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The differential formation of excitatory (glutamate-mediated) and inhibitory (GABA-mediated) synapses is a critical step for the proper functioning of the brain. An imbalance in these synapses may lead to various neurological disorders such as autism, schizophrenia, Tourette's syndrome and epilepsy (1-4). Synapses are formed through communication between the appropriate synaptic partners (5-8). However, the molecular mechanisms that mediate the formation of specific synaptic types are not known. Here we show that two members of the fibroblast growth factor (FGF) family, FGF22 and FGF7, promote the organization of excitatory and inhibitory presynaptic terminals, respectively, as target-derived presynaptic organizers. FGF22 and FGF7 are expressed by CA3 pyramidal neurons in the hippocampus. The differentiation of excitatory or inhibitory nerve terminals on dendrites of CA3 pyramidal neurons is specifically impaired in mutants lacking FGF22 or FGF7. These presynaptic defects are rescued by postsynaptic expression of the appropriate FGF. FGF22-deficient mice are resistant to epileptic seizures, and FGF7-deficient mice are prone to them, as expected from the alterations in excitatory/inhibitory balance. Differential effects of FGF22 and FGF7 involve both their distinct synaptic localizations and their use of different signalling pathways. These results demonstrate that specific FGFs act as target-derived presynaptic organizers and help to organize specific presynaptic terminals in the mammalian brain., Target (postsynaptic cell)-derived 'presynaptic organizers' promote the local differentiation of presynaptic axons into functional nerve terminals at sites of synaptic contact (5-8). This presynaptic differentiation includes the clustering of synaptic [...]

Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient ([nNOS.sup.-/-]), eNOS-deficient ([eNOS.sup.-/-]), and W/[W.sup.v] mice (lacking certain c-Kit-expressing ICC) with or without L-arginine or [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of [eNOS.sup.-/-] was higher and W/Wv was lower than wild-type and [nNOS.sup.-/-] mice. L-Arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, [nNOS.sup.-/-], and W/[W.sup.v] mice. However, neither L-arginine nor L-NAME affected basal tone in [eNOS.sup.-/-] mice. In vitro, L-arginine decreased basal tone in wild-type and [nNOS.sup.-/-] IAS but not in [eNOS.sup.-/-] or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, [eNOS.sup.-/-], and W/[W.sup.v] mice but absent in all [nNOS.sup.-/-] mice. EFS-induced IAS relaxation was also reduced in [nNOS.sup.-/-] IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR. basal internal anal sphincter tone; electrical field stimulation; internal anal sphincter; nitric oxide synthases; rectoanal inhibitory reflex

Neurons in the brain must establish a balanced network of excitatory and inhibitory synapses during development for the brain to function properly. An imbalance between these synapses underlies various neurological and psychiatric disorders. The formation of excitatory and inhibitory synapses requires precise molecular control. In the hippocampus, the structure crucial for learning and memory, fibroblast growth factor 22 (FGF22) and FGF7 specifically promote excitatory or inhibitory synapse formation, respectively. Knockout of either Fgf gene leads to excitatory-inhibitory imbalance in the mouse hippocampus and manifests in an altered susceptibility to epileptic seizures, underscoring the importance of FGF-dependent synapse formation. However, the receptors and signaling mechanisms by which FGF22 and FGF7 induce excitatory and inhibitory synapse differentiation are unknown. Here, we show that distinct sets of overlapping FGF receptors (FGFRs), FGFR2b and FGFR1b, mediate excitatory or inhibitory presynaptic differentiation in response to FGF22 and FGF7. Excitatory presynaptic differentiation is impaired in Fgfr2b and Fgfr1b mutant mice; however, inhibitory presynaptic defects are only found in Fgfr2b mutants. FGFR2b and FGFR1b are required for an excitatory presynaptic response to FGF22, whereas only FGFR2b is required for an inhibitory presynaptic response to FGF7. We further find that FGFRs are required in the presynaptic neuron to respond to FGF22, and that FRS2 and PI3K, but not PLCγ, mediate FGF22-dependent presynaptic differentiation. Our results reveal the specific receptors and signaling pathways that mediate FGF-dependent presynaptic differentiation, and thereby provide a mechanistic understanding of precise excitatory and inhibitory synapse formation in the mammalian brain. [ABSTRACT FROM AUTHOR]

Specific formation of excitatory and inhibitory synapses is crucial for proper functioning of the brain. Fibroblast growth factor 22 (FGF22) and FGF7 are postsynaptic-cell-derived presynaptic organizers necessary for excitatory and inhibitory presynaptic differentiation, respectively, in the hippocampus. For the establishment of specific synaptic networks, these FGFs must localize to appropriate synaptic locations -- FGF22 to excitatory and FGF7 to inhibitory postsynaptic sites. Here, we show that distinct motor and adaptor proteins contribute to intracellular microtubule transport of FGF22 and FGF7. Excitatory synaptic targeting of FGF22 requires the motor proteins KIF3A and KIF17 and the adaptor protein SAP102 (also known as DLG3). By contrast, inhibitory synaptic targeting of FGF7 requires the motor KIF5 and the adaptor gephyrin. Time-lapse imaging shows that FGF22 moves with SAP102, whereas FGF7 moves with gephyrin. These results reveal the basis of selective targeting of the excitatory and inhibitory presynaptic organizers that supports their different synaptogenic functions. Finally, we found that knockdown of SAP102 or PSD95 (also known as DLG4), which impairs the differentiation of excitatory synapses, alters FGF7 localization, suggesting that signals from excitatory synapses might regulate inhibitory synapse formation by controlling the distribution of the inhibitory presynaptic organizer. [ABSTRACT FROM AUTHOR]

Communication between pre- and postsynaptic cells promotes the initial organization of synaptic specializations, but subsequent synaptic stabilization requires transcriptional regulation. Here we show that fibroblast growth factor 22 (FGF22), a target-derived presynaptic organizer in the mouse hippocampus, induces the expression of insulin-like growth factor 2 (IGF2) for the stabilization of presynaptic terminals. FGF22 is released from CA3 pyramidal neurons and organizes the differentiation of excitatory nerve terminals formed onto them. Local application of FGF22 on the axons of dentate granule cells (DGCs), which are presynaptic to CA3 pyramidal neurons, induces IGF2 in the DGCs. IGF2, in turn, localizes to DGC presynaptic terminals and stabilizes them in an activity-dependent manner. IGF2 application rescues presynaptic defects of Fgf22-/- cultures. IGF2 is dispensable for the initial presynaptic differentiation, but is required for the following presynaptic stabilization both in vitro and in vivo. These results reveal a novel feedback signal that is critical for the activity-dependent stabilization of presynaptic terminals in the mammalian hippocampus. DOI: http://dx.doi.org/10.7554/eLife.12151.001

Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of signal regulatory protein-α (SIRPα) mediates presynaptic maturation. Two target-derived molecules, fibroblast growth factor 22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain in turn promotes the maturation of the presynaptic terminal. This process involves calcium/calmodulin-dependent protein kinase, matrix metalloproteinases and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has an impact on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses. [ABSTRACT FROM AUTHOR]

Abstract: We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3–3μM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03–0.3μM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca2+-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation. [Copyright &y& Elsevier]

