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It is now clear that many neuronal populations release more than one classical neurotransmitter, yet in most cases the functional role of corelease is unknown. Striatal cholinergic interneurons release both glutamate and acetylcholine, and vesicular loading of glutamate has been shown to enhance acetylcholine content. Using a combination of optogenetics and whole-cell recordings in mice, we now provide physiological evidence that optogenetic stimulation of cholinergic interneurons triggers monosynaptic glutamate- and acetylcholine-mediated currents in striatal fast-spiking interneurons (FSIs), both of which depend on the expression of the vesicular glutamate transporter 3 (VGLUT3). In contrast to corticostriatal glutamatergic inputs onto FSIs, which are mediated primarily by AMPA-type glutamate receptors, glutamate release by cholinergic interneurons activates both AMPA- and NMDA-type glutamate receptors, suggesting a unique role for these inputs in the modulation of FSI activity. Importantly, we find that the loss of VGLUT3 not only markedly attenuates glutamatergic and cholinergic inputs on FSIs, but also significantly decreases disynaptic GABAergic input onto medium spiny neurons (MSNs), the major output neurons of the striatum. Our data demonstrate that VGLUT3 is required for normal cholinergic signaling onto FSIs, as well as for acetylcholine-dependent disynaptic inhibition of MSNs. Thus, by supporting fast glutamatergic transmission as well as by modulating the strength of cholinergic signaling, VGLUT3 has the capacity to exert widespread influence on the striatal network.

In sensory systems, peripheral organs convey sensory inputs to relay networks where information is shaped by local microcircuits before being transmitted to cortical areas. In the olfactory system, odorants evoke specific patterns of sensory neuron activity that are transmitted to output neurons in olfactory bulb (OB) glomeruli. How sensory information is transferred and shaped at this level remains still unclear. Here we employ mouse genetics, 2-photon microscopy, electrophysiology and optogenetics, to identify a novel population of glutamatergic neurons (VGLUT3+) in the glomerular layer of the adult mouse OB as well as several of their synaptic targets. Both peripheral and serotoninergic inputs control VGLUT3+ neurons firing. Furthermore, we show that VGLUT3+ neuron photostimulation in vivo strongly suppresses both spontaneous and odour-evoked firing of bulbar output neurons. In conclusion, we identify and characterize here a microcircuit controlling the transfer of sensory information at an early stage of the olfactory pathway.

Striatal cholinergic interneurons are implicated in motor control, associative plasticity, and reward-dependent learning. Synchronous activation of cholinergic interneurons triggers large inhibitory synaptic currents in dorsal striatal projection neurons, providing one potential substrate for control of striatal output, but the mechanism for these GABAergic currents is not fully understood. Using optogenetics and whole-cell recordings in brain slices, we find that a large component of these inhibitory responses derive from action-potential-independent disynaptic neurotransmission mediated by nicotinic receptors. Cholinergically driven IPSCs were not affected by ablation of striatal fast-spiking interneurons but were greatly reduced after acute treatment with vesicular monoamine transport inhibitors or selective destruction of dopamine terminals with 6-hydroxydopamine, indicating that GABA release originated from dopamine terminals. These results delineate a mechanism in which striatal cholinergic interneurons can co-opt dopamine terminals to drive GABA release and rapidly inhibit striatal output neurons.

Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2 weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent a successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.

Mechanical pain contributes to the morbidity associated with inflammation and trauma, but primary sensory neurons that convey the sensation of acute and persistent mechanical pain have not been identified. Dorsal root ganglion (DRG) neurons transmit sensory information to the spinal cord using the excitatory transmitter glutamate, a process that depends on glutamate transport into synaptic vesicles for regulated exocytotic release. Here we report that a small subset of cells in the DRG expresses the low abundance vesicular glutamate transporter VGLUT3 (also known as SLC17A8). In the dorsal horn of the spinal cord, these afferents project to lamina I and the innermost layer of lamina II, which has previously been implicated in persistent pain caused by injury. Because the different VGLUT isoforms generally have a non-redundant pattern of expression, we used Vglut3 knockout mice to assess the role of VGLUT3(+) primary afferents in the behavioural response to somatosensory input. The loss of VGLUT3 specifically impairs mechanical pain sensation, and in particular the mechanical hypersensitivity to normally innocuous stimuli that accompanies inflammation, nerve injury and trauma. Direct recording from VGLUT3(+) neurons in the DRG further identifies them as a poorly understood population of unmyelinated, low threshold mechanoreceptors (C-LTMRs). The analysis of Vglut3(-/-) mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Summary: The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability. [Copyright &y& Elsevier]