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Cholinergic cells have been proposed to innervate simultaneously those cortical areas that are mutually interconnected with each other. To test this hypothesis, we investigated the cholinergic innervation of functionally linked amygdala and prefrontal cortical regions. First, using tracing experiments, we determined that cholinergic cells located in distinct basal forebrain (BF) areas projected to the different nuclei of the basolateral amygdala (BLA). Specifically, cholinergic cells in the ventral pallidum/substantia innominata (VP/SI) innervated the basal nucleus (BA), while the horizontal limb of the diagonal band of Broca (HDB) projected to its basomedial nucleus (BMA). In addition, cholinergic neurons in these two BF areas gave rise to overlapping innervation in the medial prefrontal cortex (mPFC), yet their axons segregated in the dorsal and ventral regions of the PFC. Using retrograde-anterograde viral tracing, we demonstrated that a portion of mPFC-projecting cholinergic neurons also innervated the BLA, especially the BA. By injecting retrograde tracers into the mPFC and BA, we found that 28% of retrogradely labeled cholinergic cells were double labeled, which typically located in the VP/SI. In addition, we found that vesicular glutamate transporter type 3 (VGLUT3)-expressing neurons within the VP/SI were also cholinergic and projected to the mPFC and BA, implicating that a part of the cholinergic afferents may release glutamate. In contrast, we uncovered that GABA is unlikely to be a co-transmitter molecule in HDB and VP/SI cholinergic neurons in adult mice. The dual innervation strategy, i.e., the existence of cholinergic cell populations with single as well as simultaneous projections to the BLA and mPFC, provides the possibility for both synchronous and independent control of the operation in these cortical areas, a structural arrangement that may maximize computational support for functionally linked regions. The presence of VGLUT3 in a portion of cholinergic afferents suggests more complex functional effects of cholinergic system in cortical structures.

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Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24 h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance.

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Exposure to stressors has profound effects on sleep that have been linked to serotonin (5-HT) neurons of the dorsal raphe nucleus (DR). However, the DR also comprises glutamatergic neurons expressing vesicular glutamate transporter type 3 (DRVGLUT3), leading us to examine their role. Cell-type-specific tracing revealed that DRVGLUT3 neurons project to brain areas regulating arousal and stress. We found that chemogenetic activation of DRVGLUT3 neurons mimics stress-induced sleep perturbations. Furthermore, deleting VGLUT3 in the DR attenuated stress-induced sleep perturbations, especially after social defeat stress. In the DR, VGLUT3 is found in subsets of 5-HT and non-5-HT neurons. We observed that both populations are activated by acute stress, including those projecting to the ventral tegmental area. However, deleting VGLUT3 in 5-HT neurons minimally affected sleep regulation. These findings suggest that VGLUT3 expression in the DR drives stress-induced sleep perturbations, possibly involving non-5-HT DRVGLUT3 neurons.

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A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

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In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

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The current understanding of the neuromodulatory role of the median raphe nucleus (MRN) is primarily based on its putative serotonergic output. However, a significant proportion of raphe neurons are glutamatergic. The present study investigated how glutamatergic MRN input modulates the medial prefrontal cortex (mPFC), a critical component of the fear circuitry. Our studies show that VGLUT3-expressing MRN neurons modulate VGLUT3- and somatostatin-expressing neurons in the mPFC. Consistent with this modulation of mPFC GABAergic neurons, activation of MRN (VGLUT3) neurons suppresses mPFC pyramidal neuron activity and attenuates fear memory in female but not male mice. In agreement with these female-specific effects, we observed sex differences in glutamatergic transmission onto MRN (VGLUT3) neurons and mPFC (VGLUT3) neuron-mediated dual release of glutamate and GABA. Thus, our results demonstrate a cell type-specific modulation of the mPFC by MRN (VGLUT3) neurons and reveal a sex-specific role of this neuromodulation in mPFC synaptic plasticity and fear memory.

