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

Accepted Manuscript Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals José Javier Ferrero, Jorge Ramírez-Franco, Ricardo Martín, David Bartolomé-Martín, Magdalena Torres, José Sánchez-Prieto PII:

S0028-3908(15)30032-0

DOI:

10.1016/j.neuropharm.2015.07.025

Reference:

NP 5932

To appear in:

Neuropharmacology

Received Date: 6 May 2015 Revised Date:

21 July 2015

Accepted Date: 22 July 2015

Please cite this article as: Ferrero, J.J., Ramírez-Franco, J., Martín, R., Bartolomé-Martín, D., Torres, M., Sánchez-Prieto, J., Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals, Neuropharmacology (2015), doi: 10.1016/ j.neuropharm.2015.07.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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

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Running title: mGlu7 and β adrenergic receptors cross-talk José Javier Ferrero1,2, Jorge Ramírez-Franco1, Ricardo Martín1,2, David Bartolomé-Martín1,2, Magdalena Torres1,2 and José Sánchez-Prieto1,2

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.

Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Hospital Clínico San Carlos, C/Profesor Martín Lagos s/n, Madrid 28040, Spain

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Corresponding author: José Sánchez-Prieto, Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain. Phone: 34-1-394-3891; Fax: 34-

ABSTRACT

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91-394-3909; E-mail: [email protected]

The co-existence of different 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

neurotransmitter

release.

Using

immunocytochemistry

and

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immunohistochemistry, we show that mGlu7 and β-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. These mGlu7 receptors can readily couple to

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pathways that inhibit glutamate release. However, we found that when mGlu7 receptors couple to pathways that enhance glutamate release by prolonged agonist exposure, the ensuing activation of β-adrenergic receptors results in a cross-talk between the signaling pathways activated that affects the overall release response. This interaction is the result of mGlu7 receptors inhibiting the adenylyl cyclase activated by β adrenergic receptors and thus, blocking Gi/o proteins with pertussis toxin provokes a further increase in release after receptor coactivation. A similar effect is also observed after activating β-adrenergic receptor signaling pathways downstream of adenylyl cyclase with the cAMP analogue Sp8Br or 8pCPT-2-OMecAMP (a specific activator of the guanine nucleotide exchange protein directly activated by cAMP, EPAC). Co-activation of mGlu7 and β-adrenergic receptors also enhances the PLCdependent accumulation of IP1 and the translocation of the active zone protein Munc13-1 to the

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ACCEPTED MANUSCRIPT membrane, indicating that release potentiation by these receptors involves a modulation of the release machinery.

Highlights The mGlu7 and β-adrenergic receptors are co-expressed at cerebrocortical nerve terminals.

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There is cross-talk between the signaling pathways associated to mGlu7 and β-adrenergic receptors.

Cross-talk between mGlu7 and β-adrenergic receptor signaling decreases cAMP levels.

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Suppressing mGlu7 and β-adrenergic receptor cross-talk enhances glutamate release.

Key words

G protein coupled receptors (GPCRs); β-adrenergic receptors; metabotropic glutamate receptor

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7 (mGlu7 receptor); glutamate release; cAMP

Abbreviations

β-AR, β-adrenergic receptor; mGlu7 receptor, metabotropic glutamate receptor 7; GPCRs, G protein coupled receptors; SNARE, soluble NSF attachment protein receptor; L-AP4, L-2amino-4-phosphonobutyric acid; Sp8Br, Sp-8-Br-cAMPS; HTRF, Homogeneous Time

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Resolved Fluorescence; PB, phosphate buffer; NDS, normal donkey serum; AC, adenylate cyclase; PLC, phospholipase C; PIP2, phosphatidylinositol (4,5)-bisphosphate; IP3, inositol trisphosphate; DAG, diacylglycerol; TTx, tetrodotoxin; HBM, HEPES buffered medium; BSA, bovine serum albumin; IP1, inositol monophosphate; TBS, Tris-buffered saline; PKA, cAMP-

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dependent protein kinase; Rim, Rab3-interacting molecule; EPAC, exchange protein directly activated by cAMP; IBMX, 3-isobutyl-1-methylxanthine; PDBu, phorbol dibutyrate; 8-pCPT, 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate;

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AP, action potential.

1. Introduction

Neurotransmitter release is initiated by the activation of voltage dependent Ca2+ channels and the fusion of the readily releasable pool of synaptic vesicles at the active zone. Many presynaptic G protein coupled receptors (GPCRs) inhibit release by reducing the activity of presynaptic Ca2+ channels (Choi and Lovinger, 1996; Millán et al., 2002, 2003), yet GPCRs also modulate neurotransmitter release downstream of Ca2+ channels by targeting proteins in the release machinery (Sakaba and Neher, 2003; Gerachshenko et al., 2005; Bauer et al., 2007;

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ACCEPTED MANUSCRIPT Pelkey et al., 2008; Nakajima et al., 2009; Martín et al., 2010; Zhang et al., 2011; Ferrero et al., 2013a).

The metabotropic glutamate receptor 7 (mGlu7 receptor) located in the presynaptic active zone mediates feedback inhibition of glutamate release by activating a pertussis toxin (PTx) sensitive Gi/o protein, subsequently dampening Ca2+ channel activity (Millán et al., 2002, 2003; Pelkey et

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al., 2006; Martín et al., 2007). However, mGlu7 receptors also activate phospholipase C (PLC: Perroy et al., 2000, Martín et al., 2010). Thus, prolonged exposure of mGlu7 receptors to the agonist L-AP4 enhances glutamate release in cerebrocortical nerve terminals by activating a

PTx insensitive G protein and through the subsequent hydrolysis of phosphatidylinositol (4,5)-

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bisphosphate (PIP2: Martín et al., 2010; Ferrero et al., 2011, 2013b). This enhancement of

neurotransmitter release by mGlu7 receptors is associated with the translocation of the active zone protein Munc13-1 from the soluble to particulate fraction, suggesting that this signaling

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targets the release machinery (Martín et al., 2010). The mGlu7 mediated potentiation of release occludes that induced by phorbol esters, suggesting a similar mechanism of action (Martín et al., 2011). Moreover, an increase in glutamate release after prolonged mGlu7 receptor activation was observed in hippocampal synapses (Pelkey et al., 2008). Interestingly, release potentiation after prolonged activation of mGlu7 receptors does not result from a switch in signaling as the receptor continues to inhibit Ca2+ channels (Martín et al., 2010) but rather, from receptor

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coupling to PLC-dependent signaling cascades. This phenomenon is consistent with the well established capacity of GPCRs to simultaneously activate several signaling pathways.

Beta adrenergic receptors (β-ARs) are also expressed presynaptically in axons that establish

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asymmetric, putative glutamatergic synapses (Ferrero et al., 2013a), consistent with their role as heteroreceptors that enhance glutamate release (Huang et al., 1996; Kobayashi et al., 2009; Ferrero et al., 2013a). β-ARs activate Gs proteins and increase cAMP levels. However, cAMP-

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mediated enhancement of spontaneous release is not mediated by PKA, the classic presynaptic cAMP target (Herrero and Sánchez-Prieto, 1996; Castillo et al., 2002), but rather by the guanine nucleotide exchange factor, EPAC, which has emerged as a new alternative cAMP effector (Huang and Hsu, 2006; Gekel and Neher, 2008; Ferrero et al., 2013a). Indeed, EPAC activation mimics and occludes the isoproterenol-induced increase in spontaneous release. Interestingly, βAR and EPAC activation increases PIP2 hydrolysis, and it provokes Munc13-1 translocation to the membrane (Ferrero et al., 2013a). In addition, EPAC also activates phospholipase C€ (PLC€), which can transduce signals from small GTPases through its Ras binding site (Schmidt et al., 2001; Branham et al., 2009; Dzhura et al., 2011). Accordingly, Munc13-1 can not only integrate PLC-coupled receptor signaling through its DAG binding site but also, that mediated by Gs/adenylyl-cyclase coupled GPCRs.

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ACCEPTED MANUSCRIPT The co-existence of presynaptic GPCRs at nerve terminals has received little attention (Manzoni et al., 1995; Ladera et al., 2007; Marchi and Grilli, 2010; Partovi and Frerking, 2006), even though the interplay between GPCR signaling pathways may affect neurotransmitter release. While there is currently little evidence that mGlu7 and β adrenergic receptors co-localize, both receptors are expressed at the active zone of glutamatergic nerve terminals in the cerebral cortex

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(Shigemoto et al., 1996, 1997; Kinoshita et al., 1998; Ohishi et al., 1995; Ferrero et al., 2013a). Although mGlu7 receptors inhibit adenylyl cyclase (Okamoto et al., 1994), mGlu7 receptorinduced inhibition of release is unrelated to changes in the levels of cAMP in the absence of adenylyl cyclase activity (Herrero et al., 1996), yet it is strongly dependent on cAMP when

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adenylyl cyclase is activated (Millán et al., 2002; Martín et al., 2007). Thus, activation and inhibition of adenylyl cyclase by β-ARs and mGluR7, respectively, would anticipate a

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functional interaction between their signaling pathways if these receptors were co-expressed.

