The results show that VGLUT3 serves as an important mediator of glutamate signaling, with essential roles in the auditory system and cortex. VGLUT3 KO mice are entirely deaf and the loss of auditory brainstem responses indicate a defect early in the auditory pathway, within the cochlea. Intact otoacoustic emissions exclude a problem with mechanoelectrical transduction or outer hair cell motility. Rather, the analysis of compound action potentials recorded from the auditory nerve suggests impairment of neurotransmission at the IHC-afferent synapse. However, the IHCs of VGLUT3 KO mice exhibit physiological properties very similar to WT animals. In particular, the IHCs of VGLUT3 KO mice show normal calcium currents, and at hearing onset, the normal down-regulation of calcium action potentials together with the appearance of BK potassium conductances, a process that appears to depend on thyroid hormone (Brandt et al., 2007
; Rusch et al., 2001
; Sendin et al., 2007
). Afferent nerve terminals of KO mice also exhibit sodium and potassium conductances very similar to WT. However, KO afferents lack synaptically evoked glutamate receptor currents despite normal responses to exogenous kainate, indicating a specific, presynaptic defect in glutamate release. Glutamate has been proposed as the primary transmitter at the IHC-afferent nerve synapse (Bobbin, 1979
; Comis and Leng, 1979
; Ottersen et al., 1998
). Since the only VGLUT isoform expressed by IHCs is VGLUT3, the absence of glutamate release in KO mice must reflect a loss of vesicle filling. IHCs have been reported to express VGLUT1 (Furness and Lawton, 2003
), but we could not detect specific expression of either VGLUT1 mRNA or protein by IHCs, and the VGLUT1 KO shows no measurable hearing impairment. The loss of VGLUT1 or indeed other vesicular neurotransmitter transporters does not obviously impair synaptic vesicle exo- or endocytosis (Croft et al., 2005
; Wojcik et al., 2004
), making it unlikely that the loss of VGLUT3 would affect these other aspects of the synaptic vesicle cycle.
VGLUT3 belongs to a small number of proteins involved in neurotransmitter release that have a relatively selective effect on synaptic signaling by IHCs. Otoferlin, a protein associated with hereditary deafness in humans (Yasunaga et al., 1999
), has recently been suggested to act as the principal calcium sensor for regulated exocytosis in mammalian IHCs (Roux et al., 2006
). Deletion of the otoferlin gene in mice results in profound deafness due to the loss of synchronous evoked glutamate release at the IHC-afferent synapse. Similar to the VGLUT3 KO, the IHCs of otoferlin KO mice show relatively normal ionic conductances. However, both otoferlin and VGLUT3 KO mice show an increase in IHC K+
currents at maturity, suggesting feedback of the glutamate signal onto IHCs. In contrast, mice lacking the calcium channels required for transmitter release do not exhibit the usual appearance of BK potassium currents and loss of efferents seen in both VGLUT3 KO, otoferlin KO and WT animals (Brandt et al., 2003
; Nemzou et al., 2006
), indicating that these changes do not rely on synaptic transmission, but rather on the specific activity of calcium channels.
The loss of VGLUT3 produces morphological changes in the cochlea similar to those previously observed in otoferlin KO animals (Roux et al., 2006
). VGLUT3 KO mice show a progressive reduction in the number of ganglion cells during postnatal development, as well as their projections to the brainstem. Indeed, stimulation (presumably mediated by glutamate) has been shown to provide trophic support for spiral ganglion cells (Hegarty et al., 1997
). The IHCs of both otoferlin and VGLUT3 KO mice also show abnormal ribbons, although the ribbons in otoferlin KO have defects in anchoring to the synaptic membrane, and those in VGLUT3 KO exhibit an abnormally elongated morphology. However, the number of synaptic vesicles, their association with ribbons and their morphological docking at the plasma membrane appear unaffected by the loss of VGLUT3.