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The hippocampus (HP) receives neurochemically diverse inputs from the raphe nuclei, including glutamatergic axons characterized by the expression of the vesicular glutamate transporter type 3 (VGLUT3). These raphe-HP VGLUT3 projections have been suggested to play a critical role in HP functions, yet a complete anatomical overview of raphe VGLUT3 projections to the forebrain, and in particular to the HP, is lacking. Using anterograde viral tracing, we describe largely nonoverlapping VGLUT3-positive projections from the dorsal raphe (DR) and median raphe (MnR) to the forebrain, with the HP receiving inputs from the MnR. A limited subset of forebrain regions such as the amygdaloid complex, claustrum, and hypothalamus receives projections from both the DR and MnR that remain largely segregated. This highly complementary anatomical pattern suggests contrasting roles for DR and MnR VGLUT3 neurons. To further analyze the topography of VGLUT3 raphe projections to the HP, we used retrograde tracing and found that HP-projecting VGLUT3-positive neurons (VGLUT3HP ) distribute over several raphe subregions (including the MnR, paramedian raphe, and B9 cell group) and lack co-expression of serotonergic markers. Strikingly, double retrograde tracing experiments unraveled two parallel streams of VGLUT3-positive projections targeting the dorsal and ventral poles of the HP. These results demonstrate highly organized and segregated VGLUT3-positive projections to the HP, suggesting independent modulation of HP functions such as spatial memory and emotion-related behavior.

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Visceral pain is among the most prevalent and bothersome forms of chronic pain, but their transmission in the spinal cord is still poorly understood. Here, we conducted focal colorectal distention (fCRD) to drive both visceromotor responses (VMRs) and aversion. We first found that spinal CCK neurons were necessary for noxious fCRD to drive both VMRs and aversion under naive conditions. We next showed that spinal VGLUT3 neurons mediate visceral allodynia, whose ablation caused loss of aversion evoked by low-intensity fCRD in mice with gastrointestinal (GI) inflammation or spinal circuit disinhibition. Importantly, these neurons were dispensable for driving sensitized VMRs under both inflammatory and central disinhibition conditions. Anatomically, a subset of VGLUT3 neurons projected to parabrachial nuclei, whose photoactivation sufficiently generated aversion in mice with GI inflammation, without influencing VMRs. Our studies suggest the presence of different spinal substrates that transmit nociceptive versus affective dimensions of visceral sensory information.

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After a damaging insult, hair cells can spontaneously regenerate from cochlear supporting cells within the first week of life. While the regenerated cells express several markers of immature hair cells and have stereocilia bundles, their capacity to differentiate into inner or outer hair cells, and ability to form new synaptic connections has not been well-described. In addition, while multiple supporting cell subtypes have been implicated as the source of the regenerated hair cells, it is unclear if certain subtypes have a greater propensity to form one hair cell type over another. To investigate this, we used two CreER mouse models to fate-map either the supporting cells located near the inner hair cells (inner phalangeal and border cells) or outer hair cells (Deiters', inner pillar, and outer pillar cells) along with immunostaining for markers that specify the two hair cell types. We found that supporting cells fate-mapped by both CreER lines responded early to hair cell damage by expressing Atoh1, and are capable of producing regenerated hair cells that express terminal differentiation markers of both inner and outer hair cells. The majority of regenerated hair cells were innervated by neuronal fibers and contained synapses. Unexpectedly, we also found that the majority of the laterally positioned regenerated hair cells aberrantly expressed both the outer hair cell gene, oncomodulin, and the inner hair cell gene, vesicular glutamate transporter 3 (VGlut3). While this work demonstrates that regenerated cells can express markers of both inner and outer hair cells after damage, VGlut3 expression appears to lack the tight control present during embryogenesis, which leads to its inappropriate expression in regenerated cells.