By studying mGlu7 and β-adrenergic receptors in synaptosomes, we found that these receptors are indeed co-expressed in a subpopulation of cerebrocortical nerve terminals. When the mGlu7 receptor is coupled to the signaling pathway that enhances release by prolonged agonist exposure, the ensuing activation of β-ARs further enhances glutamate release. However, the overall effects on glutamate release by co-activation of mGlu7 and β-adrenergic receptors

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involve a cross-talk between the signals that result from mGlu7 receptor-mediated inhibition of β-AR-activated adenylyl cyclase. Thus, blocking Gi/o proteins with pertussis toxin results in a supra-additive enhancement of release, which also occurs when the β-AR associated signaling is activated downstream of adenylyl cyclase with the cAMP analogue Sp8Br or an EPAC

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activator. Co-activation of mGlu7 and β adrenergic receptors also enhances the PLC-dependent accumulation of IP1 and the membrane translocation of Munc13-1, indicating that release

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potentiation by these receptors is a result of modulating the release machinery.

2. Materials and Methods

2.1. Synaptosome preparation. All animal handling was performed in accordance with European Commission guidelines (2010/63/UE) and was approved by the Animal Research Committee at the Complutense University. Synaptosomes from the cerebral cortex of adult C57BL/6 mice (2-3 months old) were purified on discontinuous Percoll gradients (GE Healthcare, Uppsala, Sweden) as described previously (Millán et al., 2002). Briefly, the tissue was homogenized in medium containing 0.32 M sucrose [pH 7.4], the homogenate was centrifuged for 2 min at 2,000 x g and 4 ºC, and the supernatant was then centrifuged again for

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ACCEPTED MANUSCRIPT 12 min at 9,500 x g. From the pellets obtained, the loosely compacted white layer containing the majority of the synaptosomes was gently resuspended in 0.32 M sucrose [pH 7.4] and an aliquot of this synaptosomal suspension (2 ml) was placed onto a 3 ml Percoll discontinuous gradient containing: 0.32 M sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3, 10 or 23 % Percoll [pH 7.4]. After centrifugation at 25,000 x g for 10 min at 4 ºC, the synaptosomes were recovered from between the 10 % and the 23 % Percoll bands, and they were diluted in a final

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volume of 30 ml of HEPES buffered medium (HBM: 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM NaH2PO4, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES [pH 7.4]). Following further centrifugation at 22,000 x g for 10 min, the synaptosome pellet was resuspended in 0.5-1 ml of HBM medium and the protein content was determined by the Biuret

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method. Finally, 0.75-1 mg of the synaptosomal suspension was diluted in 2 ml HBM and centrifuged at 10,000 x g for 10 min. The supernatant was discarded and the pellets containing the synaptosomes were stored on ice. Under these conditions the synaptosomes remain fully

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viable for at least 4-5 h.

2.2. Glutamate release. Glutamate release was assayed by on-line fluorimetry as described previously (Millán et al., 2002). Synaptosomal pellets were resuspended in HBM (0.67 mg/ml) and preincubated at 37 ºC for 1 h in the presence of 16 µM bovine serum albumin (BSA) to bind any free fatty acids released from synaptosomes during preincubation (Herrero et al., 1991).

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Adenosine deaminase (1.25 U/mg: Roche Diagnostics, Barcelona, Spain) was added for 30 min, and the synaptosomes were then washed by centrifugation for 30 s at 16,000 x g and resuspended in HBM medium. A 1 ml aliquot of the synaptosomes was transferred to a stirred cuvette containing 1 mM NADP+, 50 U glutamate dehydrogenase (Sigma, St. Louis, MO, USA)

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and 1.33 mM CaCl2, and the fluorescence of NADPH was measured in a Perkin Elmer LS-50 luminescence spectrometer at excitation and emission wavelengths of 340 and 460 nm, respectively. Data were obtained at 0.8 sec intervals and the fluorescence traces were calibrated

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by the addition of 2 nmols of glutamate at the end of each assay. Plotted traces are the mean of all individual traces corresponding to a given condition. Smoothing protocols were applied to reduce the noise using OriginPro 8 and the Savitzky-Golay method. Glutamate release was induced with the Ca2+ ionophore ionomycin, which inserts into the membrane and delivers Ca2+ to the release machinery independently of Ca2+ channel activity. In addition, ionomycin was added in the presence of the Na+-channel blocker tetrodotoxin (1 µM: Abcam, Cambridge, UK), which prevents the firing of action potentials and therefore, the AP-driven opening of voltage dependent Ca2+ channels. Tetrodotoxin was added 2 min prior to ionomycin. The ionomycin induced release was calculated by subtracting the release observed during a 10 min period in the absence of ionomycin (basal) from that observed in its presence. The concentration of ionomycin (Calbiochem, Darmstadt, Germany) was fixed in each experiment (0.5-1.0 µM) in

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ACCEPTED MANUSCRIPT order to achieve 0.5-0.6 nmols Glu/mg protein. The following drugs were administered as indicated in the figure legends: the group III mGluR agonist L-AP4 (1 mM, obtained from Tocris Bioscience, Ellisville, Mi, USA); the adenylate cyclase activator forskolin (15 µM) and the DAG-binding protein inhibitor calphostin C (0.1 µM, both obtained from Calbiochem, Darmstadt, Germany); the EPAC activator 8-pCPT-2´-O-Me-cAMP (50 µM) and the cAMP analogue Sp-8-Br-cAMPS (250 µM, both obtained from BioLog, Bremen, Germany); and the β-

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AR agonist isoproterenol (100 µM, obtained from Sigma-Aldrich, St. Louis, MO, USA).

2.3. IP1 accumulation. IP1 accumulation was determined using the IP-One kit (Cisbio, Bioassays, Bagnols sur-Cèze, France: Trinquet et al., 2006). Synaptosomes (0.67 mg/ml) in

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HBM containing 16 µM BSA and adenosine deaminase (1.25 U/mg protein) were incubated for 1 h at 37 ºC. After 25 min, 50 mM LiCl was added to inhibit inositol monophosphatase and other drugs were added as indicated in the figure legends. Synaptosomes were collected by

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centrifugation for 1 min at 4 ºC and 16,000 x g, and they were resuspended (14.3 mg/ml) in lysis buffer (50 mM HEPES, 0.8 M potassium fluoride, 0.2 % [w/v] BSA, 1 % [v/v] Triton X100 [pH 7.0] and 50 mM LiCl). The lysed synaptosomes were transferred to a 96-well assay plate and the following HTRF components were added, diluted in lysis buffer: the europium cryptate-labeled anti IP1 antibody and the d2-labeled IP1 analogue. After incubation for 1 h at room temperature (RT), the europium cryptate fluorescence and TR-FRET signals were

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measured on a FluoStar Omega microplate reader (BMG Lab Technologies, Offenburg, Germany) at 620 and 665 nm, respectively, 50 µs after excitation at 337 nm. The specific FRET signal was calculated using the following equation: ∆F% = 100 x (Rpos-Rneg)/(Rneg), where Rpos is the fluorescence ratio (665/620 nm) calculated in the wells incubated with both donor- and

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acceptor-labeled antibodies, and Rneg is the same ratio for the negative control incubated with only the donor fluorophore-labeled antibody. The FRET signal (∆F %), which is inversely proportional to the concentration of IP1 in the cells, was then transformed to the accumulated IP1

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value using a calibration curve prepared using the same plate.

2.4. cAMP accumulation. The accumulation of cAMP was determined using a cAMP dynamic 2 kit (Cisbio, Bioassays, Bagnols sur-Cèze, France). This assay was similar to that described for IP1, except that 1 mM of the cAMP phosphodiesterase inhibitor IBMX (Calbiochem, Damstard, Germany) was added for 35 min during the incubation. The HTRF assay was also similar to that described for IP1, except that an anti-cAMP antibody and a d2-labeled cAMP analogue were used.

2.5. Immunocytochemistry. Immunocytochemistry was performed using an affinity-purified goat polyclonal antiserum against β1-AR (Sigma-Aldrich, St. Louis, MO, USA), a polyclonal rabbit 6

ACCEPTED MANUSCRIPT antiserum against synaptophysin 1 (Synaptic Systems, Gottingen, Germany) and a polyclonal guinea pig antiserum against mGlu7a receptors (Shigemoto et al., 1997). As a control for the immunochemistry, the primary antibodies were omitted from the staining procedure, whereupon no immunoreactivity resembling that obtained with the specific antibodies was detected. Synaptosomes (0.67 mg/ml) were added to medium containing 0.32 M sucrose [pH 7.4] at 37 ºC, allowed to attach to poly-L-lysine coated coverslips for 1 h and then fixed for 4 min with 4

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% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at RT. Following several washes with 0.1 M PB [pH 7.4], the synaptosomes were pre-incubated for 1 h in 10 % normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA,USA) diluted in 50 mM Tris buffer [pH 7.4] containing 0.9 % NaCl (TBS) and 0.2 % Triton X-100. Subsequently, they were

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incubated overnight at 4 ºC with the appropriate primary antiserum against β1-ARs (1:100), synaptophysin (1:100) or mGlu7a receptor (1:300), diluted in TBS with 1 % NDS and 0.2 % Triton X-100. After washing in TBS, the synaptosomes were incubated with secondary

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antibodies diluted in TBS for 2 hours: Alexa fluor 488 donkey anti-rabbit IgG (1:500), Alexa fluor 594 Donkey anti-goat IgG (1:500) or Alexa fluor 594 Donkey anti-guinea pig IgG (1:500) (Molecular Probes, Eugene, OR, USA). After several washes in TBS, the coverslips were mounted with Prolong Antifade Kit (Molecular Probes, Eugene, OR, USA) and the synaptosomes were viewed on a Nikon Diaphot microscope equipped with a 100x objective, a mercury lamp light source and fluorescein-rhodamine Nikon filter sets.