How are excitatory (glutamatergic) and inhibitory (GABAergic) synapses established? Do distinct molecular mechanisms direct differentiation of glutamatergic and GABAergic synapses? In the brain, glutamatergic and GABAergic synaptic connections are formed with specific patterns. To establish such precise synaptic patterns, neurons pass through multiple checkpoints during development, such as cell fate determination, cell migration and localization, axonal guidance and target recognition, and synapse formation. Each stage offers key molecules for neurons/synapses to obtain glutamatergic or GABAergic specificity. Some mechanisms are based on intrinsic systems to induce gene expression, whereas others are based on extrinsic systems mediated by cell-cell or axon-target interactions. Recent studies indicate that specific formation of glutamatergic and GABAergic synapses is controlled by the expression or activation of different sets of molecules during development. In this review, the authors outline stages critical to the determination of glutamatergic or GABAergic specificity and describe molecules that act as determinants of specificities in each stage, with a particular focus on the synapse formation stage. They also discuss possible mechanisms underlying glutamatergic and GABAergic synapse formation via synapse-type specific synaptic organizers. [ABSTRACT FROM PUBLISHER]

Allosteric binding of calcium ion (Ca[sup 2+]) to inositol 1,4,5trisphosphate (IP[sub 3]) receptor (IP[sub 3]R) controls channel gat- ing within IP[sub 3]R. Here, we present biochemical and elec- tron microscopic evidence of Ca[sup 2+]-sensitive structural changes in the three-dimensional structure of type 1 IP[sub 3]R (IP[sub 3]R1). Low concentrations of Ca[sup 2+] and high concentrations of Sr[sup 2+] and Ba[sup 2+] were shown to be effective for the limited proteolysis of IP[sub 3]R1, but Mg[sup 2+] had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca[sup 2+] for conformational changes in IP[sub 3]R1 was <10[sup -7] M and that the IP[sub 3] scarcely affected the conformational states. The structure of IP[sub 3]R[sub 1] without Ca[sup 2+], as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "headto-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP[sub 3]R1 with Ca[sup 2+] also contains the four bridges connecting from the IP[sub 3]-binding domain toward the channel domain. These data suggest that the Ca[sup 2+]-specific conformational change structurally regulates the IP[sub 3]-triggered channel opening within IP[sub 3]R1. [ABSTRACT FROM AUTHOR]

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca2+ channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca2+ signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca2+ release by co-agonists, Ca2+, inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain. [Copyright &y& Elsevier]

The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is an IP 3 -gated ion channel that releases calcium ions (Ca 2+ ) from the endoplasmic reticulum. The IP 3 -binding sites in the large cytosolic domain are distant from the Ca 2+ conducting pore, and the allosteric mechanism of how IP 3 opens the Ca 2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP 3 R in the absence and presence of IP 3 Analyses of two distinct space group crystals uncovered an IP 3 -dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP 3 R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP 3 -controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP 3 binding to the Ca 2+ channel., Competing Interests: The authors declare no conflict of interest.

The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications., (Copyright © 2014. Published by Elsevier Inc.)

We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 μM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 μM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

At least three forms of signaling between pre- and postsynaptic partners are necessary during synapse formation. First, "targeting" signals instruct presynaptic axons to recognize and adhere to the correct portion of a postsynaptic target cell. Second, trans-synaptic "organizing" signals induce differentiation in their synaptic partner so that each side of the synapse is specialized for synaptic transmission. Finally, in many regions of the nervous system an excess of synapses are initially formed, therefore "refinement" signals must either stabilize or destabilize the synapse to reinforce or eliminate connections, respectively. Because of both their importance in processing visual information and their accessibility, retinogeniculate synapses have served as a model for studying synaptic development. Molecular signals that drive retinogeniculate "targeting" and "refinement" have been identified, however, little is known about what "organizing" cues are necessary for the differentiation of retinal axons into presynaptic terminals. To identify such "organizing" cues, we used microarray analysis to assess whether any target-derived "synaptic organizers" were enriched in the mouse dorsal lateral geniculate nucleus (dLGN) during retinogeniculate synapse formation. One candidate "organizing" molecule enriched in perinatal dLGN was FGF22, a secreted cue that induces the formation of excitatory nerve terminals in muscle, hippocampus, and cerebellum. In FGF22 knockout mice, the development of retinal terminals in dLGN was impaired. Thus, FGF22 is an important "organizing" cue for the timely development of retinogeniculate synapses.