A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.

Amyloid imaging has identified cognitively normal older people with plaques as a group possibly at increased risk for developing Alzheimer’s disease-related dementia. It is important to begin to thoroughly characterize this group so that preventative therapies might be tested. Existing cholinotropic agents are a logical choice for preventative therapy as experimental evidence suggests that they are anti-amyloidogenic and clinical trials have shown that they delay progression of mild cognitive impairment to dementia. A detailed understanding of the status of the cortical cholinergic system in preclinical AD is still lacking, however. For more than 30 years, depletion of the cortical cholinergic system has been known to be one of the characteristic features of AD. Reports to date have suggested that some cholinergic markers are altered prior to cognitive impairment while others may show changes only at later stages of dementia. These studies have generally been limited by relatively small sample sizes, long postmortem intervals and insufficient definition of control and AD subjects by the defining histopathology. We therefore examined pre- and post-synaptic elements of the cortical cholinergic system in frontal and parietal cortex in 87 deceased subjects, including non-demented elderly with and without amyloid plaques as well as demented persons with neuropathologically-confirmed AD. Choline acetyltransferase (ChAT) activity was used as a presynaptic marker while displacement of 3H-pirenzepine binding by oxotremorine-M in the presence and absence of GppNHp was used to assess postsynaptic M1 receptor coupling. The results indicate that cortical ChAT activity as well as M1 receptor coupling are both significantly decreased in non-demented elderly subjects with amyloid plaques and are more pronounced in subjects with AD and dementia. These findings confirm that cortical cholinergic dysfunction in AD begins at the preclinical stage of disease and suggest that cholinotropic agents currently used for AD treatment are a logical choice for preventative therapy.

No abstract

No abstract

No abstract

No abstract

No abstract

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. Since disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). We found that choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) were significantly higher in samples from DKO compared to wild-type (WT) mice. This was region-specific since levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKOs compared to WTs. We also found that ChAT activity was significantly reduced in cortical samples from DKOs compared to WTs. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal REM sleep and cataplexy.