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For quantification, all the images were acquired using identical settings and neutral density transmittance filters. Background subtraction was performed by applying a rolling ball algorithm (6 pixel radius), and the brightness and contrast settings were adjusted according to the negative control values using Image J 1.39f (http://rsb.info.nih.gov/ij). The number of

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stained particles larger than 0.5 µm was quantified automatically from the binary image masks, discarding aggregates. Co-localization analysis was performed automatically by measuring the

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area of coincidence of particles quantified in each pair of images within the same field.

2.6. Immunohistochemistry. Adult C57BL/6 mice (2-3 months old) were anesthetized and perfused transcardially with 4 % paraformaldehyde in 0.1 M PB [pH 7.4], and 30 µm coronal brain slices attached to poly-L-lysine coated slides were immunolabeled. After blocking with PBS containing 0.25% Triton X-100, 5 % NDS and 1 % BSA (1h; RT), the sections were incubated overnight at 4 ºC with the following antibodies diluted in PBS containing 0.25% Triton X-100 and 5 % NDS: Goat anti-β1-AR (1:75, Sigma-Aldrich); Rabbit anti-mGlu7a receptor (1:50, Shigemoto et al., 1997); and mouse monoclonal anti-synaptophysin (1:400, Synaptic System). After several washes, the sections were incubated with specific Alexa conjugated secondary antibodies (1:200, Molecular Probes) diluted in PBS containing 0.25% Triton X-100 and 5 % NDS (1h; RT): Alexa anti-mouse 488, Alexa anti-goat 594, and Alexa

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ACCEPTED MANUSCRIPT anti-rabbit 647. Finally, the sections were mounted in Prolong Antifade with DAPI (Molecular Probes) and stored at 4 ºC. Images were acquired on a confocal Olympus FV 1200 microscope.

2.7. Co-immunoprecipitation. Synaptosomes (0.67 mg/ml) were incubated for 1 h at 37 °C in HBM containing 16 µM BSA and adenosine deaminase (1.25 units/mg protein). The mGlu7 receptor agonist, L-AP4 (1mM) and the β-AR agonist, isoproterenol (100µM) were added for 10

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minutes prior to washing. Synaptosomes were washed and recovered by centrifugation at

16,000 x g, and unstimulated and stimulated synaptosomes (2.86 mg/mL) were then solubilized for 30 min on ice in radioimmunoprecipitation assay buffer containing protease inhibitors (Thermo Scientific Rockford, IL, USA) and EDTA. The solubilized extract was then

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centrifuged at 16,000 x g for 60 min, and the supernatant was processed for

immunoprecipitation, each step conducted at 4 °C with constant rotation. The supernatant was incubated overnight with a rabbit anti-RIM1 antibody (3.5 µg: Synaptic Systems) or with a

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rabbit anti-Munc13-1 antibody (3.5 µg: Synaptic Systems), and a suspension of TrueBlot antirabbit Ig IP beads (50µl, Rockland Immunochemicals, Gilbertsville, PA, USA) was then added and the mixture was incubated for a further 2 h. Subsequently, the beads were washed twice with ice-cold radioimmunoprecipitation assay buffer and twice with the same buffer but diluted 1:10 in Tris-saline (50 mM Tris-HCl [pH 7.4] and 100 mM NaCl). SDS-PAGE sample buffer (0.125 M Tris-HCl [pH 6.8], 4 % SDS, 20 % glycerol and 0.004 % bromphenol blue) was then

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added to each sample, and the immune complexes were dissociated by adding fresh dithiothreitol (DTT, 50 mM final concentration) and heating to 90 °C for 10 min. Proteins were resolved by SDS-PAGE on either 7.5 % or 10 % polyacrylamide gels, and they were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) that were then probed with mouse

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anti-Munc13-1 antibody (1:1000, Synaptic System). Antibody binding was detected with IRDye 800CW (LI-COR, Lincoln, NE, USA). The immunoreactive bands were recorded in a Odyssey

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Imager (LI-COR).

2.8. Munc13-1 translocation. Synaptosomes were resuspended (0.67 mg/ml) in HBM medium with 16 µM BSA and incubated for 30 min at 37 ºC, before adenosine deaminase (1.25 U/mg protein) was added for a further 20 min. The β-AR agonist isoproterenol (100 µM), the group III mGluR agonist L-AP4 (1 mM) and the EPAC activator 8-pCPT-2´-O-Me-cAMP (50 µM), were added for 10 minutes and the synaptosomes were then washed by centrifugation (16,000 x g for 30 sec) and resuspended (2 mg/ml) in hypo-osmotic medium (8.3 mM Tris-HCl buffer [pH 7.4]) containing a Protease Inhibitor Cocktail and EDTA (Thermo Scientific RockFord, IL, USA). The synaptosomal suspension was passed through a 22-gauge syringe to disaggregate the synaptosomes, which were then maintained at 4 ºC for 30 min with gentle shaking. The soluble and particulate fractions were separated by centrifugation for 10 min at 40,000 x g and 4 ºC, and

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ACCEPTED MANUSCRIPT the supernatant (soluble fraction) was collected while the pellet (particulate fraction) was resuspended in RIPA buffer (1 % Triton X-100, 0.5 % deoxycholate, 0.2 % SDS, 100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl [pH 7.4]). The levels of marker proteins in the soluble and particulate fractions were assessed as described previously (Ferrero et al., 2013a). The soluble and particulate fractions (3 µg of protein per lane) were diluted in Laemmli loading buffer with β-mercaptoethanol (5 % v/v), resolved by SDS-PAGE (7.5 % acrylamide, Bio-Rad)

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and analyzed in Western blots according to standard procedures. All samples were normalized to the levels of β-tubulin in the same blot. Goat anti-rabbit and goat anti-mouse secondary antibodies coupled to Odyssey IRDye 680 or Odyssey IRDye 800 (Rockland

Immunochemicals) were used to quantify the Western blots using the Odyssey System (LI-

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COR, Lincoln, NE, USA). The western blots were probed with a polyclonal rabbit anti-

Munc13-1 antiserum (1:1000; Synaptic Systems) and a monoclonal mouse anti-β-tubulin antibody (1:4000; Sigma-Aldrich), and the Munc13-1 content was expressed relative to the

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integrated intensity of the total soluble and particulate fractions.

3. Results

To test for a possible interaction between the signaling pathways activated by mGlu7 and β-

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adrenergic receptors, glutamate release was estimated after co-activation of these two receptors and the release compared to that induced by activating each receptor alone. As the mGlu7 receptor induced enhancement of release persists for at least 30 min after agonist addition (Martín et al., 2010), mGlu7 receptors were activated first, followed by the activation of β-ARs

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(Fig. 1A). Thus, the mGlu7 receptor agonist L-AP4 (1 mM) was added to synaptosomes for 10 min and after washing out, isoprotorenol was added to activate the β-ARs. The mGlu7 receptors can signal through different signaling pathways that may operate simultaneously: adenylate

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cyclase, Ca2+ channels, PLC (Millán et al., 2002, 2003; Pelkey et al., 2006; Martín et al., 2007; Perroy et al., 2000; Martín et al., 2010; Okamoto et al., 1994; Herrero et al., 1996) and therefore, the overall effect of mGlu7 receptors on release reflects this complex signaling. However, we found that in synaptosomal preparations the signaling pathways activated by mGlu7 receptors, and therefore their effects on release modulation, depended on the stimulation protocol. For example, using ionomycin to stimulate synaptosomes minimizes mGlu7 receptor signaling to Ca2+ channels because the ionophore delivers Ca2+ directly to the release machinery without the need to activate Ca2+ channels. Furthermore, these release experiments were performed in the presence of Na+-channel blocker tetrodotoxin that prevents the firing of action potentials (APs) and thus, the AP-driven opening of voltage dependent Ca2+ channels. Hence,

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ACCEPTED MANUSCRIPT the release modulation observed with ionomycin reflects mGlu7 receptor signaling through PLC activation and adenylyl cyclase inhibition, provided that adenylyl cyclase is activated (Herrero et al., 1996; Millán et al., 2002; Martín et al., 2007). We previously reported that mGlu7 receptors are the only group III mGlu receptors expressed in the cerebrocortical synaptosomes prepared from adult animals (Ladera et al., 2009) and thus, the responses to L-AP4 (1 mM) in

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this preparation are likely to be mediated exclusively by this receptor.

