JUN CUI, NADEZHDA KULAGINA, ADRIAN C. MICHAEL, Department
of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260.
Monitoring the
levels of neurotransmitters in the extracellular space of living brain is
challenging because the lifetime of these important compounds is very short.
For example, acetylcholine is rapidly metabolized in the extracellular space by
cholinesterase enzymes. For this reason, acetylcholine may be metabolized before
it reaches an implanted sampling probe, such as a microdialysis probe.
In this project, we have constructed enzyme-modified carbon fiber
microelectrodes that are suitable for the detection of choline in the
extracellular space of the living brain. The advantage of these devices is that
they are implanted into the brain. As a consequence, the microelectrode can be
placed very close to viable neuronal terminals. We have investigated whether or
not the signal observed in the brain with these microelectrodes is related to
the activity of cholinergic neurons.
Amperometric microsensors
have been developed for the in vivo detection of choline and glucose. Choline
oxidase, horseradish peroxidase and glucose oxidase were immobilized in a
crosslinked redox polymer matrix on carbon fiber electrodes. Choline
microsensors gave a detection limit of 1~3 microM. Optimized glucose
microsensors gave a detection limit of ~100 microM-200 microM and a linear
response up to 10 mM, which includes the physiologically relevant glucose
concentration. Immobilization of ascorbate oxidase and a Nafion outlayer gave
selectivity above 90% to both choline microsensors and glucose microsensors
over 400 microM ascorbate, which is the physiologically relevant concentration.
We have tested whether or not the cholinergic signal is useful as an index of
cholinergic brain activity. We have observed that when tetrodotoxin (TTX) is
infused into brain tissue near the microsensors, the signal decreases. Since
TTX causes neuronal activity to stop, this result suggests that the choline
signal may indeed provide a useful index of acetylcholine release. Furthermore,
the signals also decrease when neostigmine, a powerful cholinesterase
inhibitor, is infused into the brain tissue. Neither TTX nor neostigmine
infusions into brain tissue influence the signal recorded with glucose oxidase
modified microelectrodes. These results suggest that enzyme-modified
microelectrodes provide a useful index of acetylcholine release.