Enhanced Release Probability without Changes in Synaptic Delay during Analogue-Digital Facilitation.

Neuronal timing with millisecond precision is critical for many brain functions such as sensory perception, learning and memory formation. At the level of the chemical synapse, the synaptic delay is determined by the presynaptic release probability (Pr) and the waveform of the presynaptic action potential (AP). For instance, paired-pulse facilitation or presynaptic long-term potentiation are associated with reductions in the synaptic delay, whereas paired-pulse depression or presynaptic long-term depression are associated with an increased synaptic delay. Parallelly, the AP broadening that results from the inactivation of voltage gated potassium (Kv) channels responsible for the repolarization phase of the AP delays the synaptic response, and the inactivation of sodium (Nav) channels by voltage reduces the synaptic latency. However, whether synaptic delay is modulated during depolarization-induced analogue-digital facilitation (d-ADF), a form of context-dependent synaptic facilitation induced by prolonged depolarization of the presynaptic neuron and mediated by the voltage-inactivation of presynaptic Kv1 channels, remains unclear. We show here that despite Pr being elevated during d-ADF at pyramidal L5-L5 cell synapses, the synaptic delay is surprisingly unchanged. This finding suggests that both Pr- and AP-dependent changes in synaptic delay compensate for each other during d-ADF. We conclude that, in contrast to other short- or long-term modulations of presynaptic release, synaptic timing is not affected during d-ADF because of the opposite interaction of Pr- and AP-dependent modulations of synaptic delay.

A Noelin-organized extracellular network of proteins required for constitutive and context-dependent anchoring of AMPA-receptors.

Information processing and storage in the brain rely on AMPA-receptors (AMPARs) and their context-dependent dynamics in synapses and extra-synaptic sites. We found that distribution and dynamics of AMPARs in the plasma membrane are controlled by Noelins, a three-member family of conserved secreted proteins expressed throughout the brain in a cell-type-specific manner. Noelin tetramers tightly assemble with the extracellular domains of AMPARs and interconnect them in a network-like configuration with a variety of secreted and membrane-anchored proteins including Neurexin1, Neuritin1, and Seizure 6-like. Knock out of Noelins1-3 profoundly reduced AMPARs in synapses onto excitatory and inhibitory (inter)neurons, decreased their density and clustering in dendrites, and abolished activity-dependent synaptic plasticity. Our results uncover an endogenous mechanism for extracellular anchoring of AMPARs and establish Noelin-organized networks as versatile determinants of constitutive and context-dependent neurotransmission.

An ER Assembly Line of AMPA-Receptors Controls Excitatory Neurotransmission and Its Plasticity.

Excitatory neurotransmission and its activity-dependent plasticity are largely determined by AMPA-receptors (AMPARs), ion channel complexes whose cell physiology is encoded by their interactome. Here, we delineate the assembly of AMPARs in the endoplasmic reticulum (ER) of native neurons as multi-state production line controlled by distinct interactome constituents: ABHD6 together with porcupine stabilizes pore-forming GluA monomers, and the intellectual-disability-related FRRS1l-CPT1c complexes promote GluA oligomerization and co-assembly of GluA tetramers with cornichon and transmembrane AMPA-regulatory proteins (TARP) to render receptor channels ready for ER exit. Disruption of the assembly line by FRRS1l deletion largely reduces AMPARs in the plasma membrane, impairs synapse formation, and abolishes activity-dependent synaptic plasticity, while FRRS1l overexpression has the opposite effect. As a consequence, FRSS1l knockout mice display severe deficits in learning tasks and behavior. Our results provide mechanistic insight into the stepwise biogenesis of AMPARs in native ER membranes and establish FRRS1l as a powerful regulator of synaptic signaling and plasticity.

AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability.

AMPA-type glutamate receptors (AMPARs), key elements in excitatory neurotransmission in the brain, are macromolecular complexes whose properties and cellular functions are determined by the co-assembled constituents of their proteome. Here we identify AMPAR complexes that transiently form in the endoplasmic reticulum (ER) and lack the core-subunits typical for AMPARs in the plasma membrane. Central components of these ER AMPARs are the proteome constituents FRRS1l (C9orf4) and CPT1c that specifically and cooperatively bind to the pore-forming GluA1-4 proteins of AMPARs. Bi-allelic mutations in the human FRRS1L gene are shown to cause severe intellectual disability with cognitive impairment, speech delay and epileptic activity. Virus-directed deletion or overexpression of FRRS1l strongly impact synaptic transmission in adult rat brain by decreasing or increasing the number of AMPARs in synapses and extra-synaptic sites. Our results provide insight into the early biogenesis of AMPARs and demonstrate its pronounced impact on synaptic transmission and brain function.

Regional diversity and developmental dynamics of the AMPA-receptor proteome in the mammalian brain.

Native AMPA receptors (AMPARs) in the mammalian brain are macromolecular complexes whose functional characteristics vary across the different brain regions and change during postnatal development or in response to neuronal activity. The structural and functional properties of the AMPARs are determined by their proteome, the ensemble of their protein building blocks. Here we use high-resolution quantitative mass spectrometry to analyze the entire pool of AMPARs affinity-isolated from distinct brain regions, selected sets of neurons, and whole brains at distinct stages of postnatal development. These analyses show that the AMPAR proteome is dynamic in both space and time: AMPARs exhibit profound region specificity in their architecture and the constituents building their core and periphery. Likewise, AMPARs exchange many of their building blocks during postnatal development. These results provide a unique resource and detailed contextual data sets for the analysis of native AMPAR complexes and their role in excitatory neurotransmission.