The ionomycin-induced release of glutamate (0.59 ± 0.04 nmols/mg of protein, n=18) was

enhanced by prolonged activation of mGlu7 receptors with L-AP4 (177 ± 3.1% of the control value, n=6, p<0.001), as well as by β-AR activation with isoproterenol (172 ± 4.6%, n=9,

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p<0.001: Fig. 1B and C). Moreover, an additive response was observed when both receptors

were activated together (251 ± 4.3%, n=8, p<0.001: Fig. 1B and C). Since the release induced by receptor co-activation is similar to the sum of the release following the individual activation

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of the receptors (p>0.05), this would suggest that there is apparently no interaction between the signaling pathways at play. Thus, the additive effect of mGlu7 and β adrenergic receptors may be due to the action of these two receptors on distinct populations of nerve terminals.

3.1. Co-expression

We assessed whether mGlu7 and β-adrenergic receptors co-exist in a subpopulation of nerve

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terminals by immunocytochemistry. When synaptosomes were labeled for both synaptophysin, a marker of synaptic vesicles, and β1 adrenoreceptors, 30.6 ± 1.3% of the nerve terminals labeled for synaptophysin (7,355 nerve terminals from 25 fields) also expressed the β1-AR (Fig. 2A, B). Similarly, 30.0 ± 1.2% of the cerebrocortical nerve terminals labeled for synaptophysin (6,094

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nerve terminals from 20 fields) were also recognized by an antisera against mGlu7a receptors (Fig. 2C, D). Finally, we assessed the co-expression of the mGlu7 and β-adrenergic receptors and

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we found that of the nerve terminals labeled for the β1-adrenoreceptor (10,643 nerve terminals from 68 fields), 72.9 ± 2.6% also expressed mGlu7a receptors (Fig. 2E, F). Thus, β1-adrenergic and mGlu7 receptors are largely co-expressed in a subpopulation of nerve terminals from the cerebral cortex.

We also performed immunohistochemistry on coronal brain slices using antibodies against mGlu7 and β1-adrenergic receptors, again revealing a high degree of co-localization of the two receptors (yellow dots), which was particularly evident at higher magnifications (60x: Fig. 2G). When we studied the distribution of the presynaptic marker synaptophysin in these coronal slices to assess whether this co-localization occurred at synaptic boutons, the mGlu7 and β1-adrenergic receptors are clearly expressed presynaptically in synaptic boutons recognized by the synaptophysin antibody (Fig. 2H, I, J).

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ACCEPTED MANUSCRIPT 3.2. Cross-talk Having shown that the mGlu7 and β-adrenergic receptors are co-expressed in a subpopulation of nerve terminals, we assessed whether the additive response observed after activation of the two receptors is the result of cross-talk between the signaling pathways associated to these receptors. It has been well established that mGlu7 receptors inhibit glutamate release through Gi/o protein

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activation, coupled to the subsequent inhibition of Ca2+ channels and adenylyl cyclase

activation. It is unlikely that Ca2+ channel inhibition by the mGlu7 receptor plays a significant role in these experimental conditions because ionomycin stimulates release independently of

Ca2+ channels and because the Na+-channel blocker tetrodotoxin was present, which prevents

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AP firing and the AP-driven opening of voltage dependent Ca2+ channels. However, it is

possible that the activation of mGlu7 receptors by L-AP4 may counteract the β-AR-mediated activation of adenylyl cyclase via Gi/o proteins (Millán et al., 2002), thereby reducing cAMP

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levels and its downstream effect on glutamate release (Ferrero et al., 2013a). We found that glutamate release induced by mGlu7 and β-adrenergic receptor co-activation (175 ± 6.3%, n=6) was further enhanced (220 ± 6.2%, n=9, p<0.0001) after blocking Gi/o-dependent signaling with pertussis toxin (1.25 µg/ml, 2h: Fig. 3). This enhancement of release in the presence of PTx is consistent with the removal of an inhibitory effect on the signaling pathways associated

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to mGlu7 and β-adrenergic receptors mediated by a pertussis sensitive Gi/o protein.

The mGlu7 receptors induce a pertussis toxin sensitive decrease in cAMP that results in a decrease in glutamate release (Okamoto et al., 1994; Millán et al., 2002, 2003; Martín et al., 2007), while β-ARs activate a Gs protein to enhance cAMP levels and the release of glutamate

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(Ferrero et al., 2013a). Thus, we tested whether mGlu7 receptors counteract the increase in isoproterenol-induced release, activating mGlu7 receptors 30 seconds prior to applying isoproterenol in order to activate β-ARs (see Fig. 4A). It is important to note that this short

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agonist stimulation does not allow mGlu7 receptors to couple to PLC (Martín et al., 2010), while they do efficiently inhibit adenylyl cyclase (Millán et al., 2002) and thus, the decrease in cAMP would be expected to inhibit glutamate release. We found that mGlu7 receptor activation with L-AP4 abolished the isoproterenol-induced release, reducing it from 176 ± 3.8% (n=8, p<0.001) to 102 ± 2.9% (n=8, p>0.05: Fig. 4B, C). The ability of L-AP4 to suppress isoproterenol responses is probably related to the strong co-expression of mGlu7 and βadrenergic receptors. In fact, a similar increase in release was produced by the adenylyl cyclase activator forskolin (15 µM), which should increase cAMP in nerve terminals irrespective of whether they express mGlu7 receptors or not, and this was only partially reduced by L-AP4 (from 176 ±8.4 %, n=4 to 135 ± 5.5%, n=5, p<0.01 in the presence of L-AP4: Fig. 4D). In parallel experiments, we found that the isoproterenol-induced increase in cAMP (224 ± 25.0%,

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ACCEPTED MANUSCRIPT n=10) was also abolished by the mGlu7 receptor agonist L-AP4 (96 ± 14.1%, n=8, p<0.001: Fig. 4E), a response that was prevented by blocking Gi/o proteins with pertussis toxin (220.9 ± 29.1%, n=6 and 206 ± 16.9%, n=6, in the presence and absence of L-AP4, respectively, p>0.05: Fig. 4E). If the decrease in isoproterenol-induced release by L-AP4 is the consequence of a decrease in cAMP, then activating the β-AR signaling pathway downstream of adenylyl cyclase should prevent the effects of L-AP4. Indeed, the release induced by the specific activator of the

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guanine nucleotide exchange protein directly activated by cAMP, EPAC (176 ± 5.8%, n=4),

mimics and occludes the isoproterenol-induced potentiation of release (Ferrero et al., 2013a), and it was not altered by L-AP4 (180 ±3.2 %, n=7, p>0.05: Fig. 4F).

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Although these experiments suggest a potential interaction between the signaling pathways associated to mGlu7 and β-adrenergic receptors, it is important to note that L-AP4 was not

washed out and therefore, the inhibition of the isoproterenol-induced responses by the mGlu7

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receptor occurs in the presence of agonist. To more directly demonstrate that when synaptosomes are treated with L-AP4, mGlu7 receptors inhibit adenylyl cyclase, even after washing out L-AP4, synaptosomes were treated with L-AP4 for 10 min to couple these receptors to the PLC and then, after washing out the agonist, the β-ARs were activated with isoproterenol, assessing the effect of L-AP4-treatment on isoproterenol-induced release. Since prolonged activation with L-AP4 couples mGlu7 receptors to PLC and enhances release, the

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experiments were performed in the presence of calphostin C to abolish release enhancement via PLC, thereby only revealing the release inhibition provoked by mGlu7 receptor mediated inhibition of adenylyl cyclase signaling (Martín et al., 2010). This signaling pathway has been shown to be involved in the activation/translocation of the active zone protein Munc13-1

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(Martin et al., 2010), and Calphostin C blocks this pathway by antagonizing DAG binding to Munc13-1. We found that the isoproterenol-induced increase in release (137 ± 3.8%, n=9) was dampened in synaptosomes pre-treated with L-AP4 (121 ± 4.3%, n=9, p<0.05: Fig. 5B).

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Similarly, the isoproterenol-induced increase in cAMP (151 ± 5.6%, n=10) also decreased in synaptosomes pretreated with L-AP4 (111 ± 1.6%, n=13, p<0.001: Fig. 5C). Thus, L-AP4 pretreatment initiates the mGlu7 receptor signaling that counteracts β-AR responses, an effect that persists after L-AP4 removal.