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Vesicular glutamate transporters (VGLUTs) are responsible for the storage of glutamate into secretory vesicles. The VGLUT3 isoform is mainly expressed in neurons that secrete other classical neurotransmitters, including the cholinergic interneurons in the striatum, and VGLUT3-expressing neurons often secrete two distinct neurotransmitters. VGLUT3 is discretely distributed throughout the brain and is found in subpopulations of spinal cord interneurons, in subset of neurons in the dorsal root ganglion, and in Merkel cells. Mice with a global loss of VGLUT3 are hyperactive and the modulation of specific VGLUT3-expressing circuits can lead to changes in movement. In this study, we tested the hypothesis that increased activity of VGLUT3-expressing neurons is associated with decreased movement. Using a mouse line expressing excitatory designer receptor exclusively activated by designer drugs (hM3Dq-DREADD) on VGLUT3-expressing neurons, we showed that activation of hM3Dq signalling acutely decreased locomotor activity. This decreased locomotion was likely not due to circuit changes mediated by glutamate nor acetylcholine released from VGLUT3-expressing neurons, as activation of hM3Dq signalling in mice that do not release glutamate or acetylcholine from VGLUT3-expressing neurons also decreased locomotor activity. This suggests that other neurotransmitters are likely driving this hypoactive phenotype. We used these mouse lines to compare the effects of DREADD agonists in vivo. We observed that clozapine-N-oxide (CNO), clozapine, compound 21 and perlapine show small differences in the speed at which they prompt behavioural responses but the four of them are selective DREADD ligands.

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Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

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Adeno-associated virus (AAV)-based gene therapy has been demonstrated to be extremely effective for treating genetic hearing loss over the past several years. However, successful gene therapies for hereditary deafness have not been well-studied in adult mice. To explore the possibility of gene therapy after peripheral auditory maturity, we used AAV8 to express vesicular glutamate transporter 3 (Vglut3) in the cochleae of 5w, 8w, and 20w Vglut3KO mice. Results indicated that AAV8-Vglut3 could mediate the exogenous expression of Vglut3 in all inner hair cells (IHCs). Auditory function was successfully restored, and the hearing threshold remained stable for at least 12 weeks after rescue. Moreover, the results revealed that the number of synaptic ribbons, as well as their morphology, was significantly recovered after gene therapy, potentially indicating the glutamate-dependent plasticity of IHCs. Taken together, our data introduce the possibility of gene therapy in adult mice and advance our knowledge of the role of Vglut3 in presynaptic plasticity.

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The raphe nucleus is critical for feeding, rewarding and memory. However, how the heterogenous raphe neurons are molecularly and structurally organized to engage their divergent functions remains unknown. Here, we genetically target a subset of neurons expressing VGLUT3. VGLUT3 neurons control the efficacy of spatial memory retrieval by synapsing directly with parvalbumin-expressing GABA interneurons (PGIs) in the dentate gyrus. In a mouse model of Alzheimer's disease (AD mice), VGLUT3→PGIs synaptic transmission is impaired by ETV4 inhibition of VGLUT3 transcription. ETV4 binds to a promoter region of VGLUT3 and activates VGLUT3 transcription in VGLUT3 neurons. Strengthening VGLUT3→PGIs synaptic transmission by ETV4 activation of VGLUT3 transcription upscales the efficacy of spatial memory retrieval in AD mice. This study reports a novel circuit and molecular mechanism underlying the efficacy of spatial memory retrieval via ETV4 inhibition of VGLUT3 transcription and hence provides a promising target for therapeutic intervention of the disease progression.

