From gene structure to neuronal circuits : genetic rescue of CB1 receptor function in cortical glutamatergic networks
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Abstract
A main topic of current neurobiological research is to establish causal relationships between genes, molecules and functions. Several studies using knock-out and pharmacological approaches showed the necessary role of the cannabinoid type 1 (CB1) receptor in a plethora of cellular neuronal functions and behaviors. However, caused by technological limitations, the sufficient role of the CB1 receptor in the same processes has not been established yet. To further understand the mechanisms underlying these processes, a novel mouse line for cell type-specific rescue from CB1 receptor deficiency (Stop-CB1) was generated. To this end, a loxP-flanked stop cassette was introduced into the CB1 receptor gene locus in order to repress CB1 receptor expression throughout the entire body, including the brain. By crossing this mouse line with a mouse line ubiquitously expressing Cre recombinase (Ella-Cre), complete and functional reactivation of CB1 receptor expression by excision of the stop cassette was demonstrated by histological, electrophysiological and behavioral analyses. To further address the role of intact CB1 receptor signaling in specific neuronal subpopulations, the Stop-CB1 line was crossed with a mouse line (Nex-Cre) expressing Cre recombinase selectively in cortical glutamatergic neurons and, thus, endogenous levels of CB1 receptor were reactivated cell-type specifically. Rescue of CB1 receptor expression in cortical glutamatergic neurons was sufficient to restore the alterations of global CB1 receptor deletion in food intake and was largely sufficient to restore normal levels of neuroprotection and innate anxiety. In contrast, rescue of the CB1 receptor on cortical glutamatergic neurons modified the time course of depolarization-induced suppression of excitation (DSE) in the amygdala and promoted sustained freezing behavior in auditory fear conditioning. These data revealed that the limited amount of CB1 receptor expressed in cortical glutamatergic neurons plays a key and sufficient role to modulate specific neuronal functions.
The great majority of the studies investigating the role of the CB1 receptor to advance the knowledge for treatment of human pathologies have been carried out in mice. However, the molecular structure of the mouse Cnr1 gene coding for the CB1 receptor had been poorly characterized. In this thesis, the mouse Cnr1 gene was found to be more complex than previously described. It contains 7 exons separated by 6 introns and has two additional retained introns in the coding exon (exon 7). The Cnr1 gene produces several transcript variants in hippocampus, caudate putamen and cerebellum with different 5’ UTR exon assemblies. Splicing of the retained introns in the coding exon generates two novel splice variants with shortened N-termini that have different signaling efficiencies. These findings on the exon-intron structure of the mouse Cnr1 gene add to a better understanding of regulatory processes and allelic variations contributing to pathological phenotypes observed in the rodent model.