Inhibitory Synapse Dynamics: Coordinated Presynaptic and Postsynaptic Mobility and the Major Contribution of Recycled Vesicles to New Synapse Formation

Neurotransmission at different synapses is highly variable, and cell-adhesion molecules like α-neurexins (α-Nrxn) and their extracellular binding partners determine synapse function. Although α-Nrxn affect transmission at excitatory and inhibitory synapses, the contribution of neurexophilin-1 (Nxph1), an α-Nrxn ligand with restricted expression in subpopulations of inhibitory neurons, is unclear. To reveal its role, we investigated mice that either lack or overexpress Nxph1. We found that genetic deletion of Nxph1 impaired GABA B receptor (GABA B R)-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami, a region where Nxph1 is normally expressed at high levels. To test the conclusion that Nxph1 supports presynaptic GABA B R, we expressed Nxph1 ectopically at excitatory terminals in the neocortex, which normally do not contain this molecule but can be modulated by GABA B R. We generated Nxph1-GFP transgenic mice under control of the Thy1.2 promoter and observed a reduced short-term facilitation at these excitatory synapses, representing an inverse phenotype to the knockout. Consistently, the diminished facilitation could be reversed by pharmacologically blocking GABA B R with CGP-55845. Moreover, a complete rescue was achieved by additional blocking of postsynaptic GABA A R with intracellular picrotoxin or gabazine, suggesting that Nxph1 is able to recruit or stabilize both presynaptic GABA B R and postsynaptic GABA A R. In support, immunoelectron microscopy validated the localization of ectopic Nxph1 at the synaptic cleft of excitatory synapses in transgenic mice and revealed an enrichment of GABA A R and GABA B R subunits compared with wild-type animals. Thus, our data propose that Nxph1 plays an instructive role in synaptic short-term plasticity and the configuration with GABA receptors. synaptic transmission | thalamus | autism | neuroligin | ultrastructure

Section: Discussionmentioning

confidence: 99%

Modulation of synaptic function through the α-neurexin–specific ligand neurexophilin-1

Born

Breuer

Wang

et al. 2014

“…Images were analyzed in ImageJ (rsb.info.nih.gov/ij) using a custom plugin (rsb.info.nih.gov/ij/plugins/time-series.html). Rabbit vGAT-Oyster 550 luminal domain antibody was used to identify GABAergic neurons (23). Values are shown as mean ± SEM.…”

Section: Methodsmentioning

confidence: 99%

“…If isoflurane inhibits exocytosis purely by inhibiting AP-driven Ca 2+ entry, then the same quantitative relationship should hold. The paired isoflurane data fell on the control curve, which indicates that the effect of isoflurane on SV exocytosis can be attributed to reductions in [Ca 2+ ] i without significant contributing effects To compare the effects of isoflurane on SV exocytosis from glutamatergic or GABAergic neurons separately, we used livecell imaging of GABAergic boutons identified by vGAT-Oyster 550 immunolabeling (23) (Fig. 6A).…”

Section: Significancementioning

confidence: 99%

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Baumgart

Zhou

Hara

et al. 2015

108

Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca 2+ influx without significantly altering the Ca 2+ sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca 2+ ] i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca 2+ ([Ca 2+ ] e ). Lowering external Ca 2+ to match the isoflurane-induced reduction in Ca 2+ entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca 2+ entry without significant direct effects on Ca 2+ -exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca 2+ influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. GCaMP3 | pHlourin | mechanisms of anesthesia | live cell imaging | presynaptic T he molecular and cellular mechanisms of anesthetic-induced amnesia, unconsciousness and immobilization are incompletely understood, particularly for the modern halogenated ether derivatives like isoflurane. General anesthetics, which are essential to both medical practice and experimental neuroscience, have potent and selective effects on neurotransmission (1), including both presynaptic actions (reduced neurotransmitter release) and postsynaptic actions (modulation of receptor function). These effects contribute to anesthetic-induced reductions in neuronal interactions, which are critical to information processing and consciousness (2-4). Knowledge of the fundamental synaptic effects of anesthetics is therefore essential to a molecular and physiological understanding of anesthetic mechanisms, and to development of more selective and safer anesthetics.Although postsynaptic electrophysiological effects of anesthetics can be assessed directly using whole cell recordings and heterologous expression of putative molecular targets, their presynaptic actions have been difficult to resolve by conventional approaches that do not clearly discriminate between presynaptic and postsynaptic contributions. Direct evidence for presynaptic effects of volatile anesthetics includes selective inhibition of glutamate release from isolated nerve terminals (5, 6) and of synaptic vesicle (SV) exocytosis in intact hippocampal neurons (7). However, it remains controversial whether these effects involve direct inhibition of SV exocytosis itself or of upstrea...

“…Gephyrin forms multimeric complexes that allow the anchoring of GABA A Rs (6) via molecular mechanisms that include phosphorylation and interactions with the guanine-nucleotide exchange factor collybistin (7)(8)(9)(10)(11)(12). In addition to changes in inhibitory strength, more recent in vivo experiments revealed that inhibitory synapses are also dynamic structures that can be formed and eliminated in response to sensory experience (13)(14)(15). The mechanisms implicated in the coordinated regulation of excitatory and inhibitory plasticity remain, however, poorly understood.…”

mentioning

confidence: 99%

Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation

Flores

Nikonenko

Méndez

et al. 2014

113

Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-daspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABA A receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca 2+ /calmodulindependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phosphomutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKIIdependent phosphorylation site on gephyrin as critically important for this process.inhibition | gabaergic synapse | plasticity | hippocampus | CaMKII S everal activity-dependent plasticity and homeostatic mechanisms (1, 2) contribute to regulate synaptic strength at excitatory synapses. Similar mechanisms are also expected to finely tune the level of inhibition in response to activity in individual neurons, but the mechanisms remain poorly understood. Different forms of plasticity at GABAergic synapses have been reported based on either presynaptic or postsynaptic mechanisms (3, 4). Similar to receptors at excitatory synapses, GABA A receptors (GABA A Rs), which mediate the fast component of inhibitory transmission, display complex trafficking mechanisms that affect the surface localization and diffusion of receptors (5). The distribution and clustering of GABA A Rs at synapses is tightly regulated through interactions with the scaffolding protein gephyrin, one of the main structural constituent of inhibitory postsynaptic densities. Gephyrin forms multimeric complexes that allow the anchoring of GABA A Rs (6) via molecular mechanisms that include phosphorylation and interactions with the guanine-nucleotide exchange factor collybistin (7-12). In addition to changes in inhibitory strength, more recent in vivo experiments revealed that inhibitory synapses are also dynamic structures that can be formed and eliminated in response to sensory experience (13-15). The mechanisms implicated in the coordinated regulation of excitatory an...