Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C→T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca²⁺-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.     

Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3

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.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

Have you heard? Viral-mediated gene therapy restores hearing

Genetic loss of VGLUT3 in cochlear inner hair cells results in profound deafness. In this issue of Neuron, Akil?et?al. (2012) show that AAV-mediated introduction of wild-type VGLUT3 in the genetically deaf mouse cochlea results in significantly improved hearing.     

The expression of vesicular glutamate transporters defines two classes of excitatory synapse

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.     

Neurotransmission in the vestibular endorgans--glutamatergic transmission in the afferent synapses of hair cells.

In the sensory pathways the first synapse is that between hair cells and primary afferent neurons and its most likely neurotransmitter candidate has long been thought to be glutamate. A number of pharmacological and electrophysiological studies have lent credence to this theory (reviewed by Bledsoe et al. 1988, Bobbin 1979, Ehrenberger and Felix 1991, Puel et al. 1991; Puel 1995) as has recent neurochemical and immunocytochemical work (reviewed by Ottersen et al. 1998; Usami et al. 2000). These recent studies reveal that the afferent hair cell synapse resembles the central glutamate synapses in many ways. Of the proteins confirmed to be involved in signal transduction and transmitter metabolism at most central synapses, many are also seen in the afferent hair cell synapse, and have an analogous compartmentation. On the other hand, there are also important differences, especially those related to the molecular mechanisms that underlie transmitter release.     

Glutamate co-transmission from developing medial nucleus of the trapezoid body--lateral superior olive synapses is cochlear dependent in kanamycin-treated rats

Cochlear dependency of glutamate co-transmission at the medial nucleus of the trapezoid body (MNTB) – the lateral superior olive (LSO) synapses was investigated using developing rats treated with high dose kanamycin. Rats were treated with kanamycin from postnatal day (P) 3 to P8. A scanning electron microscopic study on P9 demonstrated partial cochlear hair cell damage. A whole cell voltage clamp experiment demonstrated the increased glutamatergic portion of postsynaptic currents (PSCs) elicited by MNTB stimulation in P9–P11 kanamycin-treated rats. The enhanced VGLUT3 immunoreactivities (IRs) in kanamycin-treated rats and asymmetric VGLUT3 IRs in the LSO of unilaterally cochlear ablated rats supported the electrophysiologic data. Taken together, it is concluded that glutamate co-transmission is cochlear-dependent and enhanced glutamate co-transmission in kanamycin-treated rats is induced by partial cochlear damage.     

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO₂ and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.     

Aromatase and 5-alpha reductase gene expression: modulation by pain and morphine treatment in male rats

Background

Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Results

Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.

Conclusions

The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.     

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.     

Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment

The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1-3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.     

Effects of subthalamic nucleus lesions and stimulation upon corticostriatal afferents in the 6-hydroxydopamine-lesioned rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.     

Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant.     

Simultaneous measurement of glutamate and dopamine release from isolated guinea pig cochlea

Glutamate is proved to be a neurotransmitter in the mammalian cochlea, transmitting signals between the inner hair cells and the afferent cochlear nerve terminals. The transmission in this synapse is modulated by the lateral olivocochlear efferent fibers by releasing dopamine and other neurotransmitters. This study undertakes to measure simultaneously the release of dopamine and glutamate from isolated guinea pig cochleae. We combined the in vitro microvolume superfusion method, that uses liquid scintillation analysis, to measure [3H]dopamine with high pressure liquid chromatography (HPLC) to determine the glutamate content of the superfusate at rest and during stimulation. The release of both neurotransmitters was significantly increased when electrical field stimulation was applied at a 10 Hz rate. The nonselective sodium-channel inhibitor tetrodotoxin (TTX) at 1 microM completely blocked the effect of stimulation, indicating the neural origin of both dopamine and glutamate. The dopamine receptor antagonist sulpiride at 100 microM and the dopamine receptor agonist bromocriptine at 20 microM did not change the release of glutamate. In contrast, both bromocriptine and sulpiride significantly increased the stimulation-evoked release of dopamine. The effect of sulpiride is most likely due to the blockade of dopamine autoreceptor. Possible explanations why bromocriptine increased the release include: (1) its partional agonist activity; (2) desensitizations of dopamine autoreceptors; or (3) the higher D1 receptor activity of bromocriptine than sulpiride. This study could provide further insights about the role of dopamine and glutamate in cochlear neurotransmission.     

Developmentally-regulated expression of tissue-specific splice variant of rat vesicular glutamate transporter 1 in retina and pineal gland

Three distinct subtypes of vesicular glutamate transporters (VGLUTs) have been identified to date that are expressed basically in a cell type-specific manner. We have found a splice variant of VGLUT1 mRNA that is expressed almost exclusively in photosensitive tissues, i.e. the retina and the pineal gland. The variant mRNA, termed VGLUT1v, contains an additional 75 base pair sequence derived from part of a second intron (designated as exon IIa) between exons 2 and 3. The variant accounted for approximately 70% and 25%of VGLUT1 mRNA in the adult retina and pineal gland, respectively. The expression of VGLUT1v was developmentally regulated in both tissues. Organ culture showed that expression of the variant in the retina increased in association with the development of rod cells, suggesting that VGLUT1v is expressed in rod cells. In situ hybridization with variant-specific probes showed expression of VGLUT1v in the inner segment layer of photoreceptor cells. On the other hand, variant expression did not parallel the development of rhodopsin-positive cells in the pineal gland. As rod cells and pinealocytes are known to release glutamate continuously at ribbon synapses, it is possible that the variant has some functional advantage over the wild-type transporter in such a specialized manner of glutamate release.     

