Presynaptic Development Research Articles

Ubiquitin ligase signalling networks shape presynaptic development, function and disease.

Ubiquitin ligases are important regulators of nervous system development, function and disease. To date, numerous ubiquitin ligases have been discovered that regulate presynaptic biology. Here, we discuss recent findings on presynaptic ubiquitin ligases that include members from the three major ubiquitin ligase classes: RING, RBR and HECT. Several themes emerge based on findings across a range of model systems. A cadre of ubiquitin ligases is required presynaptically to orchestrate development and transmission at synapses. Multiple ubiquitin ligases deploy both enzymatic and non-enzymatic mechanisms, and act as hubs for signalling networks at the synapse. Both excitatory and inhibitory presynaptic terminals are influenced by ligase activity. Finally, there are several neurodevelopmental disorders and neurodegenerative diseases associated with presynaptic ubiquitin ligases. These findings highlight the growing prominence and biomedical relevance of the presynaptic ubiquitin ligase network.

Neuronal LRP4 directs the development, maturation and cytoskeletal organization of Drosophila peripheral synapses.

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Local protein synthesis of neuronal MT1-MMP for agrin-induced presynaptic development.

Upon the stimulation of extracellular cues, a significant number of proteins are synthesized distally along the axon. Although local protein synthesis is crucial for various stages throughout neuronal development, its involvement in presynaptic differentiation at developing neuromuscular junctions remains unknown. By using axon severing and microfluidic chamber assays, we first showed that treatment of a protein synthesis inhibitor, cycloheximide, inhibits agrin-induced presynaptic differentiation in cultured Xenopus spinal neurons. Newly synthesized proteins are prominently detected, as revealed by the staining of click-reactive cell-permeable puromycin analog O-propargyl-puromycin, at agrin bead-neurite contacts involving the mTOR/4E-BP1 pathway. Next, live-cell time-lapse imaging demonstrated the local capturing and immobilization of ribonucleoprotein granules upon agrin bead stimulation. Given that our recent study reported the roles of membrane-type 1 matrix metalloproteinase (MT1-MMP) in agrin-induced presynaptic differentiation, here we further showed that MT1-MMP mRNA is spatially enriched and locally translated at sites induced by agrin beads. Taken together, this study reveals an essential role for axonal MT1-MMP translation, on top of the well-recognized long-range transport of MT1-MMP proteins synthesized from neuronal cell bodies, in mediating agrin-induced presynaptic differentiation.

Autophagy in axonal and presynaptic development.

The study of autophagy in the nervous system has predominantly centered on degeneration. Evidence is now cementing crucial roles for autophagy in neuronal development and growth, especially in axonal and presynaptic compartments. A picture is emerging that autophagy typically promotes the growth of axons and reduces presynaptic stability. Nonetheless, these are not rigid principles, and it remains unclear why autophagy does not always display these relationships during axonal and presynaptic development. Recent progress has identified mechanisms underlying spatiotemporal control of autophagy in neuronsand begun to unravel how autophagy is integrated with other cellular processes, such as proteasomal degradation and axon guidance. Ultimately, understanding how autophagy is regulated and its role in the developing nervous system is key to comprehending how the nervous system assembles its stereotyped yet plastic configuration. It is also likely to inform how we think about neurodevelopmental disorders and neurodegenerative diseases.

Presynaptic dysfunction in CASK-related neurodevelopmental disorders

CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory–inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.

Neuronal MT1-MMP mediates ECM clearance and Lrp4 cleavage for agrin deposition and signaling in presynaptic development.

Agrin is a crucial factor that induces postsynaptic differentiation at neuromuscular junctions (NMJs), but how secreted agrin is locally deposited in the context of extracellular matrix (ECM) environment and its function in presynaptic differentiation remain largely unclear. Here, we report that the proteolytic activity of neuronal membrane-type 1 matrix metalloproteinase (MT1-MMP; also known as MMP14) facilitates agrin deposition and signaling during presynaptic development at NMJs. Firstly, agrin deposition along axons exhibits a time-dependent increase in cultured neurons that requires MMP-mediated focal ECM degradation. Next, local agrin stimulation induces the clustering of mitochondria and synaptic vesicles, two well-known presynaptic markers, and regulates vesicular trafficking and surface insertion of MT1-MMP. MMP inhibitor or MT1-MMP knockdown suppresses agrin-induced presynaptic differentiation, which can be rescued by treatment with the ectodomain of low-density lipoprotein receptor-related protein 4 (Lrp4). Finally, neuronal MT1-MMP knockdown inhibits agrin deposition and nerve-induced acetylcholine receptor clustering in nerve-muscle co-cultures and affects synaptic structures at Xenopus NMJs in vivo Collectively, our results demonstrate a previously unappreciated role of agrin, as well as dual functions of neuronal MT1-MMP proteolytic activity in orchestrating agrin deposition and signaling, in presynaptic development.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS.

CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

Target-dependent retrograde signaling mediates synaptic plasticity at the Drosophila neuromuscular junction.

Neurons that innervate multiple targets often establish synapses with target-specific strengths, and local forms of synaptic plasticity. We have examined the molecular-genetic mechanisms that allow a single Drosophila motoneuron, the ventral Common Exciter (vCE), to establish connections with target-specific properties at its various synaptic partners. By driving transgenes in a subset of vCE's targets, we found that individual target cells are able to independently control the properties of vCE's innervating branch and synapses. This is achieved by means of a trans-synaptic growth factor secreted by the target cell. At the larval neuromuscular junction, postsynaptic glutamate receptor activity stimulates the release of the BMP4/5/6 homolog Glass bottom boat (Gbb). As larvae mature and motoneuron terminals grow, Gbb activates the R-Smad transcriptional regulator phosphorylated Mad (pMad) to facilitate presynaptic development. We found that manipulations affecting glutamate receptors or Gbb within subsets of target muscles led to local effects either specific to the manipulated muscle or by a limited gradient within the presynaptic branches. While presynaptic development depends on pMad transcriptional activity within the motoneuron nucleus, we find that the Gbb growth factor may also act locally within presynaptic terminals. Local Gbb signaling and presynaptic pMad accumulation within boutons may therefore participate in a "synaptic tagging" mechanism, to influence synaptic growth and plasticity in Drosophila.

Drosophila Syd-1 Has RhoGAP Activity That Is Required for Presynaptic Clustering of Bruchpilot/ELKS but Not Neurexin-1.

Syd-1 proteins are required for presynaptic development in worm, fly, and mouse. Syd-1 proteins in all three species contain a Rho GTPase activating protein (GAP)-like domain of unclear significance: invertebrate Syd-1s are thought to lack GAP activity, and mouse mSYD1A has GAP activity that is thought to be dispensable for its function. Here, we show that Drosophila melanogaster Syd-1 can interact with all six fly Rhos and has GAP activity toward Rac1 and Cdc42. During development, fly Syd-1 clusters multiple presynaptic proteins at the neuromuscular junction (NMJ), including the cell adhesion molecule Neurexin (Nrx-1) and the active zone (AZ) component Bruchpilot (Brp), both of which Syd-1 binds directly. We show that a mutant form of Syd-1 that specifically lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally. We provide evidence that Syd-1 participates with Rac1 in two separate functions: (1) together with the Rac guanine exchange factor (RacGEF) Trio, GAP-active Syd-1 is required to regulate the nucleotide-bound state of Rac1, thereby promoting Brp clustering; and (2) Syd-1, independent of its GAP activity, is required for the recruitment of Nrx-1 to boutons, including the recruitment of Nrx-1 that is promoted by GTP-bound Rac1. We conclude that, contrary to current models, the GAP domain of fly Syd-1 is active and required for presynaptic development; we suggest that the same may be true of vertebrate Syd-1 proteins. In addition, our data provide new molecular insight into the ability of Rac1 to promote presynaptic development.

Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies.

We have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.

Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.

Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease.

On the Role of Glutamate in Presynaptic Development: Possible Contributions of Presynaptic NMDA Receptors

Proper formation and maturation of synapses during development is a crucial step in building the functional neural circuits that underlie perception and behavior. It is well established that experience modifies circuit development. Therefore, understanding how synapse formation is controlled by synaptic activity is a key question in neuroscience. In this review, we focus on the regulation of excitatory presynaptic terminal development by glutamate, the predominant excitatory neurotransmitter in the brain. We discuss the evidence that NMDA receptor activation mediates these effects of glutamate and present the hypothesis that local activation of presynaptic NMDA receptors (preNMDARs) contributes to glutamate-dependent control of presynaptic development. Abnormal glutamate signaling and aberrant synapse development are both thought to contribute to the pathogenesis of a variety of neurodevelopmental disorders, including autism spectrum disorders, intellectual disability, epilepsy, anxiety, depression, and schizophrenia. Therefore, understanding how glutamate signaling and synapse development are linked is important for understanding the etiology of these diseases.

Presynaptic NMDA receptors - dynamics and distribution in developing axons in vitro and in vivo.

