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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Nov 29;174(24):4725–4737. doi: 10.1111/bph.14051

VPAC1 and VPAC2 receptor activation on GABA release from hippocampal nerve terminals involve several different signalling pathways

Diana Cunha‐Reis 1,2,3,, Joaquim Alexandre Ribeiro 1,2, Rodrigo F M de Almeida 3, Ana M Sebastião 1,2
PMCID: PMC5727335  PMID: 28945273

Abstract

Background and Purpose

Vasoactive intestinal peptide (VIP) is an important modulator of hippocampal synaptic transmission that influences both GABAergic synaptic transmission and glutamatergic cell excitability through activation of VPAC1 and VPAC2 receptors. Presynaptic enhancement of GABA release contributes to VIP modulation of hippocampal synaptic transmission.

Experimental Approach

We investigated which VIP receptors and coupled transduction pathways were involved in VIP enhancement of K+‐evoked [3H]‐GABA release from isolated nerve terminals of rat hippocampus.

Key Results

VIP enhancement of [3H]‐GABA release was potentiated in the presence of the VPAC1 receptor antagonist PG 97‐269 but converted into an inhibition in the presence of the VPAC2 receptor antagonist PG 99‐465, suggesting that activation of VPAC1 receptors inhibits and activation of VPAC2 receptors enhances, GABA release. A VPAC1 receptor agonist inhibited exocytotic voltage‐gated calcium channel (VGCC)‐dependent [3H]‐GABA release through activation of protein Gi/o, an effect also dependent on PKC activity. A VPAC2 receptor agonist enhanced both exocytotic VGCC‐dependent release through protein Gs‐dependent, PKA‐dependent and PKC‐dependent mechanisms and GABA transporter 1‐mediated [3H]‐GABA release through a Gs protein‐dependent and PKC‐dependent mechanism.

Conclusions and Implications

Our results show that VPAC1 and VPAC2 VIP receptors have opposing actions on GABA release from hippocampal nerve terminals through activation of different transduction pathways. As VPAC1 and VPAC2 receptors are located in different layers of Ammon's horn, our results suggest that these VIP receptors underlie different modulation of synaptic transmission to pyramidal cell dendrites and cell bodies, with important consequences for their possible therapeutic application in the treatment of epilepsy.


Abbreviations

aCSF

artificial CSF solution

CamKII

Ca2+/calmodulin‐dependent protein kinase II

GAT‐1

GABA transporter 1

GF‐109203X

3‐[1‐[3‐(dimethylamino)propyl]‐1H‐indol‐3‐yl]‐4‐(1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione

GRF

growth hormone‐releasing factor

H‐89

N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulfonamide

SKF89976A

(1‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid

VGCC

voltage‐gated calcium channel

VIP

vasoactive intestinal peptide

Introduction

Vasoactive intestinal peptide (VIP) modulates hippocampal synaptic transmission and excitability (Yang et al., 2010) and is present in the hippocampus exclusively in interneurones (Acsády et al., 1996a). As such, most of its actions are dependent on GABAergic transmission (Wang et al., 1997; Cunha‐Reis et al., 2004). VIP modulation of GABAergic transmission occurs both at the presynaptic component, by modulating GABA release, and at the postsynaptic component, by modulating GABAergic currents (Wang et al., 1997; Cunha‐Reis et al., 2004, 2008). These GABAergic‐dependent effects of VIP are also important in the regulation of hippocampal synaptic plasticity (Cunha‐Reis et al., 2010, 2014). VIP has also postsynaptic actions on pyramidal cells that are independent of GABAergic transmission (Cunha‐Reis et al., 2006).

In the hippocampus, two selective high‐affinity VIP receptors: VPAC1 and VPAC2 receptors that belong to the class II family of GPCRs (White et al., 2010; Yang et al., 2010) have been identified by in situ hybridization, autoradiography and immunohistochemistry (Joo et al., 2004; White et al., 2010). These receptors are encoded by two different genes, share only 55% similarity and have similar affinities for VIP. A third receptor (PAC1) binds VIP with much lower affinity (White et al., 2010; Harmar et al., 2012). All three receptors also recognize, with high affinity, the pituitary adenylate cyclase‐activating peptide (PACAP). Both VPAC1 and VPAC2 receptors are involved in the modulation of hippocampal synaptic transmission and synaptic plasticity by VIP (Cunha‐Reis et al., 2005, 2010). This suggests that both receptors may be involved in the regulation of GABA release. As neuropeptide receptors have been proposed as important targets for the development of anticonvulsive drugs (Clynen et al., 2014), studying their role in the modulation of GABA release is of particular clinical relevance.

