Neurochemistry Technique of in vivo Fixed Potential Amperometry
The majority of studies in our lab utilize in vivo fixed potential amperometry (FPA), an electrochemical method that has been valuable in elucidating the modulation of forebrain dopamine activity by other neurotransmitter systems. Like many in vivo electrochemical applications, FPA involves the implantation of a carbon fiber recording electrode. The procedure also utilizes an auxiliary electrode (stainless-steel wire) combined with a reference electrode (Ag/AgCl) placed in contact with brain tissue. An electrometer creates a circuit between these three electrodes, allowing the application of a fixed continuous potential (.8V) to the recording electrode via the auxiliary electrode, and the maintenance of a potential difference between the recording and reference electrodes. Continuously applied potential to the recording electrode allows electroactive neurochemicals (such as dopamine) to be continuously oxidized at the electrode surface. As such, FPA allows a high temporal resolution (10 000 samples/sec) for the analysis of neurochemical events in vivo. In conjunction with untreated carbon fiber electrodes (used for their rapid response times), FPA is selective for measurement of electrically-stimulated dopamine efflux in vivo. For example, this has been demonstrated by significant increases in LDT stimulation-evoked oxidation current in the NAc in response to systemic administration of the dopamine reuptake inhibitor nomifensine, while serotonin and norepinephrine reuptake blockers fail to alter the evoked response. Consequently, this technique has been utilized to explore the kinetics of dopamine neurotransmission and the biochemical basis of dopaminergic burst firing in anesthetized rats and mice. With the addition of microinfusions of various drugs (receptor antagonists), FPA lends itself to the investigation of the role of receptor types in phasic dopamine efflux as evoked by electrical stimulation. Selective measurements of elicited dopamine efflux in vivo are made in real-time, and the amplitude of the stimulated dopamine response remains stable over time and thus can be averaged within one animal to increase the signal to noise ratio.
Stimulation-evoked dopamine efflux is quantified by extraction of data points occurring within the range of 0.25 sec pre- and 2.0 sec post-stimulation from the recorded oxidation current in the striatum. The mean change in dopamine oxidation current, corresponding to stimulation-evoked dopamine efflux, is converted to a mean dopamine concentration (µM) by post-experiment in vitro calibration of the carbon fiber electrode in solutions of dopamine (2-10 µM) using a flow injection system. For each animal, changes in stimulation-evoked dopamine concentration after infusion are expressed as mean percent changes with respect to pre-infusion baseline responses (100%).
Example Surgery (from a study investigating the role of ionotropic glutamate receptors in the SNc in mediating STN stimulation-evoked striatal dopamine release)
Mice were anesthetized with urethane (1.5 g/kg, i.p.) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) within a mouse head-holder adaptor (Stoelting, Wood Dale, IL, USA), ensuring the skull was flat. Body temperature was maintained at 36 ± 0.5°C with a temperature-regulated heating pad (TC-1000; CWE Inc., New York, NY, USA). All stereotaxic coordinates (AP from bregma, ML from midline, and DV from dura, all in mm) were determined from the mouse atlas of Paxinos and Franklin (2001). In each mouse a concentric bipolar stimulating electrode (SNE-100; Rhodes Medical Co., CA, USA) was implanted into the left STN of each mouse (coord.: AP -2.0, ML +1.6, and DV -4.0). A 31 g stainless-steel guide infusion cannula was implanted into the left SNc, with the tip of the guide cannula positioned 2 mm above site (coord.: AP -3.1, ML +1.5, and DV -3.8). An Ag/AgCl reference and stainless-steel auxiliary electrode combination was placed in surface contact with contralateral cortical tissue approximately 2.0 mm posterior to bregma, and a carbon fiber recording microelectrode with an active recording surface of 250 μm (length) by 10 μm (o.d.) (Thornel Type P, Union Carbide, Pittsburgh, PA, USA) was then implanted into the left striatum (coordinates: AP +1.4, ML +1.4, and DV -2.5).
A video created by the University of Memphis to display the research of our lab. In the video, I present Deep Brain Stimulation (DBS) as a treatment for Parkinson’s disease and how our research can be applied to better the benefits of this neurosurgical approach.
Neurochemistry Lab of Dr. Charles Blaha Univ. of Memphis, Dept. of Psych.
In this lab, we are interested in a Systems Neuroscience approach for understanding the neurobiological bases of incentive-motivated behaviors, neuropsychiatric disorders, and drug addiction using state of the art in vivo electrochemical recording techniques.
The above picture shows an example set-up of in vivo fixed potential amperometry for evaluation of the effects of intra-SNc or PPT lidocaine infusions on STN electrical stimulation-evoked dopamine release in the striatum.