Dopaminergic dysfunction in the 3xTg-AD mice model of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by amyloid (Aβ) protein aggregation and neurofibrillary tangles accumulation, accompanied by neuroinflammation. With all the therapeutic attempts targeting these biomarkers having been unsuccessful, the understanding of early mechanisms involved in the pathology is of paramount importance. Dopaminergic system involvement in AD has been suggested, particularly through the appearance of dopaminergic dysfunction-related neuropsychiatric symptoms and an overall worsening of cognitive and behavioral symptoms. In this study, we reported an early dopaminergic dysfunction in a mouse model presenting both amyloid and Tau pathology. 3xTg-AD mice showed an increase of postsynaptic D2/3R receptors density in the striatum and D2/3-autoreceptors in SN/VTA cell bodies. Functionally, a reduction of anxiety-like behavior, an increase in locomotor activity and D2R hyper-sensitivity to quinpirole stimulation have been observed. In addition, microglial cells in the striatum showed an early inflammatory response, suggesting its participation in dopaminergic alterations. These events are observed at an age when tau accumulation and Aβ deposits in the hippocampus are low. Thus, our results suggest that early dopaminergic dysfunction could have consequences in behavior and cognitive function, and may shed light on future therapeutic pathways of AD.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular deposits formed from beta-amyloid (Aβ) protein and by intracellular neurofibrillary tangles of the Tau protein. These neurochemical hallmarks are accompanied by an increase in neuroinflammation which results in reactivity of astrocytes and microglial cells. However, all the therapies targeting these different actors have not allowed the emergence of effective treatments 1 . As a consequence, a better understanding in the mechanisms involved in the early stages of the pathology seems fundamental.
Beyond these AD biomarkers, a dysfunction of the dopaminergic system has been suggested. In human, an accumulation of amyloid plaques is present in the midbrain (cell bodies region of dopamine neurons) as in the ventral and dorsal striatum, major projecting regions of dopamine neurons [2][3][4] . Study of dopaminergic transmission through the evaluation of dopamine D 2/3 receptor (D 2/3 R) density is inconclusive with studies that have shown an increase, a decrease, or the absence of effects of AD on the dopaminergic system [5][6][7][8][9][10][11] . From a clinical point of view, it has been shown that AD patients present dopaminergic dysfunction-related symptoms such as Parkinson-like dysfunctions, including decreased ambulatory activity and apathy, whose frequency, severity and prevalence (from 12 to 92%) depend on the severity of the pathology 2,12-21 . It is therefore possible that dopaminergic deficits participate in the overall worsening of the clinical condition. One possible mechanism for such an effect could be the relationship of the dopaminergic system with neuroinflammation. Indeed, beyond AD, it has been shown that dopamine receptor activation could have pro-or anti-inflammatory consequences [22][23][24] . In human, however, it remains difficult to determine the onset of dopaminergic neurotransmission dysfunction. The advantage of animal studies is that neurochemical or behavioral impacts could be measured precisely even before the appearance of traditional AD markers, in order to assess the potential of this phenomenon to be used as a biomarker for early diagnosis or even as a therapeutic target. In this idea, cell death in the midbrain of neurons expressing the tyrosine hydroxylase (TH) a rate-limiting enzyme in dopamine biosynthesis, has been shown Results Increases in postsynaptic D 2/3 R and microglial reactivity in the striatum of heterozygous 3xTg-AD mice. The quantification of D 2/3 R density in the striatum of 12-month-old 3xTg-AD mice compared to age-matched control animals revealed a global upregulation of the receptor expression which did not differ between the CPu (DLS, DMS, VLS) and the NAcc (Acc, Acs) ( Fig. 1a-c, genotype: F 1,40 = 11.65, p < 0.01; striatal subdivision: F 4,40 = 49.95, p < 0.001; genotype × striatal subdivision: F 4,40 = 0.74, p > 0.05). In contrast, the quantification of the area occupied by TH and DAT (dopamine transporter, involved in presynaptic dopamine reuptake) immunostaining showed no difference in the CPu (including DLS, DMS and VLS) and the VST (Acc and Acs) between control and 3xTg-AD mice (Fig. 1d, The evaluation of the inflammatory reaction with IBA1 and GFAP as markers of microglial and astrocytic reactivity, respectively, showed increases in the IBA1 immunoreactivity and no changes in GFAP immunoreactivity in the striatum (Fig. 2a, The complexity and functional state of microglia in the striatum was assessed on a cell-by-cell basis using Sholl analysis. For that, isolated IBA1 + cells were selected (Fig. 2c) and images were converted into binary images  www.nature.com/scientificreports/ to remove the background (Fig. 2d, left panel). The number ramifications, number of processes and their length, calculated using concentric shell from the soma (Fig. 2d, right panel), reflect the cell complexity. As shown in Fig. 2e, the branching profile of microglia in 12-month-old 3xTg-AD is shifted to the left, demonstrating a reduction in intersection number (genotype: F 1,204 = 29.1, p < 0.001; intersection number: F 33,204 = 22.6, p < 0.001; genotype × intersection number: F 33,204 = 1.00, p > 0.05), which is typical of microglial reactive state.
Increases in presynaptic D 2/3 -autoreceptors in the midbrain of heterozygous 3xTg-AD mice, without modulation of inflammation markers. In the midbrain, we also observed a global upregulation of D 2/3 R density in 12-month-old 3xTg-AD mice compared to WT mice ( Fig. 3a-

Sparse expression of Aβ and Tau markers in heterozygous 3xTg-AD mice. MXO 4 and A4G8
staining were sought in the regions of dopaminergic neurons (striatum, SN/VTA), cortex and hippocampus. The examination revealed the absence of extracellular Aβ deposits (i.e. MXO4 + or A4G8 + staining) in all the studied regions except for the few plaques present in the subiculum at 12 months old ( Fig. 4a-c). The tau pathology was assessed by the use of AT8 immunostaining. No positive staining was observed in the striatum, midbrain and cortex. However, a few positive neurons were observed in the dorsal and ventral hippocampus (Fig. 4d,e). To reveal any inflammatory reaction in the hippocampus, IBA1 and GFAP staining were studied in the hippocampal subdivisions (Fig. 4f,g). No difference was observed between WT and 3xTg-AD mice (Fig. 4h,i).
Behavioral alterations in 12-month-old heterozygous 3xTg-AD mice. Two-way ANOVA indicated that 12-month-old hemizygous 3xTg-AD showed a higher spontaneous locomotor activity than WT mice, throughout the locomotor activity test (Fig. 5a  www.nature.com/scientificreports/ Administration of the D 2 R preferring agonist quinpirole induced a reduction of locomotor activity as compared to the saline injection in both WT (p < 0.001) and 3xTg-AD (p < 0.001) mice (Fig. 5b). However, hemizygous 3xTg-AD mice showed a hyper-response to the inhibitory effect of D 2 R agonist (p < 0.05, Fig. 5c).
Estimation of anxiety levels by the analysis of the exploratory behavior of mice in the different areas of the open-field (the center being the most anxiogenic area) indicated that hemizygous 3xTg-AD mice were hypoanxious with a reduction in the time past at the corners (p < 0.001), an increase in the time past in the intermediate squares of the open-field (p < 0.001) and a tendency to spend more time at the center (p = 0.0514, Fig. 5d).

Dopamine-related locomotor dysfunctions are not present in 4-month-old heterozygous 3xTg-AD mice.
To determine whether dopamine-related locomotor dysfunctions are already present at a younger age, a second cohort of 4-month-old animals performed the open-field test.
Spontaneous locomotor activity of WT and hemizygous 3xTg-AD mice aged of 4 months was recorded in the open-field test. Two-way ANOVA indicated a higher time-dependent locomotor activity in 3xTg-AD mice (genotype: F 3,336 = 129, p < 0.001; time: F 11,336 = 32.15, p < 0.001; genotype × time: F 33,336 = 3.34, p < 0.001). Post hoc test indicated that differences between hemizygous 3xTg-AD mice and WT concerned the entire 60-min time period, with different levels of significance (Fig. 6a), showing a hyperlocomotion at this age.
However, a significant inhibitory effect of quinpirole was observed in both WT (p < 0.001) and 3xTg-AD (p < 0.001) mice (Fig. 6b) and was not dependent of the genotype (p > 0.05, Fig. 6c). Consequently, the hyperresponse to the inhibitory effect of D 2 R agonist is not yet present at 4 months of age.
The time past in the centre, corners and intermediate virtual squares of the open-field did not differ between hemizygous 3xTg-AD and control mice, indicating similar anxiety levels (p > 0.05, Fig. 6d).

Discussion
Our study confirms and extends previous reports that show dysfunctions in dopaminergic system in a mouse model presenting both amyloid and Tau pathology. Our data demonstrated that 3xTg-AD mice show dopaminergic system alterations that occurs from the early stages of the pathology, marked by a low presence of intraneuronal Tau in the hippocampus and the presence of a limited number of Aβ deposits restricted to the subiculum. Regarding the dopaminergic system, we reported an increase in the density of postsynaptic D 2/3 R receptors in the striatum and in D 2/3 -autoreceptors of SN/VTA cell bodies. Functionally, there is an increase in locomotor activity at baseline and a D 2 R hyper-sensitivity to quinpirole stimulation. In the striatum, 3xTg-AD mice also show an increase in microglial cell density, which is not observed in either the hippocampus or the midbrain. This effect is accompanied at the cellular level by a reduction in complexity of the microglial cell morphology. These changes of microglial morphology and density reflect an early inflammatory response in the striatum, which could participate in altering dopaminergic functions. Finally, a reduction of anxiety-like behavior was also observed in the open-field test.