Inhibition of repulsive guidance molecule,RGMa, increases afferent synapse formation with auditory hair cells

The peripheral fibers that extend from auditory neurons to hair cells are sensitive to damage, and replacement of the fibers and their afferent synapse with hair cells would be of therapeutic interest. Here, we show that RGMa, a repulsive guidance molecule previously shown to play a role in the development of the chick visual system, is expressed in the developing, newborn, and mature mouse inner ear. The effect of RGMa on synaptogenesis between afferent neurons and hair cells, from which afferent connections had been removed, was assessed. Contact of neural processes with hair cells and elaboration of postsynaptic densities at sites of the ribbon synapse were increased by treatment with a blocking antibody to RGMa, and pruning of auditory fibers to achieve the mature branching pattern of afferent neurons was accelerated. Inhibition by RGMa could thus explain why auditory neurons have a low capacity to regenerate peripheral processes: postnatal spiral ganglion neurons retain the capacity to send out processes that respond to signals for synapse formation, but expression of RGMa postnatally appears to be detrimental to regeneration of afferent hair cell innervation and antagonizes synaptogenesis. Increased synaptogenesis after inhibition of RGMa suggests that manipulation of guidance or inhibitory factors may provide a route to increase formation of new synapses at deafferented hair cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 457–466, 2014     

Effect of Different Gentamicin Dose on the Plasticity of the Ribbon Synapses in Cochlear Inner Hair Cells of C57BL/6J Mice

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1?ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. To study effects of different doses of gentamicin on the changes of synaptic ribbons of cochlear inner hair cells (IHCs) in mice, the availability of genetic information, transgenic and knock-out animals make the C57BL/6J mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for gentamicin ototoxicity in adult C57BL/6J mouse strains. Five-week-old mice were injected intraperitoneally once daily with 50?C300?mg gentamicin base/kg body weight for 7?days. Higher doses of gentamicin appear to be associated with earlier hearing damage in C57BL/6J mice, although not necessarily with more severe damage. At 200?mg/kg, gentamicin appears to induce significant hearing damage while not significantly affect the animal??s general condition. Therefore, 200?mg/kg may be an ideal dose for ototoxicity modeling in C57BL/6J mice using gentamicin. In the early period of different dose of gentamicin effect, when the number of hair cells had not changed, the number changes of IHC ribbon synapses had taken place. Through the number of ribbon synapses changing, IHCs increased or decreased connections with spiral ganglion nerves (SGNs). The ribbon synapses played a compensatory role for gentamicin ototoxicity, while this effect was not sufficient to maintain the normal threshold of hearing.     

谷氨酸受体在噪声所致豚鼠螺旋神经节细胞损伤中的作用

本文旨在研究谷氨酸及其受体在噪声致豚鼠螺旋神经节细胞损伤中的作用。实验分为在体和离体两部分。（1）在体实验：豚鼠分为生理盐水（NS,10μL）组,NS（10μL）＋噪声组和犬尿喹啉酸（kynurenic acid,KYNA,5mmol／L,10μL）＋噪声组,每组15只。用微量注射器经完整圆窗膜表面给予NS或KYNA：暴露于白噪声110dBSPL,1h。在圆窗给药前及噪声暴露后测试听觉脑干诱发电位（auditory brainstem response,ABR）阈值及Ⅲ波幅值,听神经复合动作电位（compound action potential,CAP）阈值及N1波幅值和潜伏期,测试后取基底膜进行透射电镜观察。（2）离体实验：观察高浓度谷氨酸对急性分离的豚鼠螺旋神经节细胞的影响。结果显示,NS＋噪声组豚鼠ABR及CAP阈移显著高于KYNA＋噪声组,且Ⅲ波和NI波幅值明显降低,潜伏期明显延长。NS＋噪声组豚鼠毛细胞及传入神经末梢急性水肿和线粒体结构破坏：KYNA＋噪声组豚鼠的毛细胞和传入神经末梢无明显变化。离体胞外施加谷氨酸可引起螺旋神经节细胞逐渐出现水肿、变性,最后死亡。本实验提示,噪声暴露可引起豚鼠听功能损伤,毛细胞／传入神经突触的结构破坏和螺旋神经节细胞变性、死亡：这种损伤可能与噪声暴露引起谷氨酸的过度释放有关;谷氨酸通过其受体介导致使螺旋神经节细胞损伤,谷氨酸受体的广谱拮抗剂KYNA可减轻噪声对螺旋神经节细胞的损伤。     

Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity

Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.     

Developmental Up-Regulation of Vesicular Glutamate Transporter-1 Promotes Neocortical Presynaptic Terminal Development

Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.