Development Of Presynaptic Terminals Research Articles

GTPase-activating protein TBC1D5 coordinates with retromer to constrain synaptic growth by inhibiting BMP signaling

Formation and plasticity of neural circuits rely on precise regulation of synaptic growth. At Drosophila neuromuscular junction (NMJ), Bone Morphogenetic Protein (BMP) signaling is critical for many aspects of synapse formation and function. The evolutionarily conserved retromer complex and its associated GTPase-activating protein TBC1D5 are critical regulators of membrane trafficking and cellular signaling. However, their functions in regulating the formation of NMJ are less understood. Here, we report that TBC1D5 is required for inhibition of synaptic growth, and loss of TBC1D5 leads to abnormal presynaptic terminal development, including excessive satellite boutons and branch formation. Ultrastructure analysis reveals that the size of synaptic vesicles and the density of subsynaptic reticulum are increased in TBC1D5 mutant boutons. Disruption of interactions of TBC1D5 with Rab7 and retromer phenocopies the loss of TBC1D5. Unexpectedly, we find that TBC1D5 is functionally linked to Rab6, in addition to Rab7, to regulate synaptic growth. Mechanistically, we show that loss of TBC1D5 leads to upregulated BMP signaling by increasing the protein level of BMP type II receptor Wishful Thinking (Wit) at NMJ. Overall, our data establish that TBC1D5 in coordination with retromer constrains synaptic growth by regulating Rab7 activity, which negatively regulates BMP signaling through inhibiting Wit level.

Pathogenic GRM7 Mutations Associated with Neurodevelopmental Disorders Impair Axon Outgrowth and Presynaptic Terminal Development.

Metabotropic glutamate receptor 7 (mGlu7) is an inhibitory heterotrimeric G-protein-coupled receptor that modulates neurotransmitter release and synaptic plasticity at presynaptic terminals in the mammalian central nervous system. Recent studies have shown that rare mutations in glutamate receptors and synaptic scaffold proteins are associated with neurodevelopmental disorders (NDDs). However, the role of presynaptic mGlu7 in the pathogenesis of NDDs remains largely unknown. Recent whole-exome sequencing (WES) studies in families with NDDs have revealed that several missense mutations (c.1865G>A:p.R622Q; c.461T>C:p.I154T; c.1972C>T:p.R658W and c.2024C>A:p.T675K) or a nonsense mutation (c.1757G>A:p.W586X) in the GRM7 gene may be linked to NDDs. In the present study, we investigated the mechanistic links between GRM7 point mutations and NDD pathology. We find that the pathogenic GRM7 I154T and R658W/T675K mutations lead to the degradation of the mGlu7 protein. In particular, the GRM7 R658W/T675K mutation results in a lack of surface mGlu7 expression in heterologous cells and cultured neurons isolated from male and female rat embryos. We demonstrate that the expression of mGlu7 variants or exposure to mGlu7 antagonists impairs axon outgrowth through the mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling pathway during early neuronal development, which subsequently leads to a decrease in the number of presynaptic terminals in mature neurons. Treatment with an mGlu7 agonist restores the pathologic phenotypes caused by mGlu7 I154T but not by mGlu7 R658W/T675K because of its lack of neuronal surface expression. These findings provide evidence that stable neuronal surface expression of mGlu7 is essential for neural development and that mGlu7 is a promising therapeutic target for NDDs.SIGNIFICANCE STATEMENT Neurodevelopmental disorders (NDDs) affect brain development and function by multiple etiologies. Metabotropic glutamate receptor 7 (mGlu7) is a receptor that controls excitatory neurotransmission and synaptic plasticity. Since accumulating evidence indicates that the GRM7 gene locus is associated with NDD risk, we analyzed the functional effects of human GRM7 variants identified in patients with NDDs. We demonstrate that stable neuronal surface expression of mGlu7 is essential for axon outgrowth and presynaptic terminal development in neurons. We found that mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling and subsequent cytoskeletal dynamics are defective because of the degradation of mGlu7 variants. Finally, we show that the defects caused by mGlu7 I154T can be reversed by agonists, providing the rationale for proposing mGlu7 as a potential therapeutic target for NDDs.

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.

Spatio-temporal dynamics of neocortical presynaptic terminal development using multi-photon imaging of the corpus callosum in vivo

Within the developing central nervous system, the dynamics of synapse formation and elimination are insufficiently understood. It is ideal to study these processes in vivo, where neurons form synapses within appropriate behavioral and anatomical contexts. In vivo analysis is particularly important for long-range connections, since their development cannot be adequately studied in vitro. The corpus callosum (CC) represents a clinically-relevant long-range connection since several neurodevelopmental diseases involve CC defects. Here, we present a novel strategy for in vivo longitudinal and rapid time-lapse imaging of CC presynaptic terminal development. In postnatal mice, the time-course of CC presynaptic terminal formation and elimination was highly variable between axons or groups of axons. Young presynaptic terminals were remarkably dynamic – moving, dividing to generate more boutons, and merging to consolidate small terminals into large boutons. As synaptic networks matured, presynaptic mobility decreased. These rapid dynamics may be important for establishing initial synaptic contacts with postsynaptic partners, refining connectivity patterns or modifying synapse strength during development. Ultimately, this in vivo imaging approach will facilitate investigation of synapse development in other long-range connections and neurodevelopmental disease models.

Ablation of the presynaptic organizer Bassoon in excitatory neurons retards dentate gyrus maturation and enhances learning performance

Bassoon is a large scaffolding protein of the presynaptic active zone involved in the development of presynaptic terminals and in the regulation of neurotransmitter release at both excitatory and inhibitory brain synapses. Mice with constitutive ablation of the Bassoon (Bsn) gene display impaired presynaptic function, show sensory deficits and develop severe seizures. To specifically study the role of Bassoon at excitatory forebrain synapses and its relevance for control of behavior, we generated conditional knockout (Bsn cKO) mice by gene ablation through an Emx1 promoter-driven Cre recombinase. In these animals, we confirm selective loss of Bassoon from glutamatergic neurons of the forebrain. Behavioral assessment revealed that, in comparison to wild-type littermates, Bsn cKO mice display selectively enhanced contextual fear memory and increased novelty preference in a spatial discrimination/pattern separation task. These changes are accompanied by an augmentation of baseline synaptic transmission at medial perforant path to dentate gyrus (DG) synapses, as indicated by increased ratios of field excitatory postsynaptic potential slope to fiber volley amplitude. At the structural level, an increased complexity of apical dendrites of DG granule cells can be detected in Bsn cKO mice. In addition, alterations in the expression of cellular maturation markers and a lack of age-dependent decrease in excitability between juvenile and adult Bsn cKO mice are observed. Our data suggest that expression of Bassoon in excitatory forebrain neurons is required for the normal maturation of the DG and important for spatial and contextual memory.

Astrocyte lipid metabolism is critical for synapse development and function in vivo.

The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682.

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.

Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

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.

Area-specific effects of brain-derived neurotrophic factor (BDNF) genetic ablation on various neuronal subtypes of the mouse brain

The effects of brain-derived neurotrophic factor (BDNF) on the development of presynaptic terminals and of neuronal subtypes in various brain areas were studied in BDNF-knockout (BDNF−/−) mice at postnatal days 15–17. Western analysis revealed no changes in the overall amount of a variety of synaptic proteins in BDNF−/− mice as compared to wild type mice. In addition, the complex between the vesicular proteins, synaptophysin and synaptobrevin, as well as their respective homodimers were unaltered. Moreover, no changes in the density of neurons were found in, e.g., the CA3 region of the hippocampus and the nucleus nervi facialis of BDNF−/− mice. However, cholinergic cells were reduced by 20% in the medial septum of BDNF−/− mice associated with a decrease in the activity of choline acetyltransferase and protein levels of nerve growth factor in the hippocampus by 16% and 44%, respectively. In the striatum, however, the total number of cholinergic cells were comparable in both groups, although the activity of choline acetyltransferase was decreased by 46%. In GABAergic interneurons, the expression of neuropeptides in various brain areas was differentially affected by BDNF deletion as revealed by immunohistochemistry. In the hippocampus and cortex of BDNF−/− mice, the density of neuropeptide Y-, somatostatin-, and parvalbumin-immunoreactive cells was drastically reduced, whereas the density of calretinin-positive cells was increased. The extent of these changes in neuropeptide-containing cells varied among hippocampal subregions. In the striatum, only the density of parvalbumin-immunoreactive cells was decreased by approximately 45%. In conclusion, BDNF deficiency is accompanied by a differential dysregulation in the expression of neuropeptides and calcium-binding proteins in otherwise intact GABAergic and glutamatergic neurons in a region-specific manner.