The involvement of dopaminergic system in Alzheimer's disease has been suggested in humans as well as in various animal models 2,18,25,40,41 . However, the precocious nature of these alterations remains poorly understood. www.nature.com/scientificreports/ In this study, the use of heterozygous mice makes it possible to obtain a slower evolution of the pathology 37-39 and therefore to better observe the early phenomena, related to the age of the animals. At 12 months, a stage when mice show low amyloid and Tau accumulation in the hippocampus, we observed a general increase in D 2/3 R levels in the striatum and in the cell body regions of dopamine neurons. In humans, there is no consensus on such an alteration in D 2/3 R density [5][6][7][8][9][10][11] . However, these studies were carried out in patients whose pathological stage is, in comparison with our model, more advanced. As a decrease in dopamine synthesis in AD human brain has been described 42,43 the increase in D 2/3 R density could be due to a compensatory phenomenon in response to this decrease in dopamine availability. Interestingly, such a compensation phenomenon has been observed in Parkinson's disease in unmedicated patients 44 , and in an experimental model of reduction in dopamine system activity 45,46 .
In the striatum, we observed an increase in D 2/3 R which could be linked with the hyper-locomotion of mice at baseline. In D 1 R and D 2 R knockout mice 47 , a decrease in locomotor activity was observed, which also corroborates the locomotor activity changes with the modulation of dopamine receptors expression. In addition, an increase in inflammation was observed through microglial reactivity. Indeed, microglial cells density is increased while their complexity is decreased, which reflects proliferation and activation of these cells. Importantly, microglial reactivity in the striatum appears to occur very early since within the hippocampus, one of the first region to develop amyloid plaques, astrocytes and microglial cells are not yet altered at this early stage. This further suggests that the glial cell alterations in brain regions associated to the dopaminergic system may play a role in the DA dysfunctions. Reciprocal links between dopaminergic function and the presence of inflammation have been suggested in some other studies. For example, stimulation of D 1 and D 2 dopamine receptors induces antiinflammatory effects while stimulation of D 3 and D 5 dopamine receptors promotes inflammation 22,23 . In turn, inflammation-mediated effects appear to be amplified by the stimulation of D 3 R 24 . Not only expressed by neurons, dopamine receptors are also present on microglial cells and astrocytes, in animals as well as in humans [48][49][50][51] , which implies direct control of glial cell activity by dopamine. Thus, it is possible that the increase in D 2/3 R also affects glial receptors, which could participate in the modulation of inflammation. Overall, microglial activation could participate in a disorganization of the dopaminergic system.
At the level of the dopamine neurons cell bodies, D 2/3 -autoreceptors play a role in inhibiting dopamine neurons and therefore locomotion 52 . In response to an injection of the preferential D 2 R agonist, locomotor activity decreases, reflecting the activation of D 2/3 -autoreceptors. Twelve-month-old 3xTg-AD mice show increased inhibition of the locomotor activity, which may be correlated with neurochemical increases in D 2/3 -autoreceptors. This increase in D 2/3 -autoreceptors density could result in a stronger tonic inhibition of dopamine neurons. This idea is consistent with the decrease in the response of dopaminergic neurons to electrical and chemical stimulation 25,41,53 . However, the production and reuptake of dopamine by neurons does not still appear to be impacted in our 3xTg-AD mice, as shown by a stable level of TH and DAT, two key enzymes in dopamine level regulation. Interestingly, behavioral hyper-response to quinpirole is also observed early in a rat model of the pathology 41 . In another AD model, an increase in locomotion was also shown in the presence of constant www.nature.com/scientificreports/ dopamine levels compared to controls 28 . These observations validate the direct involvement of D 2/3 -autoreceptors in locomotion control, without necessarily a modulation of dopamine levels. Beyond nigrostriatal and mesolimbic pathways, another pathway for activating striatal dopamine release should also be considered. In fact, neuronal activity in the hippocampus induces striatal dopamine release 54 . Demonstrations of hippocampal role in controlling locomotor activity have also been reported 55,56 . However, at the age of 12 months, our mouse model shows levels of Tau and amyloid in the hippocampus, even if relatively low, which could in themselves have pathological effects that cannot be totally excluded. It is therefore possible that amyloid and Tau accumulation in the hippocampus modify the transmission between the hippocampus and the striatum, resulting in a decrease of dopamine release which would generate an up-regulation of postsynaptic D 2/3 R. This hypothesis is consistent with a previous study demonstrating that homozygous 3xTg-AD mice display a decrease of cortical dopamine release at 10-month-old that was not present at 3-month-old. This decrease is accompanied by a loss of TH + neurons, not yet visible in our heterozygous mice 57 . It could be consequently interesting to repeat this study at an advanced stage, when mice present a more important amyloid and tau pathology to observe if dopaminergic deficits worsen with the progression of the pathology.
