In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release

Abstract

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fastscan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 μM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

Caged Zn2+ Photolysis in Zebrafish Whole Brains Reveals Subsecond Modulation of Dopamine Uptake

“…Fish were maintained in a tank system as previously described. 45 Water quality was monitored, and parameters were maintained: conductivity, 800 μS cm −1 ; pH, 7.2; and temperature, 28 °C. A light/dark cycle of 14 h on and 10 h off was used.…”

“…Fast scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes is a method of choice for measuring the release/uptake of DA in living brain tissue. , FSCV provides sub-s temporal resolution and good selectivity. ,− However, combining FSCV with perfusion or direct injection of free Zn 2+ through tissues is not an ideal approach for investigating sub-s interactions due to the time needed for Zn 2+ concentrations to reach equilibrium (minutes to hours). We have previously combined caged compound photolysis with FSCV to quantify glutamate photoactivation in living brain tissue . The combination of FSCV with newly developed caged metals, such as caged Zn 2+ compounds, will allow us to directly probe the effects of free metals on DA release and uptake from presynaptic terminals on sub-s time scales.…”

Impaired Dopamine Release and Latent Learning in Alzheimer’s Disease Model Zebrafish

“…The electrochemical measurement was carried out the same day as the behavioral probe trial. The procedure to harvest the zebrafish brain was described elsewhere. ,, Briefly, the examined fish was euthanized by a rapid cooling method, followed by decapitation. Immediately following euthanasia, the whole brain was harvested using previously described methods and transferred to a perfusion chamber.…”

The Discovery, Development and Demonstration of Three Caged Compounds†

“…It is often important to accurately quantify the amount of caged substrate that has been photoreleased and quanta absorbed in opaque environments, such as the brain. Johnson and coworkers (45) solved this problem by developing a method using fast scan cyclic voltammetry (FSCV) in conjunction with photolysis of pHP‐glu to temporally track the concentration of the photoreleased glu in real time in a living whole zebrafish brain, which is opaque. The method required distinguishing between p‐hydroxyphenylacetic acid produced during photolysis and unreacted pHP‐glu by subtracting their two CVs.…”