Influence of the target on distribution and functioning of the varicosities of Helix pomatia metacerebral cell C1 in dissociated cell culture

The serotonergic metacerebral giant cell (C1) of Helix pomatia was isolated with its bifurcate axon and plated in culture under five conditions: (i) with no target; (ii) with the appropriate target B2 near the stump of the bigger branch (CBC); (iii) with B2 near the stump of the smaller branch (CC); (iv) with a wrong target (C3) near the stump of the CBC branch and (v) with B2 and C3 positioned near the CBC and CC stump, respectively. The counting of anti-serotonin antibody-labelled varicosities of the C1 neuron showed that the presence of the appropriate target in either axonal domain both down-regulated the number of varicosities of the contralateral neuritic field, and increased their average size, whereas the wrong target induced an overall reduction of the number of C1 neuron varicosities, and inhibited the evoked transmitter release. The action potential-evoked calcium concentration increase in the neuritic terminals of the C1 neuron cultured alone, or in presence of the appropriate target, reached a value significantly higher than that reached in presence of the wrong target. These results provide evidence that the postsynaptic neuron regulates both morphological and functional development of presynaptic terminals.

GAP-43 distribution is correlated with development of growth cones and presynaptic terminals.

GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.

Postnatal development of presynaptic terminals in the gigantocellular tegmental field (FTG) of the rat

Presynaptic boutons of the gigantocellular tegmental field (FTG) of the brain stem reticular formation were analyzed by stereological techniques for changes in numerical and volumetric density during early postnatal development. Electron microscopic analysis revealed that presynaptic boutons increased in numerical density and volumetric fraction during the first 15 postnatal days. By day 15, the presynaptic boutons had reached the numerical density and volumetric fraction seen in the adult tissue. Myelination was not evident until day 10 and by day 30 contributed over 45% to the total volume of the FTG tissue. Presynaptic boutons were differentiated into those with spherical vesicles and those with predominantly polymorphic (i.e. oblong) vesicles. Both types of presynaptic boutons were present at day 1, but over the next 30 days the percentage of boutons with polymorphic vesicles increased to over 40%. These results revealed that the FTG region of the rat demonstrates its synaptogenic period during the earliest postnatal days. The differentiation of the synapse into two types within the FTG region suggests a morphological basis for alterations in function during early maturation.

The effect of spinal cord transection on the development of cholinergic and adrenergic sympathetic neurons

Summary The role of central pathways in the maturation of cholinergic and adrenergic sympathetic neurons was studied in the neonatal rat. The ontogeny of the sixth lumbar (L-6) ganglion was examined after midthoracic spinal transection in 0–11-day-old animals. Initial experiments defined the normal development of presynaptic choline acetyltransferase (ChAc) and postsynaptic tyrosine hydroxylase (T-OH) activities in L-6 ganglia. The pattern of maturation was similar to that previously described for the superior cervical ganglion, but was delayed, suggesting a rostrocaudal gradient of sympathetic development. Spinal transection prevented the normal development of presynaptic terminals and postsynaptic neurons in the L-6 ganglion. ChAc activity failed to increase during the first postoperative week, and at 2 months of age was less than 50% of normal. Postsynaptic T-OH and DOPA decarboxylase activities also failed to increase normally after spinal transection. These abnormalities were not attributable to direct damage to presynaptic intermediolateral column neurons in the spinal cord, since histological examination did not reveal neuronal dropout, neuronophagia or gliosis. These observations suggest that spinal pathways regulate the maturation of proximate presynaptic cholinergic neurons and second-order postsynaptic adrenergic neurons in the autonomic system.

Stereology: applications to biomedicalresearch.

Stereology: applications to biomedicalresearch.

Somatic sensory properties of bulbar reticular neurons.

Somatic sensory properties of bulbar reticular neurons.

Convergence and interaction of afferent impulses on single units of reticular formation.

Convergence and interaction of afferent impulses on single units of reticular formation.