Determination of L-serine-O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection.

L-Serine-O-phosphate (L-SOP), the precursor of l-serine, is an agonist at group III metabotropic glutamate receptors. Despite the interest in L-SOP, very few articles have reported its brain levels. Here we report a convenient and reproducible method for simultaneous analysis of L-SOP and several other important amino acids in brain tissue using high-performance liquid chromatography (HPLC) with fluorimetric detection after derivatization with o-phthaldialdehyde and N-isobutyl-L-cysteine. Analyses were carried out in rat whole brain and cerebellum and in mouse whole brain, forebrain, amygdala, and prefrontal cortex. The method should be useful for future comprehensive neurochemical and pharmacological studies on neuropsychiatric disorders.

The effects of doxycycline administration on amino acid neurotransmitters in an animal model of neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia (HI) is a major contributor to many neurological, psychiatric and behavioral disorders. Previous studies in our laboratory have shown that a one-time dose of doxycycline (DOXY), even when given 3h after HI insult, was neuroprotective and significantly reduced microglial activation and cleaved caspase-3 protein expression in the immature brain. In light of these data, the goal of this study was to investigate the effects of DOXY administration on amino acid neurotransmitters. Post-natal-day 7 rats received DOXY (10mg/kg) or vehicle (VEH) concomitant with the onset of HI, and were euthanized 30 min, 1, 2 or 4h post-HI (n>or=6). Extracted brains were either immediately dissected for frontal cortex, striatum and hippocampal regions, or removed in their entirety and flash frozen in isopentane for histological analyses. Dissected regions were homogenized and aliquots were prepared for high performance liquid chromatography (HPLC) analyses of amino acid levels and brain levels of DOXY. HPLC extraction revealed that systemic administration of DOXY resulted in mean drug levels of 867.1+/-376.1 ng/g of brain tissue. Histological analyses revealed microglial activation, caspase-3 activation and neuronal degeneration consistent with a mild injury in the regions most vulnerable to HI. We found that HI caused significant, time-dependent, regional changes in brain amino acids including glutamate, GABA, alanine, aspartate, asparagine, serine, glutamine, glycine and taurine. HI significantly increased glutamate levels in the hippocampus (HI+VEH=15.8+/-3.1 ng/microg versus control=11.8+/-1.4 ng/microg protein) 4h post-HI (p<0.05). Pups treated with DOXY had lower glutamate levels (13.1+/-2.4 ng/microg) when compared to VEH-treated pups (15.8+/-3.1 ng/microg), however these values failed to reach significance. In addition, DOXY-treated pups had significantly lower alanine (HI+VEH=1.1+/-0.2 ng/microg versus HI+DOXY=0.5+0.1 ng/microg) and serine (HI+VEH=1.4+/-0.4 ng/microg versus HI+DOXY=0.7+0.1 ng/microg) levels in the hippocampus, 4h post-HI. Similar normalizations and significant reductions in alanine and serine were seen in the cortex and striatum. These results show that in addition to its previously reported and well-documented anti-inflammatory and anti-apoptotic properties, DOXY has significant effects on amino acid neurotransmitters.