VPAC1 and VPAC2 receptors are positively coupled to adenylate cyclase by Gs activation in most tissues (White et al., 2010; Harmar et al., 2012). Postsynaptic excitatory actions of VIP in hippocampal pyramidal cells involve either cAMP or cAMP‐dependent mechanisms (Haas and Gähwiler, 1992; Haug and Storm, 2000; Ciranna and Cavallaro, 2003), and VIP‐mediated enhancement of mIPSCs in hippocampal cell cultures occurs through a PKA‐dependent mechanism (Wang et al., 1997). However, VPAC1 and VPAC2 receptors can also couple to other signalling/G protein‐dependent mechanisms in different brain preparations (Fatatis et al., 1994; Gressens et al., 1998; Nielsen et al., 2002). Furthermore, VPAC1 receptor couples to Gi/o proteins in the hippocampus (Shreeve, 2002), and VPAC1‐mediated enhancement of synaptic transmission in the CA1 area of the hippocampus is dependent on both PKA and PKC activity (Cunha‐Reis et al., 2005). This suggests that VIP receptors can couple to different signalling pathways in the hippocampus.

Hippocampal inhibitory and excitatory circuits are tightly interconnected, making it difficult to discriminate between the actions of VIP on release of inhibitory and excitatory transmitters in vivo or in a slice preparation. Isolated nerve terminal preparations are virtually free of connections with other neuronal components and have been successfully used to evaluate presynaptic modulation of neurotransmitter release (Ghijsen and Leenders, 2005). In the present work, we used this preparation to evaluate the VIP receptors and transduction pathways involved in the presynaptic enhancement of GABA release caused by VIP.

Methods

Animals

All animal care and experimental procedures were performed in accordance with the Portuguese law and the European Community (86/609/EEC) and ARRIVE guidelines. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Male outbred Wistar rats (5–6 weeks old) were purchased from Harlan Iberica (Barcelona, Spain) and housed in the local animal house until use. Animals were maintained under a 12:12 h light/dark cycle at a temperature of 22°C, with food and water ad libitum.

Preparation of isolated nerve terminals

[3H]‐GABA release experiments were carried out using hippocampal nerve terminals isolated from rats and the preparation of these nerve terminals is described below. Briefly, two hippocampi were homogenized in 4 mL ice‐cold sucrose solution (sucrose 0.32 M, EDTA 1 mM, HEPES 10 mM and BSA 1 mg·mL−1, pH 7.4) and centrifuged at 3000× g for 10 min at 4°C; the supernatant was then centrifuged at 14 000× g for 12 min at 4°C, and the pellet resuspended in 3 mL of a Percoll 45% (v·v−1) in modified artificial CSF solution (aCSF) without calcium (mM: NaCl 140, EDTA 1, HEPES 10 and KCl 5, glucose 5, pH 7.4).

After centrifugation at 14 000× g for 2 min at 4°C, the top layer (which corresponds to the synaptosomal fraction) was washed twice with modified aCSF without Ca2+. The pellet (synaptosomal fraction) was resuspended in complete aCSF (mM: NaCl 125, KCl 3, glucose 10, MgSO4 1.2, NaH2PO4 1, CaCl2 1.5 and HEPES 10, pH 7.4) containing 0.1 mM (aminooxy)acetic acid (AOAA) to avoid GABA catabolism by 4‐aminobutyrate aminotransferase. The protein content of the hippocampal synaptosomal fraction was determined by the Lowry method modified according to Peterson (1977) using BSA as a standard.

[3H]‐GABA release experiments

The release of [3H]‐GABA from isolated hippocampal nerve terminals was performed as previously described (Cunha‐Reis et al., 2008). Briefly, the purified synaptosomes, loaded for 20 min with [3H]‐GABA (1.6 μCi·mL−1, 18.75 nM) in the presence of unlabelled GABA (0.125 μM) were introduced into four parallel 900 μL superfusion chambers (≈0.58 mg protein per chamber) and kept in continuous superfusion (0.6 mL·min−1) with oxygenated (95% O2 and 5% CO2) Krebs solution (mM: NaCl 125, KCl 3, NaH2PO4 1, NaHCO3 25, CaCl2 1.5, MgCl2 1.2, glucose 10 and AOAA, 0.1) containing 1 μM nipecotic acid, to avoid GABA reuptake by synaptosomes. For each test, duplicate perfusion chambers were used, and the results averaged before any other calculations were performed, to ensure the reliability of single values and avoid deviations caused occasionally by inefficient bubble trapping. The superfusion system was previously coated with BSA (0.1 mg·mL−1) to prevent neuropeptide adhesion. After a 30 min equilibration period, the effluent was collected in 2 min fractions (≈1.2 mL), and samples kept for scintillation counting analysis. Synaptosomes were stimulated at 4 min (S1) and 22 min (S2) after starting sample collection, with K+ (25 mM) for 2 min (isomolar substitution of Na+ by K+ in the superfusion buffer). Tritium released by stimulation was about 50% Ca2+ dependent (see the Results section) and mainly composed of [3H]‐GABA, as shown by HPLC analysis (Cunha et al., 1997). In test conditions, VIP was present from the 16th (6 min before S2) up to the 32nd minute after starting sample collection. When testing the action of VIP in the presence of other drugs, these were present from 15 min before starting sample collection until the end of the experiment. The radioactivity measurements were normalized to the total amount of tritium retained by the synaptosomes at the end of experiment that reflected the total radioactivity loaded. The amount of radioactivity released by stimulation was calculated by integration of the area under the evoked peak of tritium release after subtraction of basal tritium release. Effects (taken as % change) were evaluated by modification of the S2/S1 ratios in test and control conditions.