If the cross-talk between the signaling pathways associated to mGlu7 and β-adrenergic receptors relies on Gi/o-mediated inhibition of adenylyl cyclase, this interaction could be overridden by activating the β-AR/Gs/AC/cAMP pathway downstream of adenylyl cyclase. We recently reported that the isoproterenol-induced increase in spontaneous release of glutamate is independent of PKA-activation as it is resistant to PKA inhibition with H-89, indicating that other cAMP targets are probably mediating this response. Indeed, 8-pCPT-2-OMe-cAMP, a

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ACCEPTED MANUSCRIPT specific activator of EPAC (the exchange protein directly activated by cAMP), mimicked and occluded isoproterenol-induced potentiation of glutamate release (Ferrero et al., 2013a). Thus, 8pCPT represents an alternative means of activating β-AR associated signaling pathway to bypass their interaction with mGlu7 receptor signaling. We found that 8pCPT increased glutamate release (175 ± 5.1%, n=6, p<0.001) to a similar extent as L-AP4 (180 ± 5.6%, n=6, p<0.001) and moreover, the activation of the EPAC-dependent signaling pathway with 8pCPT in

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synaptosomes pretreated with L-AP4 further enhanced release (317 ± 8.5%, n=8, p<0.001: Fig. 6B, C).

To activate the β-AR/Gs/AC/cAMP pathway downstream of adenylyl cyclase, we also used the

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permeable cAMP analogue Sp8Br, as opposed to the β-AR agonist isoproterenol. Sp8Br

enhanced glutamate release (177 ± 3.4%, n=10, p<0.001) to a similar extent as L-AP4 (175 ± 3.3%, n=7, p<0.001) and again, in synaptosomes pretreated with L-AP4 the addition of Sp8Br

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provoked a supra-additive effect (314 ± 8.9%, n=11, p<0.001: Fig. 6D). This response was expected to be independent of Ca2+ channel activation as these experiments were performed in the presence of the Na+-channel blocker tetrodotoxin that prevents AP firing and therefore, the AP-driven opening of voltage dependent Ca2+ channels. However, spontaneous opening of Ca2+ channels in the presence of tetrodotoxin still can occur (Millán et al., 2003) and hence, we included P/Q and N-type calcium channel blockers to prevent this. The supra-additive response

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was preserved after blocking P/Q- and N-type Ca2+ channels with ω-agatoxin-IVA and ωconotoxin GVIA and indeed, in the presence of both toxins the increase in release induced by LAP4-pretreatment (161 ± 2.4%, n=4, p<0.001) and Sp8Br (153 ± 3.8%, n=4, p<0.01) was enhanced (252 ± 5.2%, n=5, p<0.01: Fig. 6E), further underlining that glutamate release under

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these conditions is independent of Ca2+ channel activity.

3.3. IP1 accumulation

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We reported that the increase in release after prolonged activation of mGlu7 receptors depends on PLC activation, as the L-AP4-induced increase in IP1 accumulation was also impaired by the PLC inhibitor U73122 (Martín et al., 2010). The increase in release induced by isoproterenol also partially depends on PLC activity, and this response was mimicked and occluded by the EPAC activator, 8pCPT (Ferrero et al., 2013a). In these experiments, IP1 rather than IP3 accumulation was assessed as a means to monitor PLC-linked GPCRs following the LiCl inhibition of inositol monophosphatases (Trinquet et al., 2006). As mGlu7 and β-adrenergic receptors are linked to PLC activity, we determined whether their effects on release were correlated with IP1 accumulation (Fig. 7A). Isoproterenol (157 ± 4.0%, n=7) and L-AP4 (170 ± 5.5%, n=7) pre-treatment enhanced IP1 accumulation when applied separately, and a further effect was observed when they were applied together (204 ± 8.8%, n=7: Fig. 7B). The cAMP

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ACCEPTED MANUSCRIPT analogue Sp8Br was used to by-pass the Gi/o mediated interaction between mGlu7 receptor and β-AR associated signaling pathways, which also enhanced IP1 accumulation (156 ± 5.6%, n=7: Fig. 7B). Interestingly, when LAP4-treatment was combined with Sp8Br to activate the β-AR associated signaling pathways downstream of adenylyl cyclase, a further increase in IP1 accumulation was detected (249 ± 10.2%, n=7, p<0.01), more so than when L-AP4 treatment was combined with isoproterenol (204 ± 8.8%, n=7: Fig. 7B). Thus, activation of the β-

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AR/Gs/AC/cAMP pathway downstream of adenylyl cyclase results in a stronger IP1 accumulation.

3.4. Munc13-1 translocation

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The active zone protein Munc13-1 is a phorbol ester receptor essential for synaptic vesicle

priming and it plays an important role in neurotransmitter release (Betz et al., 1998; Rhee et al., 2002; Bauer et al., 2007). Munc13-1 is distributed in two biochemically distinguishable soluble

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and insoluble pools (Betz et al., 1998; Brose et al., 1995; Kalla et al., 2006). Diacylglycerol and phorbol esters increase the association of Munc13-1 with the plasma membrane (Brose and Rosenmund, 2002), and we showed previously that enhancing the release mediated by mGlu7 and β-adrenergic receptors is associated with the translocation of Munc13-1 from the cytosol to membranes (Martín et al., 2010; Ferrero et al., 2013a). Here, we tested whether the effects on release that result from the interaction between the signaling pathways associated to mGlu7 and

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β-adrenergic receptors are correlated with Munc13-1 translocation. The ratio of Munc13-1 in the soluble and particulate fractions of synaptosomes after hypo-osmotic shock (soluble/particulate ratio) was 0.41 ± 0.02 (n=7) in control nerve terminals. This value decreased significantly following exposure to L-AP4 (0.28 ± 0.02, n=7, p<0.001), isoproterenol

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(0.31 ± 0.02, n=7, p<0.05) or the EPAC activator, 8-pCPT (0.27 ± 0.02, n=7, p<0.001: Fig 7C), indicative of the translocation of the Munc13-1 protein from the soluble to the particulate fraction. Treatment with L-AP4 and isoproterenol induced a similar degree of translocation

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(0.26 ± 0.02, n=7, p<0.001). However, L-AP4 treatment followed by the addition of 8pCPT further enhanced the translocation of Munc13-1 (0.18 ± 0.01, n=7, p<0.001, compared to the controls; and p<0.05 compared L-AP4 treatment/isoproterenol: Fig. 7C).

3.5. Interaction between the Munc13-1 and RIM1α proteins Recent work identified the active zone proteins RIM and Munc13 as two key modulators of neurotransmitter release (Kaeser et al., 2011; Deng et al., 2011). As the selective disruption of the RIM-Munc13-1 interaction decreases the size of the readily releasable vesicle pool (Dulubova et al., 2005), we tested whether the activation of mGlu7 and β-adrenergic receptors enhanced the RIM1-Munc13-1 interaction by immunoprecipitating these proteins from soluble cerebrocortical synaptosome extracts known to contain Munc13-1 (Fig. 8A, Crude). The anti-

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ACCEPTED MANUSCRIPT Munc13-1 antibody immunoprecipitated a band of around 200 kDa that appeared to correspond to the Munc13-1 protein (Fig. 8A, IP: Munc13-1) and interestingly, the anti-RIM1α antibody also immunoprecipitated Munc13-1 from soluble cerebrocortical synaptosome extracts (Fig. 8A, IP: RIM1). This band was not apparent when an irrelevant rabbit IgG was used (Fig. 8A, IP: IgGr), indicating the specificity of the reaction and that the band detected indeed corresponded to Munc13-1. Moreover, when synaptosomes were pretreated with L-AP4, more Munc13-1 was

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immunoprecipitated with the anti-RIM1antibody (140 ± 6%, n=3, p< 0.01: Fig. 8B), as also

occurred when synaptosomes were exposed to the β-AR agonist isoproterenol (155 ± 6%, n=3, p< 0.05: Fig. 8B). Overall, these results suggested that activation of mGlu7 and β adrenergic

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receptors enhanced the RIM1-Munc13-1 interaction.

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4. Discussion

We have demonstrated that mGlu7 and β-adrenergic receptors are co-expressed by a subset of nerve terminals isolated from the cerebral cortex, where they modulate neurotransmitter release. The co-activation of these receptors provokes an additive response that paradoxically, reflects a negative interaction between receptor linked signaling pathways. Thus, suppression of mGlu7 receptor-mediated inhibition of adenylyl cyclase results in supra-additive release. Both these

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receptors promote the translocation of the active zone protein Munc13-1 to the membrane, and they enhance Munc13-1 and RIM-1α co-immunoprecipitation, indicating that signaling through these receptors targets the release machinery.

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4.1. β-ARs and mGlu7 receptors co-expression

The co-existence of presynaptic GPCRs at nerve terminals has received little attention, even though cross-talk between receptor-associated signaling pathways may be important to regulate

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presynaptic function (Manzoni et al., 1995; Ladera et al., 2007, Marchi and Grilli, 2010; Partovi and Frerking, 2006). For example, adenosine A1, GABAB and mGlu7 receptors co-exist in a subpopulation of cerebrocortical nerve terminals in which their responses are integrated through common intracellular signaling pathways (Ladera et al., 2007; Martín et al., 2008). Immunocytochemical and immunohistochemical data from cerebrocortical nerve terminals and tissue slices show that β-ARs and mGlu7 receptors are co-expressed in a subpopulation of nerve terminals. Indeed, the specific localization of β-ARs and mGlu7 receptors at the active zone of glutamatergic nerve terminals (Kinoshita et al., 1998; Ohishi et al., 1995; Shigemoto et al., 1996, 1997; Ferrero et al., 2013a) makes these receptors good candidates to modulate glutamate release by targeting proteins of the release machinery, such as Munc13-1 (Martín et al., 2010; Ferrero et al., 2013a) and RIM1α (Pelkey et al., 2008; Ferrero et al., 2013a).

