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Using optical recordings of neuronal [Ca2+]i in brain slices from young rats (P9-P11), we report distinct regulation of Ca2+ signalling mediated by the activity of glutamate receptors in the ventral cochlear nucleus (VCN) and the central nucleus of the inferior colliculus (CIC) in the midbrain. [Ca2+]i increases were recorded after bath-stimulation of slices with glutamate agonists of both ionotropic (AMPA/kainate or NMDA) and group I metabotropic glutamate receptors (mGluRs).NMDA-induced [Ca2+]i responses were similar in both auditory nuclei. Kainate-induced [Ca2+]i increases recorded in the VCN were over two-fold larger than those in the CIC. Blockade of kainate-induced [Ca2+]i responses in VCN neurons with 1-naphtylacetyl spermine (NAS) demonstrated that Ca2+-permeable AMPA receptors predominated in the VCN. In contrast, abundant Ca2+-impermeable AMPA receptors were found in the CIC.Both mGluR1 and mGluR5 subtypes of group I mGluRs were present in the CIC and the VCN. However, Group I mGluR [Ca2+]i responses elicited by DHPG were two fold higher in CIC than in VCN neurons. Therefore, our findings suggest that Ca2+ signalling in auditory neurons may be differentially regulated at different levels of the auditory pathway through preferential activation of different classes of glutamate receptors, which may have implications for hierarchical auditory signal processing and plasticity, at least during the early developmental stages of hearing.