Finally, medium-spiny neurons of the striatum are also involved in the regulation of anxiety 58 . In the literature, 3xTg-AD females do not appear to show changes in anxiety at the light/dark and elevated plus maze tests 59,60 . However, increases in the time past at the center of the open-field was previously observed in females 3xTg-AD mice 61 . In our study, 12-month-old mice also showed a decrease in their anxiety level as shown by their exploratory behavior in the open field. Thus, it is therefore possible that the increase in D 2/3 R contributes to this reduction in anxiety.
In conclusion, basal hyperlocomotion is consistent with an increase in postsynaptic D 2 R and the inhibitory hyper-response to quinpirole consistent with an increase in D 2/3 -autoreceptors. Alterations in D 2/3 R signaling may represent early neuroadaptative changes at least in animal models of AD. These changes may contribute to increases in inflammation processes, to the development of AD pathology and, therefore, may represent an alternative strategy of earlier intervention. This idea is still speculative but cognitive improvements observed by stimulating treatment of dopaminergic system supports this hypothesis. Therefore, early deregulation of dopaminergic system could have consequences on the course of the pathology in terms of dysfunction of locomotion, anxiety, cognitive abilities, and goal-directed behaviors. All these phenomena are also observed in humans and suggest an early dysfunction of the dopaminergic system which may represent an interesting therapeutic target for AD patients. Future studies should address the long-term effects of therapy modifying D 2/3 R activity.

Material and methods
Animals. Female wild-type (C57B1/6 J-Sv129) and hemizygous 3xTg-AD (APPswe, Tau P301L and PS1 M146V +/− ) mice aged 4 and 12 months were used. Animals were kept on 12 h light/dark cycle and provided with ad libitum access of food and water. All the experiments were approved by Ethics Committee for Animal Experimentation of the Canton of Geneva, Switzerland. All methods were performed in accordance with relevant guidelines and regulations and data are reported in accordance with Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
Microglia morphology analysis. Morphometric analysis of IBA1 + cells from the striatum was performed using z-stack images taken with a confocal microscope (Zeiss, 3 shots per section, 3 sections per animal). The Sholl analysis was performed using the ImageJ software plugging. The soma of microglial cells was manually www.nature.com/scientificreports/ drawn, and concentric circles were automatically applied (from the soma center to the end of the microglial processes with a 2 μm distance between circles). The number of intersections along the Sholl radii and the area under the curves were analyzed as previously described 62 .
In situ autoradiography. Twelve-month-old WT and 3xTg-AD mice (n = 5/genotype) were transcardially perfused with 0.9% saline under 3% isoflurane anesthesia, brains were removed and frozen in pre-cooled isopenthane. Serial coronal rostro caudal sections (30 µm) covering the striatum and the midbrain/hippocampus were cut on cryostat, mounted on gelatin-coated slides, and stored in − 20 °C. Slides were immersed in 1 × PBS (20 min), in the radioactive buffer (90 min), rinsed twice in 4 °C Tris-MgCl 2 buffer (5 min) and briefly washed in cold water. The radioactive buffer consists of Tris-MgCl 2 buffer (50 mM Tris HCl, 50 mM MgCl 2 ) containing [ 125 I]Epidepride alone or in presence of 10 µM of unlabeled ligands to determine the non-specific binding in the adjacent sections. Slides were air-dried then exposed with gamma-sensitive phosphor imaging plates (Fuji BAP-IS MS2325) for 24 h. To precisely define the anatomical landmarks, brain slices were treated with acetylcholinesterase staining. Autoradiograms were revealed using Fujifilm BAS-1800II phosphoimager in presence of homemade 125 I calibration curves and analyzed using Aida Software V4.06 (Raytest Isotopenmessgerate GmbH), where the ROI were drawn manually. Based on an acetylcholinesterase staining to obtain good anatomical landmarks, the following ROIs were used: CPu (dorsolateral, ventrolateral, dorsomedian), NAcc (Shell and Core), SNc, SNr, VTA. For each ROI, the specific binding ratio (SBR) was calculated as follows: (radioactivity in ROI/radioactivity in ROI with 10 µM of unlabeled radiotracer) −1. For each animal and targets, 6-8 sections were analyzed and values in bilateral ROI were averaged. Statistics. Two-way ANOVAs with the LSD post-hoc test was used to analyse locomotor behaviors (genotype × time), autoradiograms (genotype × ROI), immunofluorescence (genotype × ROI) and area under the curve (genotype × distance from soma). Comparison of anxiety, habituation levels and quinpirole effects were performed using a two-tailed t-test. Data are expressed as mean ± SEM.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.