During cortical development, N-methyl-D-aspartate (NMDA) receptors (NMDARs) facilitate presynaptic terminal formation, enhance neurotransmitter release and are required in presynaptic neurons for spike-timing-dependent long-term depression (tLTD). However, the extent to which NMDARs are found within cortical presynaptic terminals has remained controversial, and the sub-synaptic localization and dynamics of axonal NMDARs are unknown. Here, using live confocal imaging and biochemical purification of presynaptic membranes, we provide strong evidence that NMDARs localize to presynaptic terminals in vitro and in vivo in a developmentally regulated manner. The NR1 and NR2B subunits (also known as GRIN1 and GRIN2B, respectively) were found within the active zone membrane, where they could respond to synaptic glutamate release. Surprisingly, NR1 also appeared in glutamatergic and GABAergic synaptic vesicles. During synaptogenesis, NR1 was mobile throughout axons - including growth cones and filopodia, structures that are involved in synaptogenesis. Upon synaptogenic contact, NMDA receptors were quickly recruited to terminals by neuroligin-1 signaling. Unlike dendrites, the trafficking and distribution of axonal NR1 were insensitive to activity changes, including NMDA exposure, local glutamate uncaging or action potential blockade. These results support the idea that presynaptic NMDARs play an early role in presynaptic development.

Cellular and molecular mechanisms underlying presynapse formation

Synapse formation is a highly regulated process that requires the coordination of many cell biological events. Decades of research have identified a long list of molecular components involved in assembling a functioning synapse. Yet how the various steps, from transporting synaptic components to adhering synaptic partners and assembling the synaptic structure, are regulated and precisely executed during development and maintenance is still unclear. With the improvement of imaging and molecular tools, recent work in vertebrate and invertebrate systems has provided important insight into various aspects of presynaptic development, maintenance, and trans-synaptic signals, thereby increasing our understanding of how extrinsic organizers and intracellular mechanisms contribute to presynapse formation.

Loss ofsyd-1from R7 Neurons Disrupts Two Distinct Phases of Presynaptic Development

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. We show that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. We find that the earlier defect coincides with a failure to localize synaptic vesicles, suggesting that it reflects a failure in presynaptic assembly. We then analyze the relationship between syd-1 and Liprin-α in R7s. We find that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: we show that Liprin-α promotes Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. We show that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. We therefore conclude that the two defects are caused by distinct molecular mechanisms. We find that Trio overexpression rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity.

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.

Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

BackgroundNeocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear.ResultsHere, we use confocal imaging to demonstrate that NMDA receptor (NMDAR) activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia.ConclusionsBased on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

NAB-1 instructs synapse assembly by linking adhesion molecules and F-actin to active zone proteins

During synaptogenesis, macromolecular protein complexes assemble at the pre- and postsynaptic membrane. Extensive literature identifies numerous transmembrane molecules sufficient to induce synapse formation and several intracellular scaffolding molecules responsible for assembling active zones and recruiting synaptic vesicles. However, little is known about the molecular mechanisms coupling membrane receptors to active zone molecules during development. Using C.elegans, we identify an F-actin network present at nascent presynaptic terminals required for presynaptic assembly. We unravel a sequence of events where specificity-determining adhesion molecules define the location of developing synapses and locally assemble F-actin. Next, an adaptor protein NAB-1/Neurabin binds to F-actin and recruits active zone proteins, SYD-1 and SYD-2/Liprin-α by forming a tripartite complex. NAB-1 localizes transiently to synapses during development and is required for presynaptic assembly. Together, we identify a role for the actin cytoskeleton during presynaptic development and characterize a molecular pathway where NAB-1 links synaptic partner recognition to active zone assembly.

The role of sensory experience in presynaptic development is cortical area specific.

The postsynaptic response to a stimulus is dependent on the history of previous activity at that synapse. This short-term plasticity (STP) is a key determinant of neural network function. During postnatal development, many excitatory intracortical synapses switch from strong depression during early postnatal life, to weaker depression and in some cases facilitation in adulthood. However, it is not known whether this developmental switch is an innate feature of synaptic maturation, or whether it requires activity. We investigated this question in the barrel and visual cortex, two widely studied models of experience-dependent plasticity. We have previously defined the time course over which presynaptic development occurs in these two cortical areas, enabling us to make the first direct comparison of the role of sensory experience during synaptic development. We found that maturation of STP in visual cortex was unaffected by dark rearing from before eye opening. In marked contrast, total whisker deprivation completely blocked the developmental decrease in presynaptic release probability (Pr), and the concomitant increase in paired pulse ratio (PPR), which occur in barrel cortex during the third and fourth postnatal weeks. However, the developmental increase in the steady state response to a train of stimuli was unaffected by whisker deprivation. This supports a mechanistic link between Pr and the PPR, but dissociates Pr from the steady state amplitude during repetitive stimulation. Our findings indicate that sensory experience plays a greater role in presynaptic development at L4 to L2/3 excitatory synapses in the barrel cortex than in the visual cortex.