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Introduction: The neurotransmitter serotonin is a key regulator of neurotransmission, mood, and behavior and is essential in neurodevelopment. Dysfunction in this important neurotransmitter system is connected to behavioral disorders such as depression and anxiety. We have previously shown that the developing serotonin system is sensitive to perinatal exposure to Western-style diet (WSD). Methods: To advance our hypothesis that perinatal WSD has a long-term impact on the serotonergic system, we designed a fluorescent immunohistochemistry experiment using antibodies against tryptophan hydroxylase 2 (TPH2) and vesicular glutamate transporter 3 (VGLUT3) to probe protein expression in the raphe subnuclei in 13-month-old Japanese macaques (Macaca fuscata; n = 22). VGLUT3 has been shown to be coexpressed in TPH2+ cells in the dorsal raphe (DR) and median raphe nucleus (MnR) of rodent raphe nuclei and may provide information about the projection site of serotonergic fibers into the forebrain. We also sought to improve scientific understanding of the heterogeneity of the serotonin production center for the central nervous system, the midbrain raphe nuclei. Results: In this immunohistochemical study, we provide the most detailed characterization of the developing primate raphe to date. We utilize multi-level modeling (MLM) to simultaneously probe the contribution of WSD, offspring sex, and raphe anatomical location, to raphe neuronal measurements. Our molecular and morphological characterization revealed that the 13-month-old macaque DR is remarkably similar to that of adult macaques and humans. We demonstrate that vesicular glutamate transporter 3 (VGLUT3), which rodent studies have recently shown can distinguish raphe populations with distinct projection targets and behavioral functions, likewise contributes to the heterogeneity of the primate raphe. Discussion: This study provides evidence that perinatal WSD has a long-term impact on the density of serotonin-producing neurons, potentially limiting serotonin availability throughout the brain. Due to the critical involvement of serotonin in development and behavior, these findings provide important insight into the mechanisms by which maternal nutrition and metabolic state influence offspring behavioral outcomes. Finally, these findings could inform future research focused on designing therapeutic interventions to optimize neural development and decrease a child's risk of developing a mental health disorder.

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Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15-16 were exposed to 1-12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔC m showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3 KO mice showed a noise-induced reduction in ΔC m and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.

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A pericellular basket is a presynaptic configuration of numerous axonal boutons outlining a target neuron soma and its proximal dendrites. Recent studies show neurochemical diversity of pericellular baskets and suggest that neurotransmitter usage together with the dense, soma-proximal boutons may permit strong input effects on different timescales. Here we review the development, distribution, neurochemical phenotypes, and possible functions of pericellular baskets. As an example, we highlight pericellular baskets formed by projections of certain Pet1/Fev neurons of the serotonergic raphe nuclei. We propose that pericellular baskets represent convergence sites of competition or facilitation between neurotransmitter systems on downstream circuitry, especially in limbic brain regions, where pericellular baskets are widespread. Study of these baskets may enhance our understanding of monoamine regulation of memory, social behavior, and brain oscillations.

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High-intensity noise can cause permanent hearing loss; however, short-duration medium-intensity noise only induces a temporary threshold shift (TTS) and damages synapses formed by inner hair cells (IHCs) and spiral ganglion nerves. Synaptopathy is generally thought to be caused by glutamate excitotoxicity. In this study, we investigated the expression levels of vesicle transporter protein 3 (Vglut3), responsible for the release of glutamate; glutamate/aspartate transporter protein (GLAST), responsible for the uptake of glutamate; and Na+/K+-ATPase α1 coupled with GLAST, in the process of synaptopathy in the cochlea. The results of the auditory brainstem response (ABR) and CtBP2 immunofluorescence revealed that synaptopathy was induced on day 30 after 100 dB SPL noise exposure in C57BL/6J mice. We found that GLAST and Na+/K+-ATPase α1 were co-localized in the cochlea, mainly in the stria vascularis, spiral ligament, and spiral ganglion cells. Furthermore, Vglut3, GLAST, and Na+/K+-ATPase α1 expression were disrupted after noise exposure. These results indicate that disruption of glutamate release and uptake-related protein expression may exacerbate the occurrence of synaptopathy.

