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A novel solubilising agent (0.l% sodium cholate-lM NaCl) has been developed which will solubilise 10-30% of muscarinic cholinergic receptors from bovine caudate nucleus. Using the muscarinic antagonist quinuclidinyl benzilate (QNB), a single saturable binding component was found with an equilibrium constant of ZOOpM, approximately 10-fold higher than the membrane receptor and 4-fold higher than the ratio k_-l/k_l determined kinetically in the soluble material. This latter difference may indicate that the binding of QNB to the solubilised receptor is not a simple second-order process. Inhibition constants for a variety of muscarinic agonists and antagonists were 10 to 20-fold higher than in the membrane state and non-muscarinic ligands were without effect. The decrease in affinity was shown to be due to the presence of high salt. Evidence was presented that the apparent increase in Hill coefficient for muscarinic agonist binding to soluble material was not due to a differential solubilisation of muscarinic receptors or to a conformational change of high to low affinity agonist sites during the solubilisation. Instead the Hill coefficient of the soluble material decreased as the percentage of total binding sites solubilised increased. The stability of receptor binding at different temperatures was shown to be dependent on the protein: cholate (w/w) ratio. Results from gel filtration, affinity chromotography and immunization studies are also reported. The results of this thesis are discussed in the light of the possible importance of phospholipids for receptor activity.

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Graduation date: 1997

Graduation date: 1998

Graduation date: 1994

Excitatory M5 muscarinic acetylcholine receptors are expressed at the highest level in the brain, in the ventromedial hypothalamus (VMH). M5 knockout (KO) mice emit fewer ultrasonic vocalizations (USVs) during mating, and show 40-50% lesser morphine-induced locomotion as compared to wild-type (WT) mice. Following viral transfection of M5 muscarinic receptors in the ventral tegmentum (VTA), KO mice depict restored mating-induced USVs and enhanced morphine-induced locomotion. The VMH is important for motivational processes, such as, feeding and producing USVs in rats. With a Herpes simplex virus (HSV), the M5 receptor gene was transfected into the VMH of WT mice. M5 transfection into neurons of the VMH increased locomotion in mice injected with 10mg/kg morphine. When a D2-selective dopamine blocker was injected into the same mice, locomotion was drastically reduced. There were no significant differences in amount of USVs produced. VMH may exert its effects on morphine through a DA dependent VTA pathway.

Excitatory M5 muscarinic acetylcholine receptors are expressed at the highest level in the brain, in the ventromedial hypothalamus (VMH). M5 knockout (KO) mice emit fewer ultrasonic vocalizations (USVs) during mating, and show 40-50% lesser morphine-induced locomotion as compared to wild-type (WT) mice. Following viral transfection of M5 muscarinic receptors in the ventral tegmentum (VTA), KO mice depict restored mating-induced USVs and enhanced morphine-induced locomotion. The VMH is important for motivational processes, such as, feeding and producing USVs in rats. With a Herpes simplex virus (HSV), the M5 receptor gene was transfected into the VMH of WT mice. M5 transfection into neurons of the VMH increased locomotion in mice injected with 10mg/kg morphine. When a D2-selective dopamine blocker was injected into the same mice, locomotion was drastically reduced. There were no significant differences in amount of USVs produced. VMH may exert its effects on morphine through a DA dependent VTA pathway.