Spatiotemporal patterns of proteins associated with GABA synthesis and transport in the developing auditory brainstem

Abstract

During an early developmental period, some glycinergic synapses in the brainstem and spinal cord release predominately GABA, which activates GABAA receptors on the postsynaptic membrane. The function of this early GABAergic transmission is unknown but presumed to contribute to synapse maturation. Classically, the enzyme glutamic acid decarboxylase (GAD), which synthesizes GABA from glutamate, has been considered the sole source of GABA in neurons. GABAergic neurons typically express one or both of the two known isoforms of this enzyme, GAD65 and GAD67. However, co-transmitting synapses in the midbrain were recently reported to acquire GABA through other means – GABA transporters (GAT1 and GAT3) and/or aldehyde dehydrogenase (ALDH1A1). To determine the source of GABA in immature glycinergic neurons of the auditory brainstem, we immunostained for GADs, GATs, and ALDH1A1, co-staining with markers for glial cell and synaptic terminals to verify cellular and subcellular location. GAD65 was expressed in synaptic terminals whereas GAD67 was localized to neuronal cell bodies, proximal dendrites, and presumabed synaptic terminals. However, during the peak period of GABA transmission in the first postnatal week, expression levels of both GAD65 and GAD67 were surprisingly low. Although GAT1 and GAT3 expression levels coincided with the peak period of GABA transmission, neither GAT was localized to neuronal cell bodies. In contrast, ALDH1A1 was expressed during the first postnatal week and was localized to neuronal cell bodies. These results suggest that immature glycinergic neurons of the auditory brainstem may not acquire GABA through classical GABA synthesis or GABA reuptake, but perhaps are able to synthesis GABA through the putrescine degradation pathway mediated by ALDH1A1.