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The overconsumption of sugar-sweetened food and beverages underpins the current rise in obesity rates. Sugar overconsumption induces maladaptive neuroplasticity to decrease dietary control. Although serotonin and glutamate co-localisation has been implicated in reward processing, it is still unknown how chronic sucrose consumption changes this transmission in regions associated with executive control over feeding-such as the prefrontal cortex (PFC) and dentate gyrus (DG) of the hippocampus. To address this, a total of 16 C57Bl6 mice received either 5% w/v sucrose or water as a control for 12 weeks using the Drinking-In-The-Dark paradigm (n = 8 mice per group). We then examined the effects of chronic sucrose consumption on the immunological distribution of serotonin (5-HT), vesicular glutamate transporter 3 (VGLUT3) and 5-HT+/VGLUT3+ co-localised axonal varicosities. Sucrose consumption over 12 weeks decreased the number of 5-HT-/VGLUT3+ and 5-HT+/VGLUT3+ varicosities within the PFC and DG. The number of 5-HT+/VGLUT3- varicosities remained unchanged within the PFC but decreased in the DG following sucrose consumption. Given that serotonin mediates DG neurogenesis through microglial migration, the number of microglia within the DG was also assessed in both experimental groups. Sucrose consumption decreased the number of DG microglia. Although the DG and PFC are associated with executive control over rewarding activities and emotional memory formation, we did not detect a subsequent change in DG neurogenesis or anxiety-like behaviour or depressive-like behaviour. Overall, these findings suggest that the chronic consumption of sugar alters serotonergic neuroplasticity within neural circuits responsible for feeding control. Although these alterations alone were not sufficient to induce changes in neurogenesis or behaviour, it is proposed that the sucrose consumption may predispose individuals to these cognitive deficits which ultimately promote further sugar intake.

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KEY POINTS: M1 intrinsically photosensitive retinal ganglion cells (ipRGCs) are known to encode absolute light intensity (irradiance) for non-image-forming visual functions (subconscious vision), such as circadian photoentrainment and the pupillary light reflex. It remains unclear how M1 cells respond to relative light intensity (contrast) and patterned visual signals. The present study identified a special form of contrast sensitivity (suppressed-by-contrast) in M1 cells, suggesting a role of patterned visual signals in regulating non-image-forming vision and a potential role of M1 ipRGCs in encoding image-forming visual cues. The study also uncovered a synaptic mechanism and a retinal circuit mediated by vesicular glutamate transporter 3 (vGluT3) amacrine cells that underlie the suppressed-by-contrast response of M1 cells. M1 ipRGC subtypes (M1a and M1b) were revealed that are distinguishable based on synaptic connectivity with vGluT3 amacrine cells, receptive field properties, intrinsic photo sensitivity and membrane excitability, and morphological features, suggesting a division of visual tasks among discrete M1 subpopulations. ABSTRACT: The M1 type ipRGC (intrinsically photosensitive retinal ganglion cell) is known to encode ambient light signals for non-image-forming visual functions such as circadian photo-entrainment and the pupillary light reflex. Here, we report that a subpopulation of M1 cells (M1a) in the mouse retina possess the suppressed-by-contrast (sbc) trigger feature that is a receptive field property previously found only in ganglion cells mediating image-forming vision. Using optogenetics and the dual patch clamp technique, we found that vesicular glutamate transporter 3 (vGluT3) (vGluT3) amacrine cells make glycinergic, but not glutamatergic, synapses specifically onto M1a cells. The spatiotemporal and pharmacological properties of visually evoked responses of M1a cells closely matched the receptive field characteristics of vGluT3 cells, suggesting a major role of the vGluT3 amacrine cell input in shaping the sbc trigger feature of M1a cells. We found that the other subpopulation of M1 cells (M1b), which did not receive a direct vGluT3 cell input, lacked the sbc trigger feature, being distinctively different from M1a cells in intrinsic photo responses, membrane excitability, receptive-field characteristics and morphological features. Together, the results reveal a retinal circuit that uses the sbc trigger feature to regulate irradiance coding and potentially send image-forming cues to non-image-forming visual centres in the brain.

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Vesicular glutamate transporters (VGLUTs) were long thought to be specific markers of glutamatergic excitatory transmission. The discovery, two decades ago, of the atypical VGLUT3 has thoroughly modified this oversimplified view. VGLUT3 is strategically expressed in discrete populations of glutamatergic, cholinergic, serotonergic, and even GABAergic neurons. Recent reports show the subtle, but critical, implications of VGLUT3-dependent glutamate co-transmission and its roles in the regulation of diverse brain functions and dysfunctions. Progress in the neuropharmacology of VGLUT3 could lead to decisive breakthroughs in the treatment of Parkinson's disease (PD), addiction, eating disorders, anxiety, presbycusis, or pain. This review summarizes recent findings on VGLUT3 and its vesicular underpinnings as well as on possible ways to target this atypical transporter for future therapeutic strategies.

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