Ralph N. Adams

Liquid chromatographic analysis of catecholamines routine assay for regional brain mapping

Abstract A thoroughly tested and highly reliable catecholamine assay is described which routinely determines 20–100 picograms of norepinephrine and dopamine in small punches of brain tissue weighing 0.50 to 50 mg. The assay was designed for regional brain mapping. It employs liquid chromatography with electrochemical detection and involves a minimum of sample pretreatment. Its realistic performance is illustrated by typical experimental data. Modifications for larger or whole brain samples as well as details of construction and operation of this system are given.

Nafion-coated electrodes with high selectivity for CNS electrochemistry

A major improvement in the selectivity of small graphite electrodes used for in vivo electrochemistry is described. The electrodes are coated with Nafion, a perfluorosulfonated polymer. This coating is practically impermeable to ascorbic acid and anionic biogenic amine metabolites and only slightly responsive to neutral metabolites. Thus it becomes selective for the cationic primary neurotransmitters, dopamine, norepinephrine and 5-hydroxytryptamine. Responses of Nafion-coated and untreated electrodes in vivo are compared.

An Electrochemical Detector for Liquid Chromatography with Picogram Sensitivity

Abstract A simple liquid chromatograph with an extremely sensitive electrochemical detector is described. The detector, which can be constructed for lees than

Graphite paste electrodes: Effects of paste composition and surface states on electron-transfer rates

5.00 (excluding electronics), has an actual dead volume of < 1 μl and operates well in the 50–100 picogram region. Routine quantitative work at the 5–10 nanogram level is described. Potential applications to the analysis of biogenic amines are discussed.

New high performance liquid chromatographic analysis of brain catecholamines

Abstract A new understanding of the nature of electrode reactions at graphite paste electrodes has been obtained by studying electron-transfer rates of redox systems under conditions of carefully controlled electrode composition and pretreatment. Dry graphite gives electron-transfer rates which give an almost Nernstian response and approach those obtained with platinum. The addition of any pasting liquid decreases these rates materially. Both electrochemical and chemical oxidative pretreatments of such pastes increase the electron transfer in the direction of the “dry” graphite limit. This effect appears to correlate with the increased hydrophilic nature of the electrode surface and concomitant removal of organic layers from the electrode surface.

Effect ofl-glutamate on the release of striatal dopamine: in vivo dialysis and electrochemical studies

Abstract A new high performance liquid chromatography analysis has been developed for catecholamines in brain tissue. The method retains alumina separation of the catecholamines. Quantitative read-out is by direct liquid chromatography, replacing the tedious trihydroxyindole chemistry and fluorescence measurements. The analysis is rapid and accurate and agrees well with existing literature data. The equipment is inexpensive and the technique can be utilized for routine analyses after 1–2 weeks of practice. The method is directly applicable to whole small animal brains and, depending on the NE and DA levels, to dissected sub-portions.

Monitoring 5-hydroxytryptamine release in the brain of the freely moving unanaesthetized rat using in vivo voltammetry

Microdialysis and in vivo voltammetry combined with K(+)-selective microelectrodes were utilized to study the effect of L-glutamate (GLU) on the in vivo release of dopamine (DA) from the rat striatum. Perfusion of 500 nM-5 mM GLU through the microdialysis probe was without an effect on DA outflow whereas 10 mM GLU resulted in a significant (295%) increase in the basal level of DA. This increase was blocked in the presence of 2-amino-5-phosphonopentanoic acid, an N-Methyl-D-aspartate (NMDA) receptor antagonist. Repetitive local applications of 10 mM GLU were also required to observe an increase in extracellular DA measured by in vivo voltammetry. These signals were accompanied with a massive increase in extracellular K+ and a large negative shift in the field potential resembling the ionic changes seen after the phenomenon spreading depression. These studies suggest that high concentrations of GLU are required to enhance the extracellular concentration of DA in vivo. Further, pathophysiological conditions such as spreading depression may be responsible for the observed increase in extracellular DA concentration.

Diffusion coefficients of neurotransmitters and their metabolites in brain extracellular fluid space

The possibility of using in vivo voltammetry to monitor 5-hydroxytryptamine (5-HT) release from brain tissue in freely moving unanaesthetized rats has been examined. A potential (+0.2 to +1.0 V) was applied to a micrographite electrode stereotaxically placed within a specific brain region and current changes following the oxidation of electroactive compounds in the vicinity of the electrode tip were recorded. Administration of p-chloroamphetamine (5 mg/kg) produced a large increase in current in the striatum and this could be prevented by pretreatment with p-chlorophenylalanine (150 mg/kg X 2) to deplete brain 5-HT or Fluoxetine (10 mg/kg) which prevents the uptake of p-chloroamphetamine by 5-HT neurones. Fluoxetine (10 mg/kg) caused a small but long lasting increase in current. Stimulation of the median raphe nucleus produced a marked and rapid rise in current in the hippocampus but a much smaller one in the striatum. This response could also be prevented by 24 h pretreatment with p-chlorophenylalanine (150 mg/kg). Seven days after p-chlorophenylalanine administration raphe stimulation again produced an increase in current. Rats under barbiturate anaesthesia showed no clear increase in current either after p-chloroamphetamine or raphe stimulation, indicating that barbiturates may affect neurotransmitter release. The results suggest that 5-HT release can be monitored in the freely moving unanaesthetized rat using in vivo voltammetry, and that a moderate decrease in brain 5-HT concentration leads to a substantial inhibition of drug or stimulation induced release of 5-HT.

Voltammetry in brain tissue: Chronic recording of stimulated dopamine and 5-hydroxytryptamine release

Diffusion coefficients of catecholamine neurotransmitters, their metabolites and related species was measured in brain extracellular fluid using in vivo voltammetric techniques. Nanoliter volumes of the species were pressure-ejected into the rat caudate nucleus and their concentration profiles were determined at nearby voltammetric detector electrodes. Thorough testing was carried out to show that the present methodology gave results which agreed with brain diffusion coefficients measured previously by ion-selective microelectrode techniques. All of the species which are anionic at pH 7.4 have brain diffusion coefficients about one-third of their solution counterparts in accord with earlier studies of diffusion in tortuous media. However, the brain diffusion coefficients of all the cation species are about three-times slower than those of the anions. This phenomenon is believed to be caused by ion binding with the polyanionic glycosaminoglycans and related species in brain tissue. In vitro model experiments lend support to this interpretation. This new information on biogenic amines and their metabolites provides meaningful predictions of the spatio-temporal concentration distribution of these species in the extracellular fluid.

Detailed mapping of ascorbate distribution in rat brain

Abstract Micro voltammetric electrodes which continuously monitor stimulated release of dopamine and 5-hydroxytryptamine in unanesthetized, essentially unrestrained rats are described. The results demonstrate that the electrochemical technique correctly follows dopamine efflux, especially in the case of amphetamine-stimulated dopamine release in the caudate. While the method is still exploratory, its value in pharmacological manipulations of neurotransmitter release, etc. is already clearly evident.