and Fmr1-KO mice. TH is a rate-limiting enzyme that converts tyrosine to DA precursor L-DOPA. We first examined the origins of the DA system, i.e. the SNc and VTA in the midbrain, and their major projections to the dorsal striatum (dSTR) and NAc via the respective nigrostriatal and mesocorticolimbic pathways . Diagrams in Fig. 1A depict the distribution of the soma and axons of dopaminergic neurons identified by TH labelling in the four nuclei. Higher-magnification images illustrate the specific locations of these neurons within the SNc and VTA (Fig. 1B). By measuring the intensities of anti-TH staining and normalizing them to the average values of the WT group, we found that TH expression in 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 TH expression were observed in the brain areas between Fmr1-KO and WT mice (p>0.05; Mann-Whitney U test).
As the STR (mostly dSTR) is a key substrate of the DA outputs and crucially involved in ASD , we subsequently focused on the dopaminergic innervations in this region. We noticed a different pattern of TH-positive axons in Fmr1-KO mice as compared to WT and BTBR animals (Fig. 2A). Quantitative assessments of fractal dimensions  unraveled significant “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 mice displayed no such differences from the WT cohort (p>0.05) (Fig. 2B).
Beside the dopaminergic afferents, the STR receive glutamatergic and GABAergic inputs, and their interactions are essential for the functionality of the basal ganglia. The main excitatory projections come from the cortex and thalamus, which end with terminal boutons that are immunoreactive to VGLUT1 and VGLUT2, respectively [43, 44]. The two populations of nerve terminals are comparable in the amount and in a similar spatial relation with dopaminergic axons . The inhibitory synapses largely arise from different types of interneurons in the STR and medium spiny neurons in the “direct” (striatonigral) and “indirect” (striatopallidal) pathways . Staining GABAergic neurons with an anti-GAD67 antibody showed no difference in the intensity of 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, using VGLUT1 as a marker for glutamatergic synapses, we did not detect any 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 the analysis of co-labelled VGLUT1 and TH signals, we uncovered an increased number of VGLUT1-possitive boutons co-localized with the TH-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 to estimate the quantities of TH, VGLUT1 and GAD67 proteins. In agreement with the immunohistochemical findings (Fig. 1), the total amount of TH in the STR of BTBR mice was substantially lower than that in the WT brains (U=0, Z=-2.309, p=0.021), while VGLUT1 and GAD67 were unaltered (p>0.05) (Fig. 3A & 3B). No differences were found in the amount of TH, VGLUT1 and GAD67 between Fmr1-KO and WT groups (p>0.05). Furthermore, we measured DAT, an essential presynaptic protein that modulates DA homeostasis by the reuptake of DA. De novo mutation of DAT gene is a risk factor for ASD . Interestingly, the DAT levels dropped in both BTBR (U=0, Z=-2.309, p=0.021) and Fmr1-KO (U=0, Z=-2.309, p=0.021) mice, as compared to the WT controls. These changes in the protein expression were confirmed by normalized quantities of TH, VGLUT1, GAD67 and DAT in the BTBR and Fmr1-KO STR relative to the WT cohorts (Table 1).
Taken together, BTBR mice exhibited a global reduction of TH expression in the cell body and axon projections of dopaminergic neurons in multiple nuclei, suggesting severe detriments along the DA pathways. Fmr1-KO animals did not have such alterations yet showed abnormal morphology of TH-positive axons in the STR. Both strains evidenced more VGLUT1 in close proximity to the TH signals, indicating an altered regulation of the excitatory inputs by DA. Lastly, the decreased amount of striatal DAT implies deficient DA reuptakes in the two models.