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ACCEPTED MANUSCRIPT 4.2 β-AR and mGlu7 receptor cross-talk It is known that mGlu7 receptors couple to multiple signaling pathways, inhibiting Ca2+ channels and adenylyl cyclase either through Gi/Go proteins (Choi and Lovinger, 1996; Millán et al., 2002, 2003; Martín et al., 2007) or through PLC-dependent pathways resistant to pertussis toxin (Perroy et al., 2000; Martín et al., 2010). The inhibition of Ca2+ channels and of adenylyl

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cyclase reduce glutamate release at nerve terminals (Millán et al., 2002), while coupling to PLC enhances release (Martín et al., 2010). However, release enhancement requires prolonged

receptor stimulation, suggesting a weaker coupling of the receptor to this pathway (Martín et al.,

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2010; Ferrero et al., 2011).

We obtained direct evidence that mGlu7 and β-adrenergic receptors enhance glutamate release by activating a PLC-dependent pathway, both receptors augmenting PIP2 hydrolysis and DAG

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generation, which in turn activates/translocates the active zone protein Munc13-1 to the membrane. While several lines of evidence support the direct coupling of mGlu7 receptors to PLC, the coupling of β-ARs to PLC-dependent signaling pathways is less straightforward. βARs are known to activate Gs proteins and to increase cAMP levels. However, some presynaptic responses of cAMP are not mediated by its classic target, protein kinase A (Herrero and Sánchez-Prieto, 1996; Castillo et al., 2002). Rather, these responses could be mediated by

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EPAC, the guanine nucleotide exchange factor activated by cAMP that has emerged as an alternative cAMP effector (Gekel and Neher, 2008; Ferrero et al., 2013a). EPAC has been characterized as a GEF for the small GTPases, Rap1 and Rap2 (Kawasaki et al., 1998), which bind to and activate PLC-ε in HEK-293 cells that express adenylyl cyclase-coupled to β2-ARs

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(Schmidt, 2001). PLC-ε is expressed in the brain (Thomas et al., 1991; Kelley et al., 2001), although it is currently unknown whether it is located at nerve terminals. However, since EPAC activation mimics and occludes the isoproterenol-induced increase in spontaneous release of

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glutamate (Ferrero et al., 2013a), and β-AR and EPAC activation increases PIP2 hydrolysis and induces Munc13-1 translocation (Ferrero et al., 2013a), a cAMP-dependent and PLC-dependent signaling pathway would appear to operate at cerebrocortical nerve terminals.

Here we show that co-activation of mGlu7 and β-adrenergic receptors produces a synergic enhancement of release that involves cross-talk between receptor signaling pathways. Indeed, suppression of mGlu7 receptor mediated inhibition of adenylyl cyclase shifts the additive response into a supra-additive one. In this interaction, mGlu7 receptors couple to a PLCdependent pathway to enhance glutamate release, and also to adenylyl cyclase in order to inhibit the enzyme and reduce glutamate release, consistent with the capacity of these receptors to simultaneously activate several signaling pathways. Thus, prolonged stimulation of mGlu7

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ACCEPTED MANUSCRIPT receptors to engage the pathways that enhance release can co-exist with the receptor signaling that inhibits Ca2+ channels in cerebrocortical synaptosomes (Martín et al., 2010). In addition, activation of group III mGluRs reduces the release probability without changing the overall response at the Calyx of Held. Modeling this receptor response suggests that group III mGluR activation may also increase the size of the readily releasable pool (Billups et al., 2005).

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We also found that IP1 accumulation, measured as an alternative to IP3 to monitor PLC-linked GPCRs (Trinquet et al., 2006), parallels the release induced by β-AR and mGlu7 receptor co-

activation. Thus, suppression of mGlu7 receptor-mediated inhibition of adenylyl cyclase leads to supra-additive enhancement of glutamate release and to greater IP1 accumulation, consistent

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with the ability of β-ARs to initiate signaling by augmenting cAMP, as well as through the

downstream activation of PLC and IP1 accumulation (Ferrero et al., 2013a). Furthermore, these data also underline the role of PLC activity and diacylglycerol as determinants of release

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capacity. PKC and Munc13 are the presynaptic targets of diacylglycerol to enhance transmitter release (Rhee et al., 2002; Bauer et al., 2007; Wierda et al., 2007; Lou et al., 2007). Experiments with phorbol esters have shown that the potentiation of evoked neurotransmitter release depends on PKC activity, while the potentiation of spontaneous transmitter release depends on second messenger pathways acting through Munc13 proteins (Lou et al., 2007). Moreover, the increase in IP1 accumulation resulting from the suppression of mGlu7 receptor-mediated inhibition of

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adenylyl cyclase also leads to further Munc13-1 translocation to membranes, supporting the hypothesis that the β-AR and mGlu7 receptor response depends on Munc13-1 activity. Munc13 is a phorbol ester receptor essential for synaptic vesicle priming that plays an important role in neurotransmitter release (Betz et al., 1998; Rhee et al., 2002; Bauer et al., 2007). Munc13

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promotes SNARE complex assembly and it is regulated by diacylglycerol, Ca2+/calmodulin and Ca2+/phospholipid, which bind to the Ca2+/calmodulin site, Ca2+ and C2B domains (Junge et al., 2004; Dimova et al., 2006; Rodriguez-Castañeda et al., 2010; Shin et al., 2010). We found that

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activation of the Munc13-1 dependent pathway, either with the β-AR agonist isoproterenol (Ferrero et al., 2013a) or the mGlu7 receptor agonist L-AP4 (Ferrero et al., unpublished data), results in the approximation of synaptic vesicles to the presynaptic membrane. This is consistent with the role of Munc13 proteins in synaptic vesicle docking, as Munc13-deficient neurons show no synaptic vesicle fusion due to the loss of synaptic vesicle membrane attachment (Imig et al., 2014). Hence, Munc13-1 activation/translocation via DAG binding to its C1 domain could not only integrate the signaling initiated by PLC-coupled receptors like the mGlu7 receptor but also, that by Gs/adenylyl cyclase coupled GPCRs, such as the β-AR.

4.3. Signaling through the mGlu7 receptor in the absence of an agonist

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ACCEPTED MANUSCRIPT One interesting finding of this study is the ability of mGlu7 receptors to maintain signaling after agonist removal. First, we found that the cross-talk between signaling pathways activated by mGlu7 and β-adrenergic receptors persists after a 10 minute exposure with L-AP4 and its subsequent wash-out. Indeed, mGlu7 receptors still diminished isoproterenol-induced release and cAMP after agonist wash-out. As L-AP4 is a hydrophilic agonist that is easily removed by washing the synaptosome preparation, this signaling might occur without the agonist in the

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extracellular medium. The removal of surface GPCRs by endocytosis controls receptor function and after ligand-induced endocytosis, receptors are recycled to the plasma membrane or they are delivered to lysosomes (von Zastrow and Williams, 2012). Endocytosis of mGlu7 receptors can be triggered by prolonged agonist exposure (Lavezzari and Roche, 2007; Pelkey et al., 2005,

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2007), and the mechanisms that regulate mGlu7 receptor trafficking highlight the importance of both PKC phosphorylation and receptor binding to the PICK1 protein in cell surface expression of the mGlu7 receptor (Suh et al., 2008). However, there is little data available regarding the

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ability of the internalized receptor to activate transduction pathways. Recently, internalized receptors were shown to contribute to the overall cAMP cellular response (Iranejad et al., 2013). Thus, it is possible that cross-talk between signaling pathways activated by mGlu7 and βadrenergic receptors relies on the ability of mGlu7 receptors to trigger intracellular signaling pathways during agonist induced internalization, adding further complexity to the signaling

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mediated by these receptors (Iacovelli et al., 2014).

In conclusion, the role that mGlu7 and β adrenergic receptors play in the control of glutamate release is modulated through the interplay between the signaling pathways associated to these receptors. Thus, the release enhancement observed after co-activation of the two receptors is

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attenuated by mGlu7 receptor signaling via Gi/o proteins, and the subsequent inhibition of adenylyl cyclase and reduction in cAMP (Fig. 9). These signaling pathways mediate a cross-talk

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that could prevent strong glutamate release and potential neurotoxicity.

Acknowledgements

We thank María del Carmen Zamora for her excellent technical assistance and Dr F. Ciruela (Universitat de Barcelona) for his advice regarding the co-immunoprecipitation experiments. This work was financed by grants from the Spanish ‘MINECO‘(BFU2010-16947 and BFU 2013-43163R to JS-P), the ‘Instituto de Salud Carlos III’ (RD12/0014) and the ‘Comunidad de Madrid’ (CAM-I2M2 2011-BMD-2349 to JS-P and MT). We thank Dr M. Sefton for editorial assistance. JJF holds a FPU fellowship from Spanish MECD.

