HPLC/EC Detection and Quantification of Acetylcholine in Dialysates - Drugs of Abuse: Neurological Reviews and Protocols - page 428

required for its oxidation at this surface. Platinum is an excellent surface, being
inert and requiring a relatively low applied potential for the oxidation of peroxide.
Moreover we have not found the reported drawback of a long equilibration time
for platinum electrodes to be problematic. However, platinum is soft and easily
scratched. The required applied potential of +0.5 V also contributes signifi cantly
to background noise, especially due to the continuous activity of the enzyme
reactor. A newer electrode surface that is constructed from a glassy carbon
electrode coated with a peroxidase-redox polymer is now commercially avail-
able from BAS. This surface requires a low applied potential and should thus
reduce background noise. Maintenance of this surface may be signifi cantly more
problematic than for platinum surfaces.
8. The analytical methods described in the preceding provide a highly sensitive
assay for ACh. However the detection of ACh in dialysate may still require the
application of an ACh esterase inhibitor in vivo due to the very low concentration
of ACh in certain CNS regions. Although this chapter discusses only the analysis
of ACh and not its extraction from brain by microdialysis, the two techniques
are highly interdependent. The need for esterase inhibitors can be minimized
by careful surgical technique. Recovery of ACh is especially sensitive to local
tissue damage, which can be minimized by extremely slow lowering of the
microdialysis probe. In this way esterase inhibitor concentrations can be kept low
and the interpretation of changes in ACh levels becomes more straightforward.
The use of high concentrations of the esterase inhibitor neostigmine has been
repeatedly shown not only to elevate basal ACh levels but also to alter the
responsiveness of the striatal cholinergic system to drug challenge (12-14) .
This result highlights the variable interdependence of basal ACh levels and the
magnitude of changes in ACh level subsequent to experimental manipulation.
For this reason our laboratory always reports basal ACh levels and the magnitude
of changes in ACh levels in absolute terms.
Acknowledgments
The authors acknowledge the pioneering efforts of Dr. Peter DeBoer toward
establishing and refining the methodology described in this chapter. The
assistance of Dr. J. M. Tepper with the digital photography is also greatly
appreciated. This work was supported USPHS Grants DA08086 and NS19608
and the Tourette's Syndrome Association.
References
1. Loewi, O. (1921) Ueber humorale Uebertragbarkeit der Herznervenwirkung.
Pfl ugers Arch. 189, 239-242.
2. Dale, H. H., Feldberg, W., and Vogt, M. (1936) Release of acetylcholine at
voluntary motor nerve endings. J. Physiol. (Lond.) 86, 353-380.
3. Dale, H. H. (1914) The action of certain esters and ethers of choline and their
relation to muscarine. J. Pharmacol. 6, 147-190.