ß-Adrenergic Receptors/Epac Signaling Increases the Size of the Readily Releasable Pool of Synaptic Vesicles Required for Parallel Fiber LTP.

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca2+-induced increase in presynaptic cAMP that is mediated by Ca2+-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by ß-adrenergic receptors (ßARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that ßARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these ßARs occluded PF-PC LTP, while the ß1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2-/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the ßAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP.SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to ß-adrenergic receptor (ßAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by ßARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this ßAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of ßARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.

Bidirectional modulation of glutamatergic synaptic transmission by metabotropic glutamate type 7 receptors at Schaffer collateral-CA1 hippocampal synapses.

KEY POINTS: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu7 ) receptors that reduce Ca2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission. ABSTRACT: Neurotransmitter release is driven by Ca2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu7 ) receptors provoke a reduction in Ca2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.

CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.

Munc13- and SNAP25-dependent molecular bridges play a key role in synaptic vesicle priming.

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N-ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.

Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals.

The co-existence of presynaptic G protein coupled receptors, GPCRs, has received little attention, despite the fact that interplay between the signaling pathways activated by such receptors may affect the neurotransmitter release. Using immunocytochemistry and immuhistochemistry we show that mGlu7 and ß-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. mGlu7 receptors readily couple to pathways that inhibit glutamate release. We found that when mGlu7 receptors are also coupled to pathways that enhance glutamate release by prolonged exposure to agonist, and ß-adrenergic receptors are also activated, a cross-talk between their signaling pathways occurs that affect the overall release response. This interaction is the result of mGlu7 receptors inhibiting the adenylyl cyclase activated by ß adrenergic receptors. Thus, blocking Gi/o proteins with pertussis toxin provokes a further increase in release after receptor co-activation which is also observed after activating ß-adrenergic receptor signaling pathways downstream of adenylyl cyclase with the cAMP analog Sp8Br or 8pCPT-2-OMe-cAMP (a specific activator of the guanine nucleotide exchange protein directly activated by cAMP, EPAC). Co-activation of mGlu7 and ß-adrenergic receptors also enhances PLC-dependent accumulation of IP1 and the translocation of the active zone protein Munc13-1 to the membrane, indicating that release potentiation by these receptors involves the modulation of the release machinery.

Potentiation of mGlu7 receptor-mediated glutamate release at nerve terminals containing N and P/Q type Ca2+ channels.

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca(2+) channels, which are less tightly coupled to glutamate release than P/Q-type Ca(2+) channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca(2+) channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca(2+) channels and in nerve terminals containing P/Q-type Ca(2+) channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca(2+). By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Ca(v)2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca(2+). We conclude that release potentiation at 1.3 mM [Ca(2+)](e) occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca(2+)](e) represents the release from nerve terminals containing P/Q-type Ca(2+) channels. Although, mGlu7 receptor mediated potentiation is independent of Ca(2+) channel activity, as it was induced by the Ca(2+) ionophore ionomycin, release potentiation is influenced by the Ca(2+) channel type and/or the associated release machinery.

Inhibitors of diacylglycerol metabolism reduce time to the onset of glutamate release potentation by mGlu7 receptors.

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery.