Fig. 3: The expression of compulsive adjunctive behaviour is associated with a decrease in Arc mRNA levels across the Locus Coeruleus.

A Schematic representation of the unilateral infusion sites (pink dots) of a CAV2-GFP retrograde virus into the medial NacS at four different anteroposterior levels (relative to Bregma, coordinates shown next to each brain atlas template extracted from the Paxinos & Watson’s rat brain atlas [94]) encompassing the target territory in which infusion of atomoxetine was shown to recapitulate the effect systemic administration of this noradrenaline reuptake inhibitor on impulse control (blue dot). B While labelling of noradrenergic neurons in the A2 nucleus was never observed, infusions in the target territory or adjacent to it caudally, but not in other areas of the NacS resulted in GFP immunofluorescence positive cells across the LC. C Schematic representation of the distribution of the coronal sections of the LC ranging from −9.60 to −9.96 (relative to Bregma) that have been processed with RNAscope assays systematically to quantify the number of copies of target mRNAs across the entire LC, as informed by the outcome of the tracing experiment (A, B). D Representative superimage of a 12μm-thick coronal section, taken at ×40 magnification showing the LC, where cell bodies of tyrosine hydroxylase expressing cells are located, delineated in white and a yellow outline of the edge of the fourth ventricle as anatomical reference, processed with RNAscope targeting the tyrosine hydroxylase, zif268 and Arc mRNAs. Representative 63x images of the LC from HD (E) and LD (F) rats that had, or not, the opportunity to express their adjunctive polydipsic behaviour during the challenge session that preceded the harvesting of the brains, with the same green, red and magenta, fluorescent probes as in (D) targeting TH, zif268 and Arc mRNA, respectively. The white and orange arrows point the presence of Arc and zif268 mRNA molecules, respectively. G The individual tendency to eventually express compulsive adjunctive polydipsic drinking behaviour was associated with a lower level of Arc mRNA expression in the LC in that HD rats showed fewer ARC mRNA copies in this monoaminergic nucleus than LD rats. Preventing these HD rats from expressing their compulsive adjunctive polydipsic drinking behaviour by removing their bottle resulted in an increase of Arc mRNA copies in the LC, an effect not observed in LD rats. H At the population level, the number of Arc mRNA copies in the LC was found to be correlated with the level of polydipsic drinking during the challenge session [Spearman correlation, r = −0.692, p = 0.038]. (*p < 0.05).