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1.
ACS Chem Neurosci ; 14(18): 3460-3471, 2023 09 20.
Article in English | MEDLINE | ID: mdl-37681686

ABSTRACT

Cholinergic signaling, i.e., neurotransmission mediated by acetylcholine, is involved in a host of physiological processes, including learning and memory. Cholinergic dysfunction is commonly associated with neurodegenerative diseases, including Alzheimer's disease. In the gut, acetylcholine acts as an excitatory neuromuscular signaler to mediate smooth muscle contraction, which facilitates peristaltic propulsion. Gastrointestinal dysfunction has also been associated with Alzheimer's disease. This research focuses on the preparation of an electrochemical enzyme-based biosensor to monitor cholinergic signaling in the gut and its application for measuring electrically stimulated acetylcholine release in the mouse colon ex vivo. The biosensors were prepared by platinizing Pt microelectrodes through potential cycling in a potassium hexachloroplatinate (IV) solution to roughen the electrode surface and improve adhesion of the multienzyme film. These electrodes were then modified with a permselective poly(m-phenylenediamine) polymer film, which blocks electroactive interferents from reaching the underlying substrate while remaining permeable to small molecules like H2O2. A multienzyme film containing choline oxidase and acetylcholinesterase was then drop-cast on these modified electrodes. The sensor responds to acetylcholine and choline through the enzymatic production of H2O2, which is electrochemically oxidized to produce an increase in current with increasing acetylcholine or choline concentration. Important figures of merit include a sensitivity of 190 ± 10 mA mol-1 L cm-2, a limit of detection of 0.8 µmol L-1, and a batch reproducibility of 6.1% relative standard deviation at room temperature. These sensors were used to detect electrically stimulated acetylcholine release from mouse myenteric ganglia in the presence and absence of tetrodotoxin and neostigmine, an acetylcholinesterase inhibitor.


Subject(s)
Acetylcholine , Alzheimer Disease , Animals , Mice , Acetylcholinesterase , Hydrogen Peroxide , Reproducibility of Results , Cholinesterase Inhibitors , Choline , Colon
2.
Anal Chem ; 95(2): 1027-1037, 2023 01 17.
Article in English | MEDLINE | ID: mdl-36524968

ABSTRACT

This research reports on the preparation of a boron-doped diamond microelectrode modified with platinum nanoparticles and Nafion and its application for detecting nitric oxide (NO) in vitro in the mouse colon. Platinum nanoparticle deposition was performed potentiodynamically using a 2.0 mmol L-1 potassium hexachloroplatinate solution and cycling from -0.2 to 1.3 V vs Ag/AgCl at 0.01 V s-1 for 10 cycles. The Nafion overlayer was applied by immersion in a solution containing 2.5% (w/v) colloidal Nafion and drying overnight at 55 °C in a humid environment. The optimal microelectrode preparation conditions were chosen based on the electrode response for NO oxidation as well as rejection of nitrite (NO2-) oxidation, the main interferent in the electrochemical detection of NO in biological media. Detection figures of merit include a sensitivity of 16.7 ± 2.7 mA M-1 cm-2 (n = 3 electrodes), a detection limit of 0.5 µmol L-1 (S/N = 3), and an electrode response reproducibility of 2.5% (RSD). Electrical stimulation and continuous amperometry were used to measure NO release from myenteric ganglia in wild-type male and female mice in response to an increasing number of electrical stimuli to study nitrergic signaling in the colon. We also present preliminary data regarding the use of optogenetics to selectively stimulate nitrergic myenteric neurons using blue light stimulation with a goal of understanding how inhibitory neuromuscular signaling is involved in the myenteric plexus circuitry that controls intestinal motility.


Subject(s)
Metal Nanoparticles , Nitric Oxide , Female , Male , Animals , Mice , Microelectrodes , Boron , Platinum , Diamond , Reproducibility of Results , Electrodes
3.
Neurogastroenterol Motil ; 34(12): e14439, 2022 12.
Article in English | MEDLINE | ID: mdl-36458522

ABSTRACT

BACKGROUND: Alterations in gastrointestinal (GI) function and the gut-brain axis are associated with progression and pathology of Alzheimer's Disease (AD). Studies in AD animal models show that changes in the gut microbiome and inflammatory markers can contribute to AD development in the central nervous system (CNS). Amyloid-beta (Aß) accumulation is a major AD pathology causing synaptic dysfunction and neuronal death. Current knowledge of the pathophysiology of AD in enteric neurons is limited, and whether Aß accumulation directly disrupts enteric neuron function is unknown. METHODS: In 6-month-old 5xFAD (transgenic AD) and wildtype (WT) male and female mice, GI function was assessed by colonic transit in vivo; propulsive motility and GI smooth muscle contractions ex vivo; electrochemical detection of enteric nitric oxide release in vitro, and changes in myenteric neuromuscular transmission using smooth muscle intracellular recordings. Expression of Aß in the brain and colonic myenteric plexus in these mice was determined by immunohistochemistry staining and ELISA assay. KEY RESULTS: At 6 months, 5xFAD mice did not show significant changes in GI motility or synaptic neurotransmission in the small intestine or colon. 5xFAD mice, but not WT mice, showed abundant Aß accumulation in the brain. Aß accumulation was undetectable in the colonic myenteric plexus of 5xFAD mice. CONCLUSIONS: 5xFAD AD mice are not a robust model to study amyloidosis in the gut as these mice do not mimic myenteric neuronal dysfunction in AD patients with GI dysmotility. An AD animal model with enteric amyloidosis is required for further study.


Subject(s)
Amyloidosis , Female , Male , Animals , Mice , Synaptic Transmission , Neurons , Submucous Plexus , Myenteric Plexus , Disease Models, Animal
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