Ricardo Martín

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABAB-activated GIRK channels in Purkinje cells

Activation of G protein‐gated inwardly‐rectifying K+ (GIRK or Kir3) channels by metabotropic gamma‐aminobutyric acid (B) (GABAB) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABAB receptors in cerebellar Purkinje cells at post‐ and pre‐synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co‐immunoprecipitation analysis demonstrated that GIRK subunits are co‐assembled with GABAB receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment‐dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post‐synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post‐synaptic association of GIRK subunits with GABAB receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre‐synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co‐localize with GABAB receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABAB receptors. The association of GIRK channels and GABAB receptors with excitatory synapses at both post‐ and pre‐synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.

The Metabotropic Glutamate Receptor mGlu7 Activates Phospholipase C, Translocates Munc-13-1 Protein, and Potentiates Glutamate Release at Cerebrocortical Nerve Terminals

At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca2+ influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca2+ ionophore ionomycin, suggesting a mechanism that is independent of Ca2+ channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function.

mGluR7 inhibits glutamate release through a PKC-independent decrease in the activity of P/Q-type Ca2+ channels and by diminishing cAMP in hippocampal nerve terminals

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine‐tuning of neurotransmitter release. Here we report that, in hippocampal nerve terminals from adult rats, the inhibition of glutamate release by the group III mGluR agonist L‐2‐amino‐4‐phosphonobutyrate (L‐AP4) is largely mediated by mGluR7. In this preparation, P/Q‐type Ca2+ channels support the major component of glutamate release while the remaining release is supported by N‐type Ca2+ channels. The release associated with P/Q channels was modulated by mGluR7, either in the presence of ω‐conotoxin‐GVIA or after decreasing the extracellular Ca2+ concentration [Ca2+]o to abolish the contribution of N‐type Ca2+ channels. Under these conditions, L‐AP4 (1 mm) reduced the evoked glutamate release by 35 ± 2%. This inhibition was largely prevented by pertussis toxin, but it was insensitive to inhibitors of protein kinase C (bisindolylmaleimide) and protein kinase A (H‐89). Furthermore, this inhibition was associated with a reduction in the Ca2+ influx mediated by P/Q channels in the absence of any detectable change in cAMP levels. However, L‐AP4 decreased the levels of cAMP in the presence of forskolin. The activation of this additional signalling pathway was very efficient in counteracting the facilitation of glutamate release induced by forskolin. Thus, mGluR7 mediates the inhibition of glutamate release at hippocampal nerve terminals primarily by inhibiting P/Q‐type Ca2+ channels, although augmenting the levels of cAMP reveals the ability of the receptor to decrease cAMP.

Non-additive potentiation of glutamate release by phorbol esters and metabotropic mGlu7 receptor in cerebrocortical nerve terminals.

J. Neurochem. (2011) 116, 476–485.

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors.

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca2+ channels and adenylyl cyclase. Through Ca2+ imaging and immunocytochemistry, we have recently shown that adenosine A1, GABAB and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca2+ channels by analysing this process in nerve terminals from mice lacking the alpha1B subunit (Cav 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca2+ channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABAB and adenosine A1. Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A1, GABAB and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca2+ channel type but rather, it is associated to functional mGlu7 receptors.

Partial compensation for N-type Ca2+ channel loss by P/Q-type Ca2+ channels underlines the differential release properties supported by these channels at cerebrocortical nerve terminals

N‐type and P/Q‐type Ca2+ channels support glutamate release at central synapses. To determine whether the glutamate release mediated by these channels exhibits distinct properties, we have isolated each release component in cerebrocortical nerve terminals from wild‐type mice by specifically blocking N‐type Ca2+ channels with ω‐conotoxin‐GVIA and P/Q‐type Ca2+ channels with ω‐agatoxin‐IVA. In addition, we have determined the release properties at terminals from mice lacking the α1B subunit of N‐type channels (Cav 2.2) to test the possibility that P/Q‐type channels can compensate for the loss of N‐type Ca2+ channels. We recently demonstrated that, while evoked glutamate release depends on P/Q‐ and N‐type channels in wild‐type nerve terminals, only P/Q‐type channels participate in these knockout mice. Moreover, in nerve terminals expressing solely P/Q‐type channels, metabotropic glutamate receptor 7 (mGluR7) fails to inhibit the evoked Ca2+ influx and glutamate release. Here, we show that the failure of mGluR7 to modulate evoked glutamate release is not due to a lack of receptors, as nerve terminals from mice lacking N‐type Ca2+ channels express mGluR7. Indeed, we show that other receptor responses, such as the inhibition of forskolin‐induced release, are preserved in these knockout mice. N‐type channels are more loosely coupled to release than P/Q‐type channels in nerve terminals from wild‐type mice, as reflected by the tighter coupling of release in knockout nerve terminals. We conclude that the glutamate release supported by N‐ and P/Q‐type channels exhibits distinct properties, and that P/Q‐type channels cannot fully compensate for the loss of N‐type channels.

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 β-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 β-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 β 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 β-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 β-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.

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

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.