During the 15-min free exploration period (Fig. 1A), all five mice spent at least 25% of the time actively engaged in a specific behaviour other than sitting (“Im” or “immobility”) or transitioning between behaviours ( “null”). Unassisted rearing, ranging from 0.3% to 21.0% (CV = 0.79), exhibited the most variation of the behaviours performed by all five animals (Fig. 1B). The behaviour with the least variation was object interaction (CV = 0.12). All subjects spent more time walking near the walls of the arena than in the centre, as expected, and overall behavioural performance was consistent with other recent investigations. In the subsequent PET scan, regional distribution of 18F-fallypride-binding potential among a set of regions of interest (ROIs) was consistent with previous studies, exhibiting strongest uptake in regions such as the striatum where D2/D3 receptors are highly expressed, and whole-brain standardised uptake value (SUV) variation (Fig. 1C,D) was within the typical range.
We next used a correlation matrix to perform unbiased comparisons between the two datasets and observed both expected and unexpected outcomes (Fig. 1E).
First, unassisted rearing (canonical exploratory behaviour performed by mice) demonstrated an inverse correlation with 18F-fallypride SUV throughout the brain, suggesting either a brain-wide internalisation of D2/D3 receptors or an increase in dopamine release throughout most of the brain. We favour the latter interpretation since novel environments like the one employed in this study are known to increase the firing rate of ventral tegmental area (VTA) dopamine neurons. What our study adds to this knowledge is the potential that the degree of VTA activation corresponds to the degree of exploration within a novel environment, even when the degree of absolute novelty remains constant. Of the many ROIs demonstrating a negative correlation between 18F-fallypride SUV and unassisted rearing, the hippocampus and amygdala showed the strongest Pearson’s r. Dopamine projections to these specific regions may, therefore, be the most strongly activated by sensorium-enriching exploratory behaviours.
The association between dopaminergic signalling and the hippocampus helps confirm an already established interaction, but the strong correlation with the amygdala was more surprising. While VTA-amygdalar dopamine transmission is proposed to signal danger and increased dopamine release occurs in response to stress, more unassisted rearing in the safe novel environment employed here would not be expected to indicate more stress. Unassisted rearing is unlikely a proxy for danger in our experiment. More likely, the lack of 18F-fallypride in the amygdala reflects memory support mechanisms consistent with reduced amygdalar SUVs observed in humans during word pair association tasks.
Second, we observed that SUVs within the superior and inferior colliculi did not correlate strongly with any of the scored behaviours, suggesting D2/D3 receptors in the colliculi do not play a major role in these behaviours under the conditions used in this study. The special lack of correlations for 18F-fallypride uptake in the colliculi also points towards a functional separation of dopamine neurons that project to the colliculi compared to other target regions. Both of these ideas are consistent with the seemingly specific role for dopamine signalling in the colliculi in behavioural responses to aversive or fearful stimuli. Our experiment examined well-handled subjects in a non-fearful environment. In this context, D2/D3 receptor expression level and/or dopamine release in the colliculi would not be expected to play any major role.
Third, the strongest correlations between behaviour and 18F-fallypride SUV emerged when examining head grooming (grooming anywhere in the face and head region while standing upright on hind legs). In behavioural literature, head grooming is rarely scored on its own, even when grooming microstructure is being examined as a proxy for internal states like anxiety. However, grooming initiation is known to be governed by dopaminergic signalling, and mice always start grooming at the face. The strong correlations between global dopamine signalling and head grooming would make sense in this context. In addition, head grooming could contribute to exploration as mice sample scents collected on their paws from the environment.
Finally, we assessed the feasibility of merging small animal PET and behavioural analyses using a Pearson’s r power analysis. Without considering multiple comparisons, sample sizes of 10 to 15 animals would be needed to obtain statistical significance for stronger correlations (e.g. hippocampus 18F-fallypride and unassisted rearing). When considering multiple comparisons, the sample size required becomes highly dependent on correlative strength. For weaker correlations in the 0.6 range, multiple comparisons inflate the required sample size up to 25-30 subjects. Stronger correlations (Pearson’s r >0.8) are hardly affected by multiple comparisons, indicating sample sizes in the 10-15 range would still be adequate.