Synaptosomal disruption was evaluated by comparing LDH activity in the incubation bath with that found in the synaptosomal pellet upon its solubilization with 2% (v·v−1) Triton X‐100 (Keiding et al., 1974).

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are presented as mean ± SEM. Values of n represent the number of animals used. For each condition (test and control), duplicate perfusion chambers were used, and the results (basal release, evoked release and S2/S1 ratios) were averaged before any other calculations were performed, to ensure the reliability of the observations inefficient bubble trapping. Statistical analysis was performed using Prism version 6.01 for Windows (Graph Pad Software Inc., La Jolla, CA 92037, USA). When testing the significance of the effect of VIP versus 0%, a Student's t‐test was used. When comparing the effect of VIP in different experimental conditions, one‐way ANOVA was used followed by Dunnett's or Sidak's multiple‐comparison test as stated in the figure legends. P values of 0.05 or less were considered to represent significant differences.

Materials

VIP (Novabiochem, Madison, Wisconsin, USA), [Ac‐Tyr1, D‐Phe2] growth hormone‐releasing factor (GRF) (1‐29) (Tocris Cookson, Bristol, UK) and PG 97‐269, PG 99‐465, RO 25‐1553 and [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (a kind gift from Professor Patrick Robberecht) were made up in 0.1 mM stock solution in CH3COOH 1% (v·v−1). Cholera toxin and Pertussis toxin (Sigma/RBI, St. Louis, Missouri, EUA) were suspended in 50 mM stock in PBS. H‐89 (N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulfonamide; Calbiochem, Madison, Wisconsin, USA) and GF‐109203X (3‐[1‐[3‐(dimethylamino)propyl]‐1H‐indol‐3‐yl]‐4‐(1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione; Sigma/RBI) were made up in 5 mM stock solutions in DMSO. The maximal DMSO and CH3COOH concentrations used were devoid of effects on tritium release. SKF89976A (1‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid; Tocris Cookson), GABA, AOAA (Sigma/RBI), CdCl2 and (±)‐nipecotic acid (Sigma/RBI) were prepared in aqueous solution. [3H]‐GABA (80.0–86.0 Ci·mmol−1) was obtained from Amersham. KN‐62 was supplied by Calbiochem.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b,c,d).

Results

Under the conditions used in this study, the amount of tritium retained by the synaptosomes at the beginning of sample collection was 6.82 ± 0.46 × 1010 Bq·mg protein−1 (n = 160). In the absence of added drugs, the basal release of tritium was 4.08 ± 0.56 × 107 Bq·mg protein−1 (n = 72). Stimulation of synaptosomes with 25 mM K+ for 2 min caused an approximate sixfold increase in the amount of tritium released to (2.36 ± 0.32) × 108 Bq·mg protein−1 (n = 72) for S1 and 1.98 ± 0.17 × 108 Bq·mg protein−1 (n = 72) for S2 (S2/S1 ratio – 0.815 ± 0.007, n = 72). Addition of 0.5 mM EGTA and reduction of the extracellular calcium concentration to 200 nM during S2 decreased the S2/S1 ratio to 0.435 ± 0.012 (n = 6), inhibiting by 51.3 ± 5.0% (n = 6) evoked tritium release, compared with matched controls (S2/S1 ratio: 0.848 ± 0.009, n = 6), in agreement to what was previously observed (Kirk and Richardson, 1994). To investigate the relative contribution of voltage‐gated calcium channels (VGCCs) and GABA transporter 1 (GAT‐1) carrier reversal on [3H]‐GABA release, we used selective inhibitors. In control conditions, the S2/S1 ratio obtained was 0.818 ± 0.012 (n = 5). Addition of the VGCC inhibitor CdCl2 (200 μM) (Nachshen, 1985) before S2 decreased the S2/S1 ratio to 0.393 ± 0.017 (n = 5), thus inhibiting by 50.9 ± 1.9% (n = 5) the evoked tritium release. When the selective GAT‐1 inhibitor SKF89976A (20 μM) (Yunger et al., 1984; Borden et al., 1994) was present during S2, the S2/S1 ratio decreased to 0.592 ± 0.046 (n = 5), inhibiting evoked tritium release by 27.2 ± 4.9% (n = 5). When applied simultaneously during S2, the VGCC inhibitor and of the GAT‐1 inhibitor reduced by 73.2 ± 3.8% (n = 5) the evoked tritium release. In these experiments, synaptosomal disruption was small since only 12.7 ± 0.6% (n = 4) of the total LDH activity (EC 1.1.1.27) was found at the end of an experiment upon superfusion of the synaptosomes in closed circuit.

VIP (0.3–30 nM) enhances K+‐evoked [3H]‐GABA release in a biphasic manner (Cunha‐Reis et al., 2004). The maximum effect of VIP is obtained at 1 nM and is abolished in the presence of [Ac‐Tyr1, D‐Phe2] GRF (1‐29) (300 nM) (Cunha‐Reis et al., 2004), an antagonist of VIP receptors that does not discriminate between VIP receptor subtypes and in a concentration that can selectively block VIP receptors (Waelbroeck et al., 1985; Liu et al., 2000), but no other receptors of the family present in the hippocampus. This biphasic nature of VIP action suggests that more than one mechanism mediates VIP action on GABA release.

