Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.

Alzheimer's disease (AD) is characterized by depositions of the amyloid-β (Aβ) peptide in the brain. The disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. It is therefore imperative to develop methods that permit early detection and monitoring of disease progression. In addition, the multifactorial pathogenesis of AD has identified several potential avenues for AD intervention. Thus, evaluation of therapeutic candidates over lengthy trial periods also demands a practical, noninvasive method for measuring Aβ in the brain. Magnetic resonance imaging (MRI) is the obvious choice for such measurements, but contrast enhancement for Aβ has only been achieved using Gd(III)-based agents. There is great interest in gadolinium-free methods to image the brain. In this study, we provide the first demonstration that a nitroxide-based small-molecule produces MRI contrast in brain specimens with elevated levels of Aβ. The molecule is comprised of a  fluorene (a molecule with high affinity for Aβ) and a nitroxide spin label (a paramagnetic MRI contrast species). Labeling of brain specimens with the spin-labeled fluorene produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of spin-labeled fluorene into live mice resulted in good brain penetration, with the compound able to generate contrast 24-h post injection. These results provide a proof of concept method that can be used for early, noninvasive, gadolinium-free detection of amyloid plaques by MRI.