Tamás Szikra

Analysis of purine and pyrimidine bases, nucleosides and deoxynucleosides in brain microsamples (microdialysates and micropunches) and cerebrospinal fluid

A new chromatographic method is reported for the synchronous analysis of endogenous purine and pyrimidine bases, ribonucleosides, and deoxyribonucleosides in brain samples. An optimized gradient chromatography system with a cooled reversed-phase column allows the detection of these compounds in very low concentrations in microsamples (microdialysates and micropunches). Chromatographic peaks were identified via the retention times of known standards, with detection at two wavelengths, and also by electrospray tandem mass spectrometry, which permits the identification of certain compounds at extremely low concentrations. The method was tested on in vivo brain microdialysis samples, micropunch tissue sample and cerebrospinal fluid of rats. Extracellular concentrations of pyrimidine metabolites in brain samples and of various purine metabolites in thalamic samples are reported here first. A comparison of the results on microdialysis and cerebrospinal fluid samples suggests that the analysis of cerebrospinal fluid provides limited information on the local extracellular concentrations of these compounds. Basic dialysis experiments revealed temporarily stable baseline levels one hour after implantation of the microdialysis probes. An elevated potassium concentration in the perfusion solution caused increases in the extracellular levels of adenosine and its metabolites, and of guanosine and the pyrimidine nucleoside uridine.

Uridine is released by depolarization and inhibits unit activity in the rat hippocampus

Perfusion of 5 microM kainate through microdialysis probes induced >2-fold elevation of extracellular uridine and adenosine concentrations in the hippocampus and in the thalamus of anaesthetized rats. Administration of uridine via this route produced an estimated uridine concentration of 50-100 microM around the electrode surface. This markedly decreased the average firing rate of neurones in the hippocampus, but not in the thalamus. Activity of separated single hippocampal pyramidal cells was completely inhibited by uridine. The same amount of adenosine completely blocked neuronal activity in both hippocampus and thalamus. Uridine administration had no effect on extracellular adenosine concentration. These findings suggest an important neuromodulatory role for depolarization-released uridine in the CNS.

Post mortem degradation of nucleosides in the brain: Comparison of human and rat brains for estimation of in vivo concentration of nucleosides

There is an increasing attention paid for nucleoside metabolism and changes of nucleoside concentrations in human brain because of its pathological and physiological relevance. In order to determine the post mortem degradation of nucleosides and nucleoside metabolites, the concentrations of four nucleosides and three nucleobases were measured in rat and neurosurgical human cerebral cortical samples with 30s to 24h post mortem delay. Adenosine degradation coefficient (a multiplying factor for calculating concentrations of investigated substances for the living state) was 0.886 for human brain at 2 h post mortem time, while it was 1.976 for rats. Hypoxanthine, an adenosine degradation product had coefficients 0.564 for human brain and 0.812 for the rat brain. We provide data and degradation coefficients for the concentrations of adenosine, guanosine, inosine, uridine, uracil, hypoxanthine and xanthine with 2, 4, 6 and 24 h post mortem delay. We also report a method how to validate human neurosurgical brain samples in terms of sample preparation and statistical analysis.

Concentration of Nucleosides and Related Compounds in Cerebral and Cerebellar Cortical Areas and White Matter of the Human Brain

1. Nucleosides potentially participate in the neuronal functions of the brain. However, their distribution and changes in their concentrations in the human brain is not known. For better understanding of nucleoside functions, changes of nucleoside concentrations by age and a complete map of nucleoside levels in the human brain are actual requirements.2. We used post mortem human brain samples in the experiments and applied a recently modified HPLC method for the measurement of nucleosides. To estimate concentrations and patterns of nucleosides in alive human brain we used a recently developed reverse extrapolation method and multivariate statistical analyses.3. We analyzed four nucleosides and three nucleobases in human cerebellar, cerebral cortices and in white matter in young and old adults. Average concentrations of the 308 samples investigated (mean±SEM) were the following (pmol/mg wet tissue weight): adenosine 10.3±0.6, inosine 69.5±1.7, guanosine 13.5±0.4, uridine 52.4±1.2, uracil 8.4±0.3, hypoxanthine 108.6±2.0 and xanthine 54.8±1.3. We also demonstrated that concentrations of inosine and adenosine in the cerebral cortex and guanosine in the cerebral white matter are age-dependent.4. Using multivariate statistical analyses and degradation coefficients, we present an uneven regional distribution of nucleosides in the human brain. The methods presented here allow to creation of a nucleoside map of the human brain by measuring the concentration of nucleosides in microdissected tissue samples. Our data support a functional role for nucleosides in the brain.

Purine and Pyrimidine Nucleoside Content of the Neuronal Extracellular Space in Rat

Neurological side-effects of chemotherapeutic agents1 known to act on purine and pyrimidine metabolism enzymes raised a possible role for nucleosides and deoxynucleosides in brain cell survival as it is demonstrated in recent investigations.2–5 Some new data also demonstrated the involvement of nucleotides and nucleosides in various brain functions. Apart from the well-known transmitter function of ATP6 and neuromodulator role of adenosine7 other nucleotides and nucleosides have also been suggested to be neuroactive. Pyrimidine nucleotides have specific pyrimidinoceptors even in cells of brain origin.8 Nucleosides could have their own modulatory actions as indicated by sleep modifying effect of uridine.9 The in vivo measurement of nucleosides and related substances in behaving animals could be the following major contribution to understanding the mechanisms of neurological side-effects of chemotherapeutic agents and the functional roles of nucleosides in the brain. A recently developed method, the in vivo microdialysis technique was used in the present study. Dialysis measures the composition of local extracellular space under various experimental conditions in freely moving animals.10 The main applications of microdialysis technique is measuring synaptic neurotransmitter overflow,10 investigation of transport functions, and pharmacokinetics of various drugs,11 and also gaining information about intracellular metabolic processes.12 Microdialysis has already been applied in human patients13 with subarachnoidal haemorrhage, head trauma, Parkinson’s disease, brain tumors and epilepsy.

An in vivo eyecup preparation for the rat

A method for the preparation of an in vivo eyecup and a complex stimulating-sampling device are described; these are suitable for long-term parallel neurochemical and electrophysiological experiments on the rat retina without any additives into the eyecup. In this in vivo eyecup the extracellular microenvironment is under the normal homeostatic control of the vascular system; no continuous exchange of the eyecup fluid and no addition of glutamate is necessary to maintain stable retinal electric responses and amino acid concentrations. The eyecup viability was tested by monitoring the electroretinogram (ERG) and the amino acid contents of the eyecup fluid sampled from the preretinal space by means of microdialysis. After the initial increase the b-wave of the ERG changed by less than 10% in maximal amplitude during experiments lasting 5 h. The glutamate, glutamine, and glycine levels proved comparatively, whereas the taurine level rose continuously throughout the experimental protocol. Recovery of ERG was achieved following exposure to bright background illumination. Total exchange of the eyecup volume requires 20 min at a flow rate of 1 microl/min. The effect of L-AP4 on the ERG was successfully reproduced, which suggests the applicability of this in vivo eyecup for pharmacological experiments on the rat retina.