To test the involvement of each of the two VIP receptors, VPAC1 and VPAC2, in VIP enhancement of [3H]‐GABA release, the VPAC1 receptor selective antagonist PG 97‐269 (Gourlet et al., 1997a) and the VPAC2 receptor selective antagonist PG 99‐465 (Moreno et al., 2000) were used. In this set of experiments, VIP (1 nM) increased the K+‐evoked [3H]‐GABA release of 51.0 ± 3.2% (n = 12, Figure 1A) in the absence of other drugs. In the presence of the VPAC1 receptor antagonist, PG 97‐269 (100 nM), the excitatory effect of VIP (1 nM) on K+‐evoked [3H]‐GABA release was enhanced (F (3, 22) = 65.05; n = 5, Figure 1E) . When the VPAC2 receptor antagonist, PG 99‐465 (100 nM), was present, the effect of 1 nM VIP was reversed (F (3, 22) = 65.05; n = 5,), now inhibiting K+‐evoked [3H]‐GABA release (Figure 1E). When applied to the synaptosomes from the beginning of the experiment, PG 97‐269 (100 nM, n = 5) and PG 99‐465 (100 nM, n = 5) caused a significant decrease in the S2/S1 ratios in control conditions (Figure 1D, F (5, 30) = 45.57).

Figure 1.

Figure 1

VIP bidirectionally modulates K+‐evoked [3H]‐GABA release from hippocampal nerve terminals. (A–C) Time course of individual [3H]‐GABA release experiments in which the effect of VIP (1 nM) was tested in the absence of added drugs (A) or in the presence of either the selective VPAC1 receptor antagonist PG 97‐269 (100 nM, B) or the selective VPAC2 receptor antagonist PG 99‐465 (100 nM, C). Nerve terminals were labelled with [3H]‐GABA as described and release of [3H]‐GABA was evoked by two 2 min pulses of 25 mM KCl as indicated by the horizontal bars. VIP was added to the test chambers before S2, as indicated by the horizontal bar, whereas it was not added to the parallel control chambers. Each point represents the mean results of one experiment performed in duplicate chambers. (D) Averaged S2/S1 ratios observed when testing the action of VIP applied alone or in the presence of the VPAC1 and VPAC2 receptor selective antagonists on K+‐evoked [3H]‐GABA release. Evoked release was calculated by integration of the peak, after subtraction of the basal [3H]‐GABA release, and S2/S1 ratios were obtained. Each bar represents the mean ± SEM of the results obtained in 4–12 experiments performed in duplicate. ★ P < 0.05, significantly different from the S2/S1 ratio in the corresponding control conditions; Student's t‐test. (E) Averaged effects for the enhancement of K+‐evoked [3H]‐GABA release caused by VIP in hippocampal nerve terminals in the absence and in the presence of VPAC1 or VPAC2 receptor selective antagonists, or the broad‐range VIP receptor blocker [Ac‐Tyr1, D‐Phe2] GRF (1‐29). The effect of VIP was calculated by comparing the S2/S1 ratio obtained in test (presence of VIP during S2) and in control conditions. Each bar represents the mean ± SEM of results obtained from n=4–12 experiments. ★ P < 0.05, significantly different from 1nM VIP alone; ANOVA, followed by Dunnett's multiple‐comparison test.

The results above described suggest that VPAC1 and VPAC2 receptors exert opposing effects on [3H]‐GABA release. To confirm this hypothesis, the action of the VPAC1 selective agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (Gourlet et al., 1997b) and of the VPAC2 selective agonist RO 25‐1553 (Gourlet et al., 1997c) on K+‐evoked [3H]‐GABA release was tested. [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (1–30 nM) inhibited K+‐evoked [3H]‐GABA release in a concentration‐dependent manner (Figure 2), attaining a maximal effect at 10 nM (n = 7). RO 25‐1553 (0.3–30 nM) caused a concentration‐dependent enhancement of K+‐evoked [3H]‐GABA release (Figure 2), reaching a maximal effect at 3 nM (n = 6). For the 10 and 30 nM concentrations, a small decrease in the ability of RO 25‐1553 to cause enhancement of K+‐evoked [3H]‐GABA release was observed. This probably reflects a loss of selectivity for the VPAC2 receptor at these concentrations (Vertongen et al., 1997a).

Figure 2.