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ACCEPTED MANUSCRIPT 5. References Bauer, C. S., Woolley, R. J., Teschemacher, A. G., Seward, E. P., 2007. Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1. J. Neurosci. 27, 212-219.

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Betz, A., Ashery, U., Rickmann, M., Augustin, I., Neher, E., Sudhof, T. C., Rettig, J., Brose, N., 1998. Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. Neuron 21, 123-136. Billups, B., Graham, B. P., Wong, A. Y., Forsythe, I. D., 2005. Unmasking group III metabotropic glutamate autoreceptor function at excitatory synapses in the rat CNS. The J. Physiol. 565, 885-896.

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Branham, M. T., Bustos, M. A., De Blas, G. A., Rehmann, H., Zarelli, V. E., Trevino, C. L., Darszon, A., Mayorga, L. S., Tomes, C. N., 2009. Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis. : J. .Biol. Chem. 284, 24825-24839.

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Brose, N., Hofmann, K., Hata, Y., Sudhof, T. C., 1995. Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270, 25273-25280. Brose, N., Rosenmund, C., 2002. Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters. J. Cell. Sci. 115, 4399-4411. Castillo, P. E., Schoch, S., Schmitz, F., Sudhof, T. C., Malenka, R. C., 2002. RIM1alpha is required for presynaptic long-term potentiation. Nature 415, 327-330.

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Choi, S., Lovinger, D. M., 1996. Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons. J. Neurosci. 16, 36-45. Deng, L., Kaeser, P. S., Xu, W., Sudhof, T. C., 2011. RIM proteins activate vesicle priming by reversing autoinhibitory homodimerization of Munc13. Neuron 69, 317-331.

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Dimova, K., Kawabe, H., Betz, A., Brose, N., Jahn, O., 2006. Characterization of the Munc13calmodulin interaction by photoaffinity labeling. Biochim. Biophys. Acta 1763, 1256-1265.

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Dulubova, I., Lou, X., Lu, J., Huryeva, I., Alam, A., Schneggenburger, R., Sudhof, T. C., Rizo, J., 2005. A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity? EMBO J. 24, 2839-2850. Dzhura, I., Chepurny, O. G., Leech, C. A., Roe, M. W., Dzhura, E., Xu, X., Lu, Y., Schwede, F., Genieser, H. G., Smrcka, A. V., Holz, G. G., 2011. Phospholipase C-epsilon links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans. Islets 3, 121-128. Ferrero, J. J., Alvarez, A. M., Ramírez-Franco, J., Godino, M. C., Bartolome-Martin, D., Aguado, C., Torres, M., Lujan, R., Ciruela, F., Sanchez-Prieto, J., 2013a. beta-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1alpha interaction to potentiate glutamate release at cerebrocortical nerve terminals. J. Biol. Chem. 288, 31370-31385. Ferrero, J. J., Bartolome-Martin, D., Torres, M., Sanchez-Prieto, J., 2013b. Potentiation of mGlu7 receptor-mediated glutamate release at nerve terminals containing N and P/Q type Ca2+ channels. Neuropharmacology 67, 213-222.

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ACCEPTED MANUSCRIPT Ferrero, J. J., Torres, M., Sanchez-Prieto, J., 2011. Inhibitors of diacylglycerol metabolism reduce time to the onset of glutamate release potentation by mGlu7 receptors. Neurosci. Lett. 500, 144-147. Gerachshenko, T., Blackmer, T., Yoon, E-J., Bartleson, C., Hamm, H.E., Alford, S., 2005. Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition. Nat. Neurosci. 8, 597-605.

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Gekel, I., Neher, E., 2008. Application of an Epac activator enhances neurotransmitter release at excitatory central synapses. J. Neurosci. 28, 7991-8002. Herrero, I., Castro, E., Miras-Portugal, M. T., Sanchez-Prieto, J., 1991. Glutamate exocytosis evoked by 4-aminopyridine is inhibited by free fatty acids released from rat cerebrocortical synaptosomes. Neurosci. Lett. 126, 41-44.

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Herrero, I., Sanchez-Prieto, J., 1996. cAMP-dependent facilitation of glutamate release by betaadrenergic receptors in cerebrocortical nerve terminals. J. Biol. Chem. 271, 30554-30560.

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Herrero, I., Vazquez, E., Miras-Portugal, M. T., Sanchez-Prieto, J., 1996. Decrease in [Ca2+]c but not in cAMP Mediates L-AP4 inhibition of glutamate release: PKC-mediated suppression of this inhibitory pathway. Eur. J. Neurosci. 8, 700-709. Huang, C. C., Hsu, K. S., 2006. Presynaptic mechanism underlying cAMP-induced synaptic potentiation in medial prefrontal cortex pyramidal neurons. Mol. Pharmacol. 69, 846-856. Huang, C. C., Hsu, K. S., Gean, P. W., 1996. Isoproterenol potentiates synaptic transmission primarily by enhancing presynaptic calcium influx via P- and/or Q-type calcium channels in the rat amygdala. J. Neurosci. 16, 1026-1033.

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Iacovelli, L., Felicioni, M., Nistico, R., Nicoletti, F., De Blasi, A., 2014. Selective regulation of recombinantly expressed mGlu7 metabotropic glutamate receptors by G protein-coupled receptor kinases and arrestins. Neuropharmacology 77, 303-312. Imig, C., Min, S. W., Krinner, S., Arancillo, M., Rosenmund, C., Sudhof, T. C., Rhee, J., Brose, N., Cooper, B. H., 2014. The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84, 416-431.

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Figure legends

Figure 1 Co-activation of mGlu7 and β-adrenergic receptors reveals a pertussis toxin-sensitive interaction that reduces glutamate release.

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ACCEPTED MANUSCRIPT A) Protocol for receptor activation. B) Mean traces showing glutamate release induced by the Ca2+ ionophore ionomycin (0.5–1 µM) added 2 min after tetrodotoxin, TTx, (1 µM, control). Synaptosomes exposed to the mGlu7 receptor agonist L-AP4 (1 mM, 10 min) were then washed, recovered by centrifugation and resuspended to remove the agonist (L-AP4-treatment). The β-AR agonist isoproterenol (Iso; 100 µM) was added 40 s prior to ionomycin. Synaptosomes were also treated with L-AP4 (1 mM, 10 min) and after washing with

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isoproterenol (L-AP4-treatement + Iso). C) Diagrams summarizing the glutamate release under these conditions normalized to that induced by ionomycin (black dashed lines). The grey dashed line represents the sum of mGlu7 and β-adrenergic receptor induced release. The data represent the mean ± S.E.M. (n=6-9): NS, p>0.05; ***, p<0.001, compared to the control (symbols in the

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diagram, ANOVA with Bonferroni post-hoc test) or other conditions indicated in the figure (Student’s T-test).

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Figure 2

Co-expression of mGlu7 and β-adrenergic receptors at synaptic boutons. Immunofluorescence of synaptosomes fixed onto poly-L-lysine-coated coverslips and stained with antibodies against: β1-AR and the vesicular marker synaptophysin (A: n [number of nerve terminals] = 6094 and N [field number] = 20); the mGlu7a receptor and synaptophysin (C: n=7355 and N= 25); and the β1-AR and mGlu7a receptor (E: n=10643 and N= 68).

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Quantification of β1-AR (B) and mGlu7 receptor (D) expression in nerve terminals containing synaptophysin, and of β1-AR expression in nerve terminals containing the mGlu7a receptor (F). The data represent the mean ± S.E.M. Scale bar, 10 µm. (G), Immunohistofluorescence of brain slices labeled with DAPI and antibodies against β1-ARs and mGlu7a receptors at different

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magnifications. Note the high degree of co-localization of the two receptors, evident as yellow dots. Scale bar, 50 µm. (H, I, J), immunohistofluorescence with antibodies against the β1-AR or mGlu7a receptor and synaptophysin (used as a presynaptic marker), showing the co-localization

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of the two receptors at synaptic boutons. DAPI was used to counterstain the nuclei.

Figure 3

Blocking Gi/o proteins with pertussis toxin further enhanced the release provoked by coactivation of the mGlu7 and β-adrenergic receptors. Synaptosomes were treated with L-AP4 (1 mM, 10 min) and after washing, to remove the agonist, they were also treated with isoproterenol (Iso; 100 µM) which was added 40 s prior to ionomycin (L-AP4-treatement + Iso). Where indicated synaptosomes were preincubated with pertussis toxin (1.5 µg/ml, 2h) prior to L-AP4 and Iso treatment. Diagrams summarizing the

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ACCEPTED MANUSCRIPT glutamate release normalized to that induced by ionomycin (black dashed lines). The data represent the mean ± S.E.M. (n=6-9): ***, p<0.001, compared to the control (symbols in the diagram, ANOVA with Bonferroni post-hoc test) or other conditions indicated in the figure (Student’s T-test).