Cornichon2 dictates the time course of excitatory transmission at individual hippocampal synapses.

Cornichon2 (CNIH2), an integral component of AMPA receptor (AMPAR) complexes in the mammalian brain, slows deactivation and desensitization of heterologously reconstituted receptor channels. Its significance in neuronal signal transduction, however, has remained elusive. Here we show by paired recordings that CNIH2-containing AMPARs dictate the slow decay of excitatory postsynaptic currents (EPSCs) elicited in hilar mossy cells of the hippocampus by single action potentials in mossy fiber boutons (MFB). Selective knockdown of CNIH2 markedly accelerated EPSCs in individual MFB-mossy cell synapses without altering the EPSC amplitude. In contrast, the rapidly decaying EPSCs in synapses between MFBs and aspiny interneurons that lack expression of CNIH2 were unaffected by the protein knockdown but were slowed by virus-directed expression of CNIH2. These results identify CNIH2 as the molecular distinction between slow and fast EPSC phenotypes and show that CNIH2 influences the time course and, hence, the efficacy of excitatory synaptic transmission.

Presynaptic action potential waveform determines cortical synaptic latency.

Synaptic latency at cortical synapses is determined by the presynaptic release probability (Pr). Short- and long-term presynaptic plasticity is associated with modulation of synaptic delay. We show here that the duration and amplitude of the presynaptic action potential also determine synaptic latency at neocortical and hippocampal excitatory synapses. Blockade of voltage-gated potassium (Kv) channels with 4-aminopyridine or dendrotoxin-I, but not tetraethylammonium, induced a 1­2 ms shift in latency at excitatory synaptic connections formed by pairs of neocortical pyramidal neurons. 4-Aminopyridine or dendrotoxin-I, but not tetraethylammonium, increased the duration of the action potential recorded in the axon, suggesting that presynaptic spike duration is controlled by axonal Kv1 potassium channels. Spike width-dependent changes in latency have been identified at the mossy fibre­CA3 cell synapses and contribute to stabilization of synaptic timing during repetitive stimulation. The effects of presynaptic spike amplitude on synaptic latency were also examined. Decreasing the amplitude of the presynaptic action potential with 15­30 nm TTX reduced synaptic latency by ∼0.5 ms. The regulation of synaptic timing by potassium and sodium channel blockers could not be attributed to modulation of axonal conduction. Rather, these effects are compatible with modifications of the kinetics of the presynaptic calcium current. We conclude that synaptic latency at cortical neurons is not constant but dynamically regulated by presynaptic action potential waveform.

V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release.

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices.

Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.

Release-dependent variations in synaptic latency: a putative code for short- and long-term synaptic dynamics.

In the cortex, synaptic latencies display small variations ( approximately 1-2 ms) that are generally considered to be negligible. We show here that the synaptic latency at monosynaptically connected pairs of L5 and CA3 pyramidal neurons is determined by the presynaptic release probability (Pr): synaptic latency being inversely correlated with the amplitude of the postsynaptic current and sensitive to manipulations of Pr. Changes in synaptic latency were also observed when Pr was physiologically regulated in short- and long-term synaptic plasticity. Paired-pulse depression and facilitation were respectively associated with increased and decreased synaptic latencies. Similarly, latencies were prolonged following induction of presynaptic LTD and reduced after LTP induction. We show using the dynamic-clamp technique that the observed covariation in latency and synaptic strength is a synergistic combination that significantly affects postsynaptic spiking. In conclusion, amplitude-related variation in latency represents a putative code for short- and long-term synaptic dynamics in cortical networks.

Metabotropic glutamate receptor subtype 1 regulates sodium currents in rat neocortical pyramidal neurons.

Brain sodium channels (NaChs) are regulated by various neurotransmitters such as acetylcholine, serotonin and dopamine. However, it is not known whether NaCh activity is regulated by glutamate, the principal brain neurotransmitter. We show here that activation of metabotropic glutamate receptor (mGluR) subtype 1 regulates fast transient (I(NaT)) and persistent Na(+) currents (I(NaP)) in cortical pyramidal neurons. A selective agonist of group I mGluR, (S)-3,5-dihydroxyphenylglycine (DHPG), reduced action potential amplitude and decreased I(NaT). This reduction was blocked when DHPG was applied in the presence of selective mGluR1 antagonists. The DHPG-induced reduction of the current was accompanied by a shift of both the inactivation curve of I(NaT) and the activation curve of I(NaP). These effects were dependent on the activation of PKC. The respective role of these two regulatory processes on neuronal excitability was determined by simulating transient and persistent Na(+) conductances (G(NaT) and G(NaP)) with fast dynamic-clamp techniques. The facilitated activation of G(NaP) increased excitability near the threshold, but, when combined with the down-regulation of G(NaT), repetitive firing was strongly decreased. Consistent with this finding, the mGluR1 antagonist LY367385 increased neuronal excitability when glutamatergic synaptic activity was stimulated with high external K(+). We conclude that mGluR1-dependent regulation of Na(+) current depresses neuronal excitability, which thus might constitute a novel mechanism of homeostatic regulation acting during intense glutamatergic synaptic activity.