Figure 2

Selective VPAC1 and VPAC2 receptor activation has opposing actions on K+‐evoked [3H]‐GABA release from hippocampal nerve terminals. (A–C) Time course of individual [3H]‐GABA release experiments in which the effect of VIP (1 nM, A), the VPAC1 receptor agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC2 receptor agonist RO 25‐1553 (10 nM, C) was tested. Nerve terminals were labelled with [3H]‐GABA as described and release of [3H]‐GABA was evoked by two 2 min pulses of 25 mM KCl as indicated by the vertical bars. VIP or VIP receptor agonists were added to the test chambers before S2, as indicated by the horizontal bar, whereas were not added to the parallel control chambers. Each point represents the mean results of one experiment performed in duplicate chambers. (D) Averaged S2/S1 ratios observed when testing the action of VIP, [K15, R16, L27] VIP (1‐7)/GRF (8‐27) or RO 25‐1553 on K+‐evoked [3H]‐GABA release. The S2/S1 ratios were calculated as described . Each bar represents the mean ± SEM of the results obtained in 6–12 experiments performed in duplicate. ★ P < 0.05, significantly different from S2/S1 ratio in control conditions; ANOVA, followed by Dunnett's multiple‐comparison test. (E) Concentration–response curves for the effect of [K15, R16, L27] VIP (1‐7)/GRF (8‐27) or RO 25‐1553 on K+‐evoked [3H]‐GABA in hippocampal nerve terminals. The effect of VIP receptor agonists was calculated by comparing the S2/S1 ratio obtained in test (presence of agonists during S2) and in control conditions. Each point represents the mean ± SEM of results obtained in 6–10 experiments.

For subsequent studies, aiming to elucidate the transduction pathways activated by VIP to modulate [3H]‐GABA release, [K15, R16, L27] VIP (1‐7)/GRF (8‐27) was used in a concentration (10 nM) that caused maximal inhibition of [3H]‐GABA release. This concentration is also 10 times the EC50 for stimulation of cAMP production through rat VPAC1 receptors expressed in CHO cells (Gourlet et al., 1997b). RO 25‐1553 was used in a concentration (10 nM) that caused near maximal enhancement of [3H]‐GABA release without significant attenuation of the response. This concentration is also 10 times its IC50 for VIP binding to rat VPAC2 receptors expressed in CHO cells (Vertongen et al., 1997a) and binds negligibly to rat VPAC1 or rat PAC1 receptors (Gourlet et al., 1997c).

To investigate the transmitter release mechanisms (exocytotic vs. carrier reversal) involved in VIP enhancement of [3H]‐GABA release, we investigated the role of VGCCs and the GAT‐1 carrier‐mediated processes in the effects of VIP and VIP receptor agonists. Upon blockade of VGCCs with CdCl2 (200 μM) (Nachshen, 1985), the effect of 1 nM VIP was strongly attenuated (F (2, 19) = 17.26, n = 5, Figure 3A). Selective blockade of the GAT‐1 carrier using SKF89976A (20 μM) (Yunger et al., 1984; Borden et al., 1994) caused a smaller reduction in the effect of 1 nM VIP (F (2, 19) = 17.26, n = 5, Figure 3A). Upon blockade of VGCCs with CdCl2 (200 μM), the inhibitory effect of the VPAC1 agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM) was reduced (F (2, 12) = 15.47, n = 4, Figure 3B), and the enhancement of [3H]‐GABA release caused by the VPAC2 agonist RO 25‐1553 (10 nM) was also reduced (F (2, 14) = 20.24, n = 4, Figure 3C). Selective blockade of the GAT‐1 carrier using SKF89976A (20 μM) did not change the inhibitory effect of 10 nM [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (F (2, 12) = 15.47, n = 4, Figure 3B) and reduced the enhancement of [3H]‐GABA release caused by RO 25‐1553 (F (2, 14) = 20.24, n = 4, Figure 3C). When applied to the synaptosomes from the beginning of the experiment, CdCl2 (200 μM, n = 13) caused an enhancement in the basal release and decreased the release evoked by stimulation. CdCl2 (200 μM, n = 13), but not SKF89976A (20 μM, n = 13), caused an increase in the S2/S1 ratio in control conditions to 1.250 ± 0.008.

Figure 3.

Figure 3

VIP receptor‐mediated modulation of [3H]‐GABA release involves both VGCC‐dependent and GAT‐1 carrier‐dependent release mechanisms. Averaged effects on [3H]‐GABA release elicited by VIP (1 nM, A), the VPAC1 receptor agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC2 receptor agonist RO 25‐1553 (10 nM, C) tested alone or in the presence of either the VGCC inhibitor (CdCl2) or the GAT‐1 inhibitor (SKF89976A) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S2/S1 ratio obtained in test (presence of VIP or VIP receptor agonists during S2) and in control conditions. Each bar represents the mean ± SEM of results obtained in 4–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone; ANOVA, followed by Dunnett's multiple‐comparison test.

Because VIP‐mediated actions in the hippocampus have previously been associated with an increase of intracellular cAMP (Wright and Schoepp, 1996), the involvement of the cAMP‐mediated transducing system in VIP facilitation of [3H]‐GABA release was investigated using the PKA inhibitor, H‐89 (Chijiwa et al., 1990). When supramaximal concentrations of H‐89 (1 μM) were present during S1 and S2, VIP facilitation of [3H]‐GABA release was markedly attenuated, with VIP causing only a small increase of [3H]‐GABA release (F (10, 38) = 63.10, n = 4, Figure 4A).

Figure 4.