The appearance of VGLUT3 before birth provides a mechanism for neurotransmission before the onset of hearing, as previously shown (Eybalin et al., 2004
; Glowatzki and Fuchs, 2002
; Knipper et al., 1997
). Since spontaneous retinal activity influences development of the visual system (Eglen et al., 2003
; Firth et al., 2005
; Shatz, 1996
), the early expression of VGLUT3 by IHCs may therefore also contribute to development of the auditory system. Indeed, the transient expression of VGLUT3 at the synapse made by medial nucleus of the trapezoid body neurons onto lateral superior olivary neurons (MNTB-LSO synapse) has suggested an early role for activity in tonotopic refinement and synaptic strengthening downstream in the pathway (Gillespie et al., 2005
). Consistent with previous work, the degenerative changes observed at P10 in VGLUT3 KO mice demonstrate that early activity has a specific role in development of the auditory system.
Similar to IHCs, retinal photoreceptors and bipolar cells form ribbon synapses, but express VGLUT1 (Johnson et al., 2003
; Sherry et al., 2003
). How might the properties of the unconventional isoform VGLUT3 help to fulfill the specialized function of the IHC synapse in hearing? Although the isoforms differ little in their intrinsic transport activity, they diverge substantially in trafficking (Voglmaier et al., 2006
). The dendritic targeting of VGLUT3 in multiple neuronal populations (Fremeau et al., 2002
; Harkany et al., 2004
) may indeed correspond to its basolateral targeting in epithelial cells such as IHCs. In addition, IHCs appear to derive their synaptic vesicles from at least two sources, one a local recycling pool at the basolateral surface that involves cisternal intermediates (Lenzi et al., 2002
), and another pool particularly important for sustained, high-frequency synaptic signaling that may originate on the apical surface (Griesinger et al., 2002
; Griesinger et al., 2005
). The neuronal localization of VGLUT3 in dendrites as well as axons is indeed consistent with its trafficking to more than one pool of secretory vesicles.
The incessant interictal activity and less frequent generalized seizures observed in VGLUT3 KO mice indicate a major disturbance of cortical excitability. A distinguishing feature of these seizures is the lack of overt changes in ongoing motor behavior. Absence epilepsy in human patients and animal models involves brief (1-10 sec), highly stereotyped thalamocortical discharges, but these episodes are always accompanied by behavioral arrest and often terminate with myoclonus (Noebels and Sidman, 1979
). In contrast, VGLUT3 KO mice provide the first genetically defined mouse model of pure primary generalized non-convulsive epilepsy demonstrating essentially complete electrographic dissociation, with no prominent effects on behavior. The loss of VGLUT3 appears sufficient to synchronize a limited cellular network without allowing spread to pathways mediating tonic or clonic motor activity, providing an entry point to identify the anatomic substrate for dissociated cortical hypersynchrony.
It is interesting that the loss of one VGLUT3 allele confers interictal epileptiform abnormalities without evident electrographic seizures. VGLUT1 and 2 heterozygotes have no obvious behavioral phenotype, but a recent study of the VGLUT1 heterozygote has suggested abnormalities in anxiety and long-term memory (Tordera et al., 2007
). The VGLUT2 heterozygote shows a specific defect in nociception (Moechars et al., 2006
). Although apparently sufficient to fill vesicles under baseline conditions even when substantially reduced (Daniels et al., 2006
), VGLUT expression may thus become limiting under certain conditions, possibly related to increases in activity. The interictal abnormalities observed in VGLUT3 heterozygous mice may reflect a similar requirement for two wild type alleles, with one sufficient to prevent the development of generalized seizures. Considering the extreme demands of signaling by IHCs, it is notable that VGLUT3 heterozygotes exhibit no obvious hearing impairment, but future study may reveal more subtle abnormalities in sound processing.
The importance of VGLUT3 for neurotransmission at the IHC synapse known to use glutamate suggests that the transporter also contributes to glutamate release by neurons usually associated with a different transmitter. Indeed, VGLUT3 in the cortex localizes primarily to GABAergic interneurons, as well as to serotonergic projections from the raphe nuclei (Hioki et al., 2004
; Somogyi et al., 2004
). Interestingly, dysfunction in a subset of cortical and hippocampal interneurons due to loss of the DLX1 transcription factor produces seizures resembling those observed in VGLUT3 KO animals (Cobos et al., 2005
), suggesting that the VGLUT3 KO seizure phenotype reflects the loss of glutamate signaling by inhibitory interneurons. VGLUT3 thus has a physiologically important role in the control of cortical excitability, presumably by conferring the release of glutamate from cells usually associated with GABA. These observations strongly suggest that VGLUT3 also contributes to glutamate signaling by neurons traditionally considered to release other transmitters.