The work undertaken in this project falls under 5 sub-projects:
1. Midbrain method: I developed a method for analysing the midbrain region from preclinical PET scans. I re-analysed a control cohort of animals using a template to place a midbrain ROI, based on the mouse brain atlas. I then applied this method to the ketamine model, and showed that the ketamine model animals show elevated pre-synaptic dopamine function in both the striatum (previously demonstrated) but also the midbrain. This work was presented at the international conferences of Schizophrenia International Research Society (SIRS) and the European College of Neuropsychopharmacology (ECNP), and these findings were published in the Journal of Molecular Imaging and Biology in 2023.
2. EDiPS in mice: I conducted two pilot experiments to assess the feasibility of using the EDiPS construct (originally used in rats) in mice. I delivered the AAV-packaged construct (or a control construct) to mice. I assessed the locomotor response to amphetamine – a psychomimetic, known to release dopamine from the synapse. These results were not conclusive. However, immunohistochemistry of brain slices from these animals showed that the construct-derived proteins were indeed present, indicating the feasibility of this project. This data will be used as pilot data for future funding applications to continue this project.
3. Long-term ketamine: As part of the ketamine model, ketamine (or saline) is delivered over 5 days, and then behavioural testing or PET scanning is conducted on day 7. However, it is unclear how long this effect of ketamine treatment is maintained. I conducted the final PET scans to complete this project. Although the ketamine treatment appeared to result in increased dopamine synthesis capacity beyond the 7-day timepoint, this was not statistically increased relative to the saline-treated animals. Turther work may be conducted to clarify whether the effect in saline-treated control animals is a true effect, or an artefact of a technical issue.
4. Ketamine immunohistochemistry: I generated animals for the ketamine model (treated for 5 days with either ketamine or saline), and collected brain tissue from these animals for immunohistochemistry. I then undertook staining to examine the expression levels of two excitatatory-related proteins – VGLUT1 and VGLUT2 – in the midbrain. I found no significant differences in the expression of VGLUT1 or VGLUT2 proteins in the midbrain. Although this does not preclude changes in functional activity of excitatory transmission to this region, it suggests that glutamatergic projections from the cortex are not a key factor in this model. Future work is planned to identify potential changes in inhibitory function in this region.
5. Voltammetry: During my secondment period (3 months), I undertook ex vivo voltammetry to unpick the actions of the TAAR1 agonist ulotaront on dopamine function in the striatum. In this technique, I was able to look at dopamine release from wild-type (untreated) mouse brain slices. I firstly applied ulotaront alone, and found that it decreased normal levels of dopamine release in a dose-dependent manner. This effect was still present even when acetylcholine signalling - which modulates dopamine release – was blocked. I also used a TAAR1 antagonist to confirm that the effect of ulotaront were indeed mediated by the TAAR1, rather than via its actions at, for instance, a serotonergic receptor. This data is being included in a manuscript under preparation.
