Levels of Amino Acid Neurotransmitters during Neurogenesis and in Histotypic Cultures of Mouse Spinal Cord

Abstract

The development of spinal cord interneurons and the formation of interneuronal synaptic connections has received little attention; the most comprehensively studied developing circuit has been the connection between motoneurons and the muscle they innervate. All motoneurons are cholinergic whereas spinal interneurons are mostly glutamatergic, glycinergic or GABAergic neurons. In this study, we show quantitative data, obtained by high-pressure liquid chromatography (HPLC), on the levels of amino acid neurotransmitters during mouse spinal cord neurogenesis, from embryonic day (E) 12 until postnatal day (P) 30. At E12, high levels of glutamate, glycine and taurine were already detected but between E16 and P3, significant increments in their contents were observed, indicating the occurrence of maximum synaptogenesis during this period. Important reductions in their contents were also observed in two stages: between E12–E16 and P3–P7. These results suggest that the apoptotic death of interneurons and motoneurons in the developing brain or the synapse refinement of neural circuitry during maturation reduced the number of synapses, thereby decreasing the levels of neurotransmitters. The contents of these neurotransmitters were also analyzed in primary cultures of mouse spinal cord prepared from embryos between E13 and E19. As deduced from light microscopy, ultrastructural studies, as well as results from HPLC analysis, the cultures derived from E15–E16 embryos showed the highest degree of histotypic features and neurotransmitter contents comparable with those obtained in situ. Although glycine, GABA and taurine levels reached about 80–90% of normal in situ values, the contents of aspartate and glutamate were lower by about 40%, which could be mainly due to deafferentation of both sensory and supraspinal afferent axon terminals. These results indicate that intrinsic synaptic circuits can be maintained in histotypic spinal cord cultures prepared from E15–E16 mouse embryos. Histotypic cultures of the spinal cord will serve as a good model for studies on the pathophysiology of amino-acid-based neurotransmission and repair strategies in many CNS disorders.