Effects of intranasal DA on striatal protein expression in BTBR and Fmr1-KO mice
Knowing the DA system was dysregulated in the ASD models (Fig. 1-3), we hypothesized that application of DA might rectify their phenotypes. Because DA cannot pass the blood-brain-barrier due to its polar properties, we administrated DA via the nasal passage . BTBR and Fmr1-KO mice were randomly assigned to vehicle and DA treatments, separately. They were sacrificed 15 min after intranasal administration of either reagent. We quantified the proteins that were altered in their STR with immunoblotting. Compared to the vehicle groups, DA application increased the expression of TH in the BTBR STR (U=4, Z=2.242, p=0.025) (Fig. 4A & Table 1), but decreased it in the Fmr1-KO STR (U=0, Z=2.739, p=0.006) (Fig. 4B & Table 1). Given the basal level of TH was lower in BTBR than that in WT mice (Fig. 3), this result implicates that intranasal DA may help rectify the deficiency in the BTBR striatal circuit. In the Fmr1-KO model, the decreased amount of TH following DA delivery may relate to a modulation of FMRP-mediated DA signaling (see Discussion). Although DAT was reduced in both of the ASD strains (Fig. 3), no significant changes were found after DA administration (p>0.05, Fig. 4 & Table 1). Nevertheless, the susceptibility of TH protein to intranasal application of DA rationalizes the utility of DA for behavioral rescues.
Intranasal delivery of DA alleviates the deficits in non-selective attention, object-based attention and sociability of BTBR mice
We performed behavioral assays following intranasal delivery of vehicle or DA to BTBR mice. In the open field test, we quantified the parameters in three intervals by taking into account confounding factors (anxiety/habituation) that could influence rodent locomotor activity . Analysis of distance travelled with a repeated two-way ANOVA revealed a significant effect of “interval” (F2, 12=30.519, p<0.001), but not of “treatment” or “treatment x interval” (p>0.05; Fig. 5Ai). Since the “interval” effect was present, one-way ANOVAs with the within-subject factor “treatment” were applied separately for the periods of 0-5, 5-10, 10-15 min. No “treatment” differences in the travelling distance were detected at any time intervals (p>0.05). In the analysis of thigmotaxis, we found a significant “interval” effect (F2, 12=7.186, p=0.004), but not “treatment” or “treatment x interval” effect (p>0.05; Fig. 5Aii). Subsequent examination for each interval uncovered that DA treatment reduced thigmotactic behaviors of BTBR mice in the time bins of 0-5 min (F1, 11=6.359, p=0.028) and 5-10 min (F1, 11=5.693, p=0.036). While thigmotaxis is viewed as an index for sensorimotor function and anxiety [38, 39], the reduction of thigmotactic behaviors in the DA-treated animals cannot be readily explained by less anxiety because DA did not affect other parameters that reflect the anxiety level, including time spent in the center of the open field and performance on the elevated plus maze (see below). An alternative possibility is that DA may help control excessive thigmotaxic behaviors of BTBR mice , by refining their sensory-motor integration. Analysis of self-grooming showed a significant effect of “interval” (F2, 22=8.446, p=0.002), but not of “treatment” or “treatment x interval” (p>0.05; Fig. 5Aiii). In each interval, no “treatment” difference was detected (p>0.05). As to other assessments on time spent in the center and counts of rearing, no effects of “treatment”, “interval” or their interaction were observed (p>0.05; Fig. 5Aiv & v). By contrast, while calculating the average duration of rearing, an indicator for non-selective attention , we found a significant effect of “treatment x interval” (F2, 14=21.463, p<0.001), but not of “treatment” or “interval” (p>0.05; Fig. 5Avi). Essentially, the animals treated with DA spent more time on rearing than those receiving the vehicle in the first 5 min (F1, 10=5.146, p=0.047) but not in other intervals (p>0.05). As BTBR mice show non-selective attention deficits , we suggest that intranasal administration of DA improves their non-selective attentional processing without affecting their general locomotion or exploratory activity.
