Localization of m4 muscarinic acetylcholine receptors on striatal projection neurons using biotinylated m4-toxin

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Molecular and Cellular Pharmacology

First Committee Member

Lincoln T. Potter, Committee Chair


The predominant muscarinic receptor expressed in the striatum is the m4 subtype. Its concentration in the striatum is higher than in any other tissue. Studies to elucidate the role played by m4 receptors in striatal functions have been hampered by a lack of selective ligands capable of distinguishing the five muscarinic receptor subtypes. Recently m4-toxin, a component of the venom of Dendroaspis angusticeps, was isolated and characterized in our laboratory. This toxin has a 102-fold higher affinity for m4 receptors than m1 receptors, making it the most selective ligand available for functional studies of m4 receptors. m4-Toxin was biotinylated with N-hydroxysulfosuccinimide-biotin for detection by avidin compounds. Biotinylated m4-toxin was shown to bind specifically to m4 receptors from CHO cells in radioligand binding studies (IC50 = 2.2 x 10 -3 mug/mul). It demonstrated a slight loss in affinity for m4 receptors compared to native toxin (IC50 = 6.7 x 10 -4 mug/mul). When used to label m4 receptors in striatal tissue, biotinylated m4-toxin stained medium-sized neurons. The morphology displayed by the stained neurons was consistent with that of the GABAergic striatal projection neurons. These neurons travel in one of two pathways, the direct (striatonigral) and the indirect (striatopallidal). Neurons belonging to the two pathways were distinguished from one another after concurrent retrograde transport of FluoSpheres injected into the substantia nigra pars reticulata and globus pallidus, respectively. Only 4.0% of retrograde labeled striatal neurons incorporated both labels thereby validating this unique approach. Biotinylated m4-toxin was then used in conjunction with retrograde tracing to double-label m4-containing projection neurons. The results demonstrated a preferential distribution of m4 receptors on striatal projection neurons in the direct pathway (86%) as compared with the indirect pathway (14%). These findings provide strong evidence for a uniquely intense regulation of direct striatal projection neurons by m4 muscarinic acetylcholine receptors.


Biology, Neuroscience; Health Sciences, Pharmacology

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