An improved method for long-term measuring of hemolymph fluctuations of non-essential amino acids, GABA and histamine from freely moving crayfish.

The microdialysis method was adapted to obtain long-term hemolymph dialysates from the pericardial cavity of freely moving Procambarus clarkii crayfish, to measure fluctuations of non-essential amino acids, GABA and histamine by high-performance liquid chromatography using off-line fluorometric derivatization. Asp, Ala, Tau, GABA and histamine (HA) reached its maximal concentrations at the daybreak, whereas glutamate (Glu), Gln and Gly peaked at the end of the light period. The minimum and maximal detected amounts for each substance along the 24h cycle were (in microM): 20-300Asp, 100-200Glu, 400-700Gln, 400-600Gly, 100-200Tau, 150-300Ala, 2-10 GABA and 25-250HA. Cocktails containing the relative concentration of each amino acid, GABA and histamine resulted in a hyperpolarization that reduced the spontaneous firing of cultured peptidergic X organ neurons. Glu, GABA and histamine evoked a long-lasting hyperpolarization that suppressed the spontaneous firing, whereas Asp, Gly and Tau evoked a depolarization accompanied with neuronal firing. Finally, neither Ala nor Gln modified the resting membrane potential.