In the object-based attention test, there was no difference in the total time of object exploration between the DA and vehicle-treated groups in either the learning or the test session (p>0.05; Fig. 5B & Table 2). Yet, analysis of the test trial unfolded a significant effect of “treatment x object” (F1, 11=7.73, p=0.018), but not of “treatment” or “object” (p>0.05). Paired t-tests were then used to compare the exploration time for the old versus the novel object within each treatment. The vehicle-treated BTBR mice explored both objects indiscriminately (p>0.05), consistent with our previous report on their attention/memory deficiency . In contrast, DA-treated animals preferred the novel to the old object (t11=-2.511, p=0.029), giving a higher cognitive index (t11=3.588, p=0.004). The results imply that DA enhances object-based attention and/or short-term memory of the BTBR model.
In the three-chamber social test, the total exploration time was comparable between the treatments (p>0.05; Table 2). Significant effects of “treatment x object” (F1, 10=20.541, p=0.001) and “object” (F1, 10=17.834, p=0.002), but not of “treatment” (p>0.05), were found in the sociability trial. Specifically, the DA-treated animals explored the stranger noticeably more than the empty cup (t10=4.901, p=0.001; paired t-test; Fig. 5C), while the vehicle-treated mice did not (p>0.05). Accordingly, the DA, but not the vehicle, treatment rendered a positive sociability index (t10=8.055, p<0.001; one-sample t-test). Considering the characteristics of BTBR mice in their reduced sociability [15, 16], this result indicates a beneficial action of intranasal DA on their social impairments. As the BTBR strain has intact social novelty [15, 16], we did not continue into the social novelty trial to avoid excessive administration of DA in the same subjects within a short time.
The elevated plus maze test showed no differences in the total distance travelled, entries to and time spent in the center, open and closed arms, and counts of head-dips between the DA and vehicle treatments (p>0.05; Table 3). This suggests that DA does not amend the high non-social anxiety associated with the BTBR animals .
Biological variables, such as sex difference, could contribute to the above observations as both male (n=8) and female (n=4) BTBR mice were included. In the three-chamber social test, for example, conspecific interactions between male subjects and male strangers may have a different innate quality from those among females due to inter-male territoriality and aggression. To test the possibility, we reanalyzed the data by including the males only and found similar results from intranasal DA administration (Table 4), reinforcing the robust effects of DA on the BTBR behaviors.
Intranasal application of DA to Fmr1-KO mice rectifies their defects in object-based attention and social novelty preference
We executed the same behavioral testing in Fmr1-KO mice after vehicle or DA treatments. In the open field test, a significant 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 the analysis of distance travelled (Fig. 6A). Subsequent one-way ANOVAs showed no group difference in the travelling distance at any given time interval (p>0.05). As for thigmotaxis behavior, there was a significant 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, there was a significant “interval” (F2,28=6.895, p=0.004), but not “group” or “group x interval”, effect (p>0.05). No group differences were found at any intervals (p>0.05). Assessments on self-grooming and counts and duration of rearing indicated no significant effects of “group”, “interval” or their interaction (p>0.05).
In the object-based attention test, there were no group differences in the total time of object exploration throughout the learning and test sessions (p>0.05; Table 2). 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 more than the old one (t4=-5.423, p=0.006), whereas the vehicle group did not (p>0.05; Fig. 6B). Both cohorts had positive 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 on object-recognition impairment mediated by aberrant DA release in Fmr1-KO mice , our results indicate intranasal application of DA is an effective avenue for ameliorating the cognitive deficits in the FXS model.
In the three-chamber social test, no group differences were noticed in general explorative behaviors in any of the sessions (p>0.05; Table 2). In the sociability trial, there was a significant 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 the stranger 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 social novelty 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 explored the novel stranger more than the familiar one (t7=-3.756, p=0.007), but the vehicle-treated group did not (p>0.05). Thereby, the DA treatment elevated the social novelty index (t7=-4.45, p=0.003), however the vehicle failed to do so (p>0.05; Fig. 6C). Knowing that Fmr1-KO animals have normal social approaching but atypical social novelty preference [42, 50], we suggest that intranasal DA particularly alleviates the impaired social novelty in the autistic-like Fmr1-KO model.
In the elevated plus maze test, there were no group differences in the behavioral measurements (p>0.05; Table 3), indicating a minimal effect of DA on the anxiety level of Fmr1-KO mice.