Figure 4

VIP receptor‐mediated modulation of [3H]‐GABA release involves both PKA‐dependent, PKC‐dependent and CaMKII‐dependent mechanisms. Averaged effects on [3H]‐GABA release elicited by VIP (1 nM, A), the VPAC1 receptor agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM, B) and the VPAC2 receptor agonist RO 25‐1553 (10 nM, C) tested alone or in the presence of either the PKA inhibitor (H‐89), the PKC inhibitor (GF109203X) or the CaMKII inhibitor (KN‐62) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S2/S1 ratio obtained in test (presence of VIP or VIP receptor agonists during S2) and in control conditions. Each bar represents the mean ± SEM of results obtained in 4–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone; ANOVA, followed by Dunnett's multiple‐comparison test.

Some VIP‐mediated actions in the nervous system have been associated with an increase in PKC activity (Gressens et al., 1998) and do not depend on the Gs/adenylate cyclase/PKA transduction pathway, such as VPAC1‐mediated actions on synaptic transmission in the hippocampus, which are dependent on PKC but not PKA activity (Cunha‐Reis et al., 2005). As PKC activation is also known to enhance GABA release (Capogna et al., 1995), we investigated whether VIP facilitation of GABA release was dependent on PKC activity. By preventing PKC activation with 1 μM GF‐109203X (Toullec et al., 1991), the excitatory effect of 1 nM VIP on [3H]‐GABA release was only slightly inhibited (F (10, 38) = 63.10, n = 5, Figure 4A). However, as shown in Figure 4A, simultaneous PKA and PKC inhibition with H‐89 (1 μM) together with GF‐109203X (1 μM) abolished (F (10, 38) = 63.10) VIP facilitation of [3H]‐GABA release. Ca2+/calmodulin‐dependent protein kinase II (CamKII) is known to modulate GABA release (Sitges et al., 1995). To assess the contribution of CamKII to the effects of VIP on GABA release, we used a selective CamKII inhibitor, KN‐62. In the presence of KN‐62 (50 μM), the excitatory effect of 1 nM VIP was strongly inhibited (F (10, 38) = 63.10, n = 5, Figure 4A). In these experiments, protein kinase inhibitors did not change the basal [3H]‐GABA release or the S2/S1 ratios obtained for control chambers (no VIP added during S2), compared with the amounts obtained in the experiments where no drugs were added to the synaptosomes.

We next investigated the involvement of the Gs/adenylate cyclase/PKA transduction system in VPAC1‐mediated inhibition and VPAC2‐mediated enhancement of [3H]‐GABA release. In the presence of the selective PKA inhibitor (Chijiwa et al., 1990) H‐89 (1 μM), the inhibitory effect of 10 nM [K15, R16, L27] VIP (1‐7)/GRF (8‐27) on [3H]‐GABA release was not changed (F (6, 22) = 28.82, n = 4, Figure 4B), and the enhancement of [3H]‐GABA release caused by RO 25‐1553 was abolished (F (7, 26) = 29.59, n = 4, Figure 4C). In view of the absence of effect of the selective PKA inhibitor on the inhibitory effect of the VPAC1 agonist on [3H]‐GABA release, the involvement of PKC was also investigated. Upon selective inhibition of PKC with GF 109203X (1 μM) (Toullec et al., 1991), the inhibitory effect of [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM) on [3H]‐GABA release was attenuated (F (6, 22) = 28.82, n = 4, Figure 4B). The enhancement of [3H]‐GABA release caused by RO 25‐1553 (10 nM) was also inhibited (F (7, 26) = 29.59, n = 4, Figure 4C) in the presence of GF 109203X (1 μM). H‐89 (1 μM) and GF 109203X (1 μM) caused no significant changes in the S2/S1 ratios obtained in control conditions, although H‐89 (1 μM) caused a significant increase in both the basal and evoked release of [3H]‐GABA.

The results above suggest that activation of VPAC1 and VPAC2 receptors operates different transduction pathways in the modulation of [3H]‐GABA release. As such, we investigated the G‐protein subtypes involved in VPAC1 receptor‐mediated inhibition and VPAC2 receptor‐mediated enhancement of [3H]‐GABA release. The involvement of Gs proteins was tested using cholera toxin, which desensitizes Gs‐mediated responses by promoting ADP ribosylation of the G subunit of Gs proteins, stabilizing the dissociation of α and βγ subunits and causing persistent activation of adenylate cyclase (Cassel and Pfeuffer, 1978). In preparations treated with cholera toxin (100 ng·mL−1), the enhancement caused by 1 nM VIP on [3H]‐GABA release was turned into an inhibition (F (10, 38) = 63.10, n = 4, Figure 5A). The inhibition of [3H]‐GABA release by the VPAC1 agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM, n = 4) was not changed (F (6, 22) = 28.82) but the enhancement of [3H]‐GABA release caused by VPAC2 agonist RO 25‐1553 (10 nM) was abolished (F (7, 26) = 29.59, n = 3, Figure 5D). Upon inhibition of Gi/o proteins with Pertussis toxin (100 ng·mL−1), which ADP‐ribosylates a cysteine residue of the α subunit of Gi/Go proteins, blocking their interaction with the receptors (Yamane and Fung, 1993), the increase caused by 1 nM VIP on [3H]‐GABA release was enhanced (F (10, 38) = 63.10, n = 4, Figure 5B). Pertussis toxin (100 ng·mL−1) abolished (F (6, 22) = 28.82, Figure 5C) the inhibition of [3H]‐GABA release by [K15, R16, L27] VIP (1‐7)/GRF (8‐27) but did not change (F (7, 26) = 29.59, n = 4, Figure 5D) the enhancement of [3H]‐GABA release caused by the VPAC2 agonist RO 25‐1553 (10 nM).

