Neurosteroid regulation of cholinergic neurotransmission

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Progesterone-based neurosteroids, including progesterone and its metabolites 5a-dihydroprogesterone and 3a,5a-tetrahydroprogesterone (allopregnanolone; ALLO), are produced within the brain and induce rapid non-genomic effects at specific targets within the brain. The influence of these progesterone neurosteroids on higher-order cognitive functions appears to follow an inverted U-shaped curve, for example, as relative low levels of ALLO are beneficial whereas relative high levels of ALLO are detrimental. The mechanisms by which ALLO may impair cognitive functions are not known. We have determined that ALLO rapidly inhibits the nicotinic acetylcholine receptor (nAChR) within the prefrontal cortex, which is important because cholinergic neurotransmission at this receptor in this brain region is critical for normal cognitive functioning. Mechanistic experiments suggest that this inhibition is mediated by a combination of (i) indirect action of ALLO stimulating the membrane progesterone receptor (mPR) complex, which initiates an intracellular signalling pathway that inhibits the nAChR, and (ii) direct allosteric action of ALLO at the nAChR. The specific mechanisms of these indirect and direct actions of ALLO to regulate nAChR function, and the effects of ALLO on nAChR-dependent cognitive functions, will be investigated in the proposed research. We will test the overarching hypothesis that progesterone neurosteroids regulate cholinergic neurotransmission via a combination of indirect and direct interactions with the nAChR. We will complete three primary objectives: (1) To determine the intracellular signalling pathway linking ALLO activation of the mPR complex with ALLO inhibition of the nAChR. We will use whole-cell electrophysiology and proteomic techniques to test the postulate that the mPR G protein-coupled signalling pathway modifies the phosphorylation of the nAChR to regulate its function. (2) To determine whether ALLO is a direct allosteric modulator of the nAChR. We will use radioligand binding assays in brain synaptosomes to test the postulate that ALLO is a positive allosteric modulator of the nAChR. (3) To determine the influence of ALLO on nAChR-dependent cognitive functions. We will test the postulate that ALLO inhibits the role of nAChRs to facilitate prefrontal cortex-dependent attention and cognitive flexibility. The proposed research will contribute to the fundamental understanding of neurosteroid action within the brain. This work is important because the brain is exposed to progesterone neurosteroids throughout an organism's life, but the neurobiological consequences of this exposure are not fully known. Discoveries made within this research program will address this outstanding question and provide a foundation from which to study the neurobiology of cognitive functions during states of varying progesterone neurosteroid levels, such as development, the menstrual/estrous cycle, pregnancy, and exposure to stressful environments.