Development of chemogenetic methodology to investigate cortical mechanisms of cognition

DREADDs (designer receptors exclusively activated by designer drugs) are virally mediated engineered muscarinic receptors that no longer bind their endogenous ligand acetylcholine but instead bind the clozapine metabolite, clozapine-N-oxide (CNO). These receptors are a very useful research tool for deconstructing behaviour due to their promoter-driven cell- specificity and potential spatial resolution, the low invasiveness associated with their use, reversibility and the temporal resolution of minutes to hours. This makes them ideal tools for linking cell-specific changes to behaviours that require longer testing duration such as learning and memory tasks.
Using an array of techniques, this thesis has provided an in-depth analysis of chemogenetic functionality in the hippocampus. Immunohistochemistry and in situ hybridisation studies assessed the spread and specificity of the CaMKIIa promoter driven excitatory (Gq-DREADD) and inhibitory (Gi-DREADD) constructs in the rat dorsal hippocampus. Slice electrophysiology, in vivo local field potential (LFP)-electrocorticography (ECoG) recordings and hippocampal- dependent behavioural tasks were used to assess the functionality of the constructs. Additionally, a liquid chromatography-mass spectrometry study directly tested the ability of CNO and clozapine to enter the brain as recent reports have found evidence that CNO is back- metabolised to clozapine, that then readily enters the brain and is in fact the activator of the DREADDs.
Expression of both the Gi- and Gq-DREADD was almost entirely restricted to the principal layers of the hippocampus, with the exception of transfection of a small population of interneurons in the hilar region of the dentate gyrus. In hippocampal slices expressing Gi- DREADD, both CNO and clozapine decreased excitatory transmission. Paradoxically, both ligands also decreased excitatory transmission in slices expressing an excitatory DREADD. Follow-up experiments ruled out the potential activation of inhibitory transmission or of the mGlu2/3 receptors to be involved in this response.
In accordance with recent reports, the exposure study found evidence for back metabolism of CNO to clozapine, with the clozapine concentration in the brain after CNO injection being comparable to the clozapine concentration used to activate the DREADDs in the slice experiments (0.6 μM and 1 μM, respectively). The results also suggested a higher dose of CNO (10 mg/kg) might be needed for clozapine to enter the brain and activate the DREADDs.
In vivo LFP/ECoG recordings found decreased power in the beta, low gamma and high gamma frequencies in rats expressing a Gi-DREADD in the presence of CNO compared to saline. Somewhat surprising, there were no statistically significant changes in brain oscillations in Gq-DREADD expressing rats in the presence of CNO. This suggests that suppression of pyramidal cell activity disrupts the generation of gamma oscillations in the hippocampus by potentially modulating intraneuronal activity. Finally, object exploration, spatial short-term/working memory and locomotor activity were found to be unchanged regardless of viral construct or treatment. In addition, neither CNO (3 or 10 mg/kg) or clozapine (0.1 or 0.5 mg/kg) were found to affect behaviour in controls.
Collectively, these experiments provide evidence for DREADD functionality in the rat hippocampus. The results also suggest that despite evidence of in vivo functionality in the context of LFP signals, a behavioural phenotype might require a higher viral transfection rate. This emphasises the importance of correlating behavioural changes with neurophysiological measurements, as cell-specific manipulation might not be detected as changes in behaviour.

Keywords: chemogenetics
DREADD
Advisor: Sharp, T
Frisch Herrik, K
Bannerman, D