Figure 5.

Figure 5

Differential G‐protein coupling and downstream transduction mechanisms are involved in VPAC1 and VPAC2 receptor‐mediated modulation of [3H]‐GABA release. Averaged effects on [3H]‐GABA release elicited by VIP (1 nM, A and B), the VPAC1 receptor agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27) (10 nM, C) and the VPAC2 receptor antagonist RO 25‐1553 (10 nM, D) tested alone, in the presence of cholera toxin (ChTx) or Pertussis toxin (PTx) together with either the PKA inhibitor (H‐89) or the PKC inhibitor (GF109203X) in hippocampal nerve terminals. The effect of VIP or selective agonists was calculated by comparing the S2/S1 ratio obtained in test (presence of VIP or VIP receptor agonists during S2) and in control conditions. Each bar represents the mean ± SEM of results obtained in 3–12 experiments. ★ P < 0.05, significantly different from VIP or selective agonists alone or, VIP or agonists in the presence of either ChTx or PTx; ANOVA, followed by Sidak's multiple‐comparison test.

Aiming to elucidate the transduction pathways lying downstream of VPAC2‐mediated activation of Gs and VPAC1 mediated activation Gi, we tested the influence of PKA and PKC inhibitors on the effects of VPAC1 and VPAC2 receptor agonists observed upon treatment with either cholera toxin or Pertussis toxin. In the presence of cholera toxin (100 ng·mL−1), the inhibition caused by 1 nM VIP on [3H]‐GABA release (F (10, 38) = 63.10, n = 4, Figure 5A) was not changed by the presence of H‐89 (1 μM) but was mildly although not significantly attenuated (F (10, 38) = 63.10, n = 4, Figure 5A) in the presence of the PKC inhibitor GF109203X (1 μM). The inhibition caused by 10 nM [K15, R16, L27] VIP (1‐7)/GRF (8‐27) on [3H]‐GABA release in the presence of 100 ng·mL−1 cholera toxin (n = 3, Figure 5C) was similarly not changed (F (6, 22) = 28.82, Figure 5C) by the presence of H‐89 (1 μM) but was again only mildly but not significantly attenuated (F (6, 22) = 28.82, n = 4, Figure 5C) in the presence of the PKC inhibitor GF109203X (1 μM). Upon blockade of Gi/o proteins using Pertussis toxin (100 ng·mL−1), the enhancement caused by 1 nM VIP of [3H]‐GABA release (n = 4, Figure 5B) was abolished (F (10, 38) = 63.10, n = 3) by the presence of H‐89 (1 μM) and was markedly attenuated (F (10, 38) = 63.10, n = 3, Figure 5B) in the presence of the PKC inhibitor GF109203X (1 μM). The facilitation caused by RO 25‐1553 (10 nM) in the presence of Pertussis toxin (100 ng·mL−1) on [3H]‐GABA release (n = 4, Figure 5D) was blocked (F (7, 26) = 29.59, n = 3, Figure 5D) by H‐89 (1 μM) and was markedly attenuated (F (7, 26) = 29.59, n = 3, F (7, 26) = 29.59, Figure 5D) by the PKC inhibitor GF109203X (1 μM). The presence of cholera toxin (100 ng·mL−1) or Pertussis toxin (100 ng·mL−1) throughout the experiment increased the basal release and the release evoked by stimulation (S1 and S2). The S2/S1 ratio in control conditions in the presence of cholera toxin (100 ng·mL−1) was reduced to 0.698 ± 0.037 (n = 6) and in the presence of Pertussis toxin (100 ng·mL−1) was reduced to 0.669 ± 0.012 (n = 6). When the toxins were applied together with either H‐89 (1 μM) or GF 109203X (1 μM) throughout the experiment, the differences in basal and evoked release were not significant except when Pertussis toxin (100 ng·mL−1) was co‐applied with GF 109203X (1 μM) for which the S2/S1 ratio in control conditions was 0.683 ± 0.016 (n = 3).

Trying to elucidate which transduction pathways were involved in either VGCC‐dependent or GAT‐1‐dependent components of VPAC2‐mediated enhancement of [3H]‐GABA release, we tested the influence of PKA and PKC inhibitors on VPAC2‐mediated effects remaining after GAT‐1 blockade or VGCC blockade. When VGCCs were blocked with CdCl2 (200 μM), the remaining effect of RO 25‐1553 (11.0 ± 1.6%, n = 4) was not changed (n = 3, Figure 6A) by the PKA inhibitor H‐89 (1 μM) but was abolished (F (6, 22) = 30.95, n = 3, Figure 6A) by the PKC inhibitor GF 109203X (1 μM). When the GAT‐1 uptake carrier was blocked using SKF89976A (20 μM), the effect of RO 25‐1553 (n = 4) was abolished (F (6, 22) = 30.95, n = 3, Figure 6B) in the presence of the PKA inhibitor H‐89 (1 μM) but was attenuated (n = 3, F (6, 22) = 30.95, Figure 6B) by the PKC inhibitor GF 109203X (1 μM).

