Aims: 

Brain slice preparations using neurochemical markers are well-established models for a wide spectrum of in vitro investigations in the neuroscience. There are multiple neurotransmitters and neuromodulators in hippocampus. Changing the strength of synaptic transmission is a means to modify the communication in a neuronal network.

Methods: 

An efficient and sensitive absorbance and fluorescence detection procedure was developed for determination of multiple neurotransmitters amino acids (aspartic acid, glutamic acid, 4-aminobutanoic acid); monoamines (noradrenalin, serotonin) and neuromodulators (anandamide, somatostatin) in slice perfusate. Different parameters that influenced separation and derivatization were optimized. The method was used to investigate the dynamic changes of neurotransmitters and neuromodulators in hippocampus. The extracellular fluid of hippocampus was obtained by the microperfusion technique.

Results: 

We investigated the effects of veratridine, which induced persistent activation of voltage-gated sodium channels in hippocampal neurons. Application of this toxin in 50mM concentration for 3 min increased the extracellular levels of amino acids (aspartic, glutamic and 4-aminobutanoic acid) to 21.5, 4.9 and 21 (pmol/min/mg protein) by 10–12 min of the experiment, respectively. The increase in monoamine transmitters (noradrenalin, serotonin) and neuromodulators (anandamide, somatostatin) at the same time, reached the maximum 15–18 min after veratridine treatment at 3.8, 0.88 and 1.7, 4.8 (pmol/min/mg protein), respectively. Neurotransmitters were also analyzed in response to optogenetic activation in the hippocampus of mice microinjected with light-sensitive channelrhodopsin-2 mutant adenoviral vector (rAAV2/9-synapsin-ChR2-EGFP) to the median raphe. Light stimulation at 470nm resulted in increases in concentrations of glutamic acid (2.4 pmol/min/mg); serotonin (0.6 pmol/min/mg); somatostatin 0.05; and anandamide (0.27 pmol/min/mg).

Conclusion: 

Activity-dependent changes were detected by the simultaneous, direct neurochemical analyses of the extracellular levels of neurotransmitters and neuromodulators in the hippocampus, with distinct temporal kinetics. Using this technique valuable insight could be obtained by the interrelationship between the released compounds and thereby to the operation of hippocampal network.