Altered dopaminergic regulation of the dorsal striatum is able to induce tic-like movements in juvenile rats

Abstract

Motor tics are sudden, repetitive, involuntary movements representing the hallmark behaviors of the neurodevelopmental disease Tourette's syndrome (TS). The primary cause of TS remains unclear. The initial observation that dopaminergic antagonists alleviate tics led to the development of a dopaminergic theory of TS etiology which is supported by post mortem and in vivo studies indicating that non-physiological activation of the striatum could generate tics. The striatum controls movement execution through the balanced activity of dopamine receptor D1 and D2-expressing medium spiny neurons of the direct and indirect pathway, respectively. Different neurotransmitters can activate or repress striatal activity and among them, dopamine plays a major role. In this study we introduced a chronic dopaminergic alteration in juvenile rats, in order to modify the delicate balance between direct and indirect pathway. This manipulation was done in the dorsal striatum, that had been associated with tic-like movements generation in animal models. The results were movements resembling tics, which were categorized and scored according to a newly developed rating scale and were reduced by clonidine and riluzole treatment. Finally, post mortem analyses revealed altered RNA expression of dopaminergic receptors D1 and D2, suggesting an imbalanced dopaminergic regulation of medium spiny neuron activity as being causally related to the observed phenotype.

Fig 1. Experimental design.

The degeneration of nigrostriatal dopaminergic projections on the dorsal striatum was achieved through stereotaxic injection of the toxin 6-hydroxydopamine (6-OHDA) at postnatal day 21 (PND21). After recovery, rats were chronically administered with the DA agonist quinpirole that induced a tic-like motor phenotype that was quantified on PND48 and used for drug testing. The entire experimental procedure was concluded before puberty (~PND60-70).

Fig 2. Time course of tic-like movements score of quinpirole-treated juvenile rats lesioned in the dorsal striatum (DS) and unlesioned rats.

On observation day, tic-like movements score is taken every 30 min after quinpirole administration (0.5 mg/kg) and is calculated as the total number of body parts + average frequency + complexity + impairment scores. A significant difference between tic-like score in quinpirole treated lesioned and sham rats is indicated as *p< 0.05.

Fig 3. Average frequency scores of the body parts involved in tic-like movements.

Average frequency scores of limb, neck, mouth movements and complex tic-like movements during the phase of maximal abnormal motor manifestation (60-120min after quinpirole administration) on dorsal striatum-lesioned rats (A) or sham-lesioned rats (B).

Fig 4. Time course of tic-like movements score in aDS and cDS.

Tic-like movements score is taken every 30 min after quinpirole administration and is calculated as the total of number of body parts + average frequency + complexity + impairment scores. Significant difference is indicated as ****p<0.001.

Fig 5. Phenotype components in DS, aDS and cDS lesioned juvenile rats.

The average frequency scores, the average complexity score and the average impairment score are calculated during the phase of maximal phenotype score (60- 120mins after quinpirole administration) for the different body parts involved in tic-like movements: limb (A), neck (B), mouth (C), complex tic-like movements (D) and impairment score (E). Significant difference is indicated as *p<0.05 **p<0.01 ****p<0.001.

Fig 6. In situ hybridization reveals DrD1 RNA expression in aDS and cDS of 6-OHDA lesioned rats, evaluated at PND25 and PND50.

In all panels, red dots result from a chromogenic reaction indicating the presence of the target RNA, while blue dots represent the nuclear marker; aDS PND25 (top left), cDS PND25 (bottom left), aDS PND50 (top right), cDS PND50 (bottom right). The orange circle approximately indicates the area taken into account for quantitative analysis.

Fig 7. In situ hybridization reveals DrD2 RNA expression in aDS and cDS of 6-OHDA lesioned rats, evaluated at PND25 and PND50.

In all panels, red dots result from a chromogenic reaction indicating the presence of the target RNA, while blue dots represent the nuclear marker; aDS PND25 (top left), cDS PND25 (bottom left), aDS PND50 (top right), cDS PND50 (bottom right). The orange circle approximately indicates the area taken into account for quantitative analysis.

Fig 8. Percentage of DRD1 and DRD2 positive cells in DS.

The number of positive cells for DRD1 RNA and nuclear staining (A) or DRD2 RNA and nuclear staining (B) in the lesioned and in the corresponding area on the control side of PND25 and PND50 rats is reported in percentages of the total number of nuclear staining-positive cells. Significant total difference is indicated as ****p<0.001, **p<0.01, *p<0.05.

Fig 9. Total cell density in the lesioned area at PND25 and PND50.

The total cell density in the area that had been interested by the lesion was calculated as number of nuclear staining-positive cells/ um² of tissue in 18 samples taken from rats sacrificed after the lesion at PND25 or at PND50 after phenotype validation. Significant difference is indicated as **p<0.01, ****p<0.001.

Fig 10. Preliminary analysis of DrD1 and DrD2 RNA positive cells in aDS and cDS of rats sacrificed at PND50.

Cells positive for DrD1 RNA and nuclear staining (A) or DrD2 RNA and nuclear staining (B) were quantified in lesioned versus control side in aDS (n = 2) or cDS (n = 3) of DS lesioned rats and are reported as percentage of positive cells compared to total cells. Significant difference is indicated as **** p<0.001.

Fig 11. Time course of tic-like movements score after clonidine and riluzole administration.

Clonidine (0.05 mg/kg IP) was administered together with quinpirole (A). Riluzole (6mg/kg IP) was administered 60min after quinpirole (indicated by an arrow) (B). Significant reduction of phenotype score compared to the score obtained during quinpirole treatment by the same animals is indicated as *p<0.05, **p<0.01.