Figure 4

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Acute mGlu7 receptor activation with L-AP4 reduces the isoproterenol-induced increase in glutamate release and cAMP.

A) Protocol for receptor activation. B) Mean traces showing glutamate release induced with the Ca2+ ionophore ionomycin (0.5–1 µM) in the presence of tetrodotoxin (TTx; 1 µM), added 2

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min prior to ionomycin. L-AP4 (1 mM), and isoproterenol (Iso, 100 µM) added 100 s and 40 s min prior to ionomycin, respectively. The diagrams summarize the effect of L-AP4 on the glutamate release induced by forskolin (15 µM, Fsk: C), isoproterenol (100 µM, Iso: D) or 8-

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pCPT-2´-O-Me-cAMP (50 µM, 8-pCPT: F). Release was normalized to that induced by ionomycin alone (black dashed line). E) L-AP4 reduces the isoprotrenol-induced increase in cAMP in a pertussis toxin-sensitive manner. The phosphodiesterase inhibitor, 3-isobutyl-1methylxanthine (IBMX, 1 mM) was added 35 min prior to measuring cAMP, while the mGlu7 receptor agonist L-AP4 (1 mM) and the β-AR agonist isoproterenol (Iso; 100 µM) were added 11 and 10 min prior to cAMP measurement, respectively. Pertussis toxin (PTX, 1.5 µg/ml) was

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added 1 h and 15 min prior to IBMX. The results are presented as the fold increase compared to the basal cAMP levels in control synaptosomes (black dashed line). The data represent the mean ± S.E.M. (n=4-10): NS, p> 0.05; *, p<0.05; **, p<0.01; ***, p< 0.001 compared to the control (symbols inside the diagram, ANOVA with Bonferroni post-hoc test) or other conditions

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indicated in the figure (Student’s T-test).

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Synaptosomes treated with the mGlu7 receptor agonist L-AP4 further dampen isoproterenolinduced release and limit cAMP after agonist washout. (A) The scheme shows the synaptosome treatments. Synaptosomes treated with L-AP4 (1mM, 10 min) released less glutamate (B) and accumulated less cAMP (C) than was induced by isoproterenol. To measure cAMP, the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1 mM) was added 15 min prior to L-AP4 treatment (1 mM, 10 min), and again after washout. The β-AR agonist isoproterenol (Iso; 100 µM) was then added and 10 min later cAMP was assayed. The DAG-binding protein inhibitor Calphostin C (0.1 µM) was added 20 min prior to L-AP4 treatment in the release experiments. Glutamate release was induced with the Ca2+ ionophore ionomycin (0.5–1 µM) in the presence of tetrodotoxin (TTx; 1 µM) added 2 min prior to ionomycin. The results are presented as the increase relative to the basal cAMP levels or the

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ACCEPTED MANUSCRIPT glutamate release from control synaptosomes (black dashed line). The data represent the mean ± S.E.M. (n=9-13): NS, p> 0.05; *, p<0.05; ***, p< 0.001 compared to the controls (symbols inside the diagram, ANOVA with Bonferroni post-hoc test) or the other conditions indicated in the figure (Student’s T-test).

Figure 6

prevents the interaction that inhibits glutamate release.

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Activation of the β-AR/Gs/AC/cAMP signaling pathway downstream of adenylyl cyclase

(A) Scheme showing the synaptosome treatments. B) Mean traces showing glutamate release

induced by the Ca2+ ionophore ionomycin (0.5–1 µM) added 2 min after TTx (1 µM, control).

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Synaptosomes exposed to the mGlu7 receptor agonist L-AP4 (1mM, 10 min) were then washed, recovered by centrifugation and re-suspended to remove the agonist (L-AP4-treatment). The EPAC protein activator 8-pCPT-2´-O-Me-cAMP was added 40 s prior to ionomycin, both to

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untreated synaptosomes (8-pCPT) and to synaptosomes treated with L-AP4 (1 mM, 10 min: LAP4 treatment + 8-pCPT). C) Diagrams summarizing the glutamate release under these conditions, normalizing release to that induced by ionomycin (black dashed lines). D) Diagrams summarizing the glutamate release induced by L-AP4 treatment alone, the cAMP analogue Sp8-Br-cAMPS (Sp8Br, 250 µM) alone (added 40s prior to ionomycin: Sp8Br), or a combination of both (L-AP4-treatment + Sp8Br), in the absence (D) or presence of Ca2+ channel toxins (E).

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The P/Q-type (ω-Agatoxin-IVA, 0.2 µM) and N-type (ω-Conotoxin-GVIA, 2 µM) Ca2+ channel blockers were added 20 min prior to L-AP4 treatment. The control release corresponds to that induced by ionomycin alone (black dashed line). The grey dashed line represents the sum of release induced by 8-pCPT or Sp8Br and L-AP4. The data represent the mean ± S.E.M. (n=4-8):

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**, p<0.01; ***, p< 0.001 compared to the controls (symbols inside the diagram, ANOVA with Bonferroni post-hoc test) or other conditions indicated in the figure (Student’s T-test).

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Figure 7

Activation of the β-AR/Gs/AC/cAMP signaling pathway downstream of adenylyl cyclase with 8-pCPT or Sp8Br further enhances IP1 accumulation and Munc13-1 translocation. A) Scheme showing the synaptosome treatments. IP1 accumulation (B) and Munc13-1 translocation (C) in synaptosomes treated with L-AP4 (1mM, 10 min) and then washed to remove the agonist (L-AP4-treatment). The β-AR agonist isoproterenol (Iso; 100 µM), or the cAMP analogue Sp-8-Br-cAMPS (Sp8Br, 250 µM) or the specific Epac protein activator 8pCPT-2´-O-Me-cAMP (8-pCPT, 50 µM) were added 10 min prior to measuring IP1 accumulation or Munc13-1 translocation. In IP1 experiments, LiCl (50 mM) was added 15 min prior to L-AP4 and it was maintained until the IP1 was measured (See Materials and Methods). The results are presented as the relative increase compared to the basal IP1 levels in control

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ACCEPTED MANUSCRIPT synaptosomes (black dashed line). The data represent the mean ± S.E.M. (n=9-13): **, p<0.01 ***, p<0.001 compared to the controls (symbols inside the diagram, ANOVA with Bonferroni post-hoc test) or other conditions indicated in the figure (Student’s T-test). In the Munc13-1 translocation experiments, the protein content was assayed in the particulate (P) and soluble (S) fractions of control and treated synaptosomes (top panel C). The sum of the soluble and particulate fractions was taken as 100%. The ratio of the Munc13-1 content in the soluble versus

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particulate fractions is shown (bottom panel in C). The data represent the mean ± S.E.M.: *, p<0.05; ***, p<0.001 compared to either the soluble or particulate fraction, or the

soluble/particulate ratio, in control synaptosomes (symbols inside the diagram, ANOVA with Bonferroni post-hoc test). Other comparisons are as indicated in the figure (Student’s T-test).

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Western blots (n=7-8) were performed from 4 synaptosome preparations.

Figure 8

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Activation of the mGlu7 and β-adrenergic receptors promote the interaction between Munc13-1 and RIM1.

Synaptosomes exposed for 10 min to the mGlu7 receptor agonist L-AP4 (1 mM) or to the β-AR agonist isoproterenol (Iso, 100 µM) were solubilized and immunoprecipitated with a rabbit antiFLAG antibody (3.5 µg; IP: IgGr), a mouse anti-Munc13-1 antibody (3.5 µg; IP: Munc13-1) or a rabbit anti-RIM1 (3.5 µg; IP: RIM1). Extracts (Crude) and immunoprecipitates (IP) were

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analyzed in Western blots (IB) probed with the rabbit Munc13-1 antibody.IRDye 800 (1:15,000) were used as a secondary antibody. B) Quantification of the L-AP4 and Isoproterenol-induced Munc13-1-RIM1 interaction. The ratio between the Munc13-1 immunoprecipitated with anti-RIM1 and the total Munc13-1 (IP ratio) was calculated and

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normalized to the IP ratio found in the untreated synaptosomes (Control). The data are expressed as the mean ± S.E.M of three independent experiments: *, p<0.05; **, p<0.01 for the

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comparisons indicated in the figure (Student’s T-test).

Figure 9

The interplay between mGlu7 and β-adrenergic receptor signaling. The mGlu7 receptor stimulates a pertussis toxin-resistant G protein to enhance PLC activity, thereby increasing PIP2 hydrolysis and the generation of diacylglycerol, which in turn binds to and activates/translocates the non-kinase DAG-binding protein Munc13-1 that promotes glutamate release. The β-AR stimulates the Gs protein and adenylyl cyclase, thereby increasing cAMP levels. In turn, cAMP activates EPAC, which can promote PLC-dependent PIP2 hydrolysis to produce DAG and activate/translocate Munc13-1. However, the activity of the cAMP-dependent pathway is down-regulated by mGlu7 receptor signaling via Gi/o proteins and the subsequent inhibition of adenylyl cyclase, and by a reduction in cAMP levels. PLC,

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