Figure 6.

Figure 6

VPAC2 receptor‐mediated enhancement of [3H]‐GABA release through VGCC‐dependent and GAT‐1 carrier‐dependent mechanisms involves different protein kinases. Averaged effects on [3H]‐GABA release elicited by VPAC2 receptor antagonist RO 25‐1553 (10 nM) tested alone, in the presence of VGCC inhibitor (CdCl2) or the GAT‐1 inhibitor (SKF89976A) together with either the PKA inhibitor (H‐89) or the PKC inhibitor (GF109203X) in hippocampal nerve terminals. The effect of RO 25‐1553 (10 nM) was calculated by comparing the S2/S1 ratio obtained in test (presence of RO 25‐1553 during S2) and in control conditions. Each bar represents the mean ± SEM of results obtained in 3–9 experiments. ★ P < 0.05, significantly different from RO 25‐1553 in the absence of other drugs (first column), or VIP or agonists in the presence of either CdCl2 or SKF89976A (second column): ANOVA, followed by Sidak's multiple‐comparison test .

Discussion

The main findings of the work here described are that (i) VIP, through activation of both VPAC1 and VPAC2 receptors, exerts opposing actions on GABA release from hippocampal nerve terminals, VPAC1 receptor inhibiting and VPAC2 receptor enhancing GABA release; (ii) VPAC2 receptor activation not only enhances VGCC‐dependent GABA exocytosis through a Gs‐dependent, PKA‐dependent and PKC‐dependent mechanism but also enhances GAT‐1 carrier‐mediated GABA outflow through a Gs‐dependent and PKC‐dependent (but not PKA‐dependent) mechanism; (iii) VPAC1 receptor activation inhibits VGCC‐dependent GABA exocytosis through a Gi/o, PKA‐independent and partly PKC‐dependent mechanism; and (iv) both VPAC1 and VPAC2 receptors are present in GABAergic hippocampal nerve terminals.

The work described in this paper suggests that VPAC2 receptor activation is the trigger for the prevailing VIP enhancement of GABA release, because VIP was not able to enhance GABA release in the presence of the VPAC2 selective antagonist PG 97‐269, and the selective VPAC2 receptor agonist RO 25‐1553 enhanced GABA release. This VPAC2‐mediated action appears to be mediated by sequential activation of Gs proteins, adenylate cyclase and PKA (see Figure 7) as the enhancement of GABA release caused by the VPAC2 receptor activation was abolished by desensitizing Gs‐mediated responses with cholera toxin or by inhibiting PKA activity (Figures 4C and 5D). PKA is a known modulator of the exocytotic pathway in several types of secretory cells (Evans and Morgan, 2003), where it is involved in enhancement of both the priming and fusion steps of vesicle exocytosis through phosphorylation of several proteins of the exocytotic machinery. As VIP and the selective VPAC2 receptor agonist enhance GABA release through a Gs‐dependent and PKA‐dependent mechanism that also depends largely on VGCC activity (Figure 3A), regulation of PKA activity and its actions on the exocytotic machinery is likely to be the main mechanism involved in VPAC2 receptor‐mediated enhancement of GABA release. VPAC2‐mediated actions on GABA release through exocytosis were also partly dependent on PKC activity. This serine/threonine kinase is another key modulator of the exocytotic machinery (Morgan et al., 2005), having multiple exocytotic protein targets. The fact that PKA inhibition alone, independently of PKC activation levels, could block the enhancement of GABA release caused by VIP or the VPAC2 receptor agonist in the presence of CdCl2 suggests that the PKC‐dependent step in VPAC2‐mediated enhancement of exocytotic GABA release lies downstream of PKA regulation.

Figure 7.

Figure 7

Schematic representation of the main mechanisms involved in VPAC2‐mediated VIP enhancement of GABA release from hippocampal nerve terminals. AC, adenylate cyclase; G, Gs protein α subunit; VPAC2 R, VPAC2 receptor.

VPAC2 receptor activation also enhances GABA outflow through the GAT‐1 nerve terminal carrier as shown by the attenuation of VIP‐mediated and VPAC2 agonist‐mediated enhancement of GABA release in the presence of the selective inhibitor of the GAT‐1 carrier, SKF89976A. This GABA release mechanism is thought to be more important in pathological conditions that induce elevation of extracellular K+ such as hypoxia/ischaemia, trauma or epileptic seizures (Raiteri et al., 2002; Allen et al., 2004). Although a minor component of VIP enhancement of GABA release under the stimulation conditions that were used in this work, the ability of VIP to enhance GAT‐1‐mediated GABA release might constitute an important regulatory mechanism in the control of GABA outflow under such stressful or pathological conditions. VPAC2 receptor mediated enhancement of GABA release through the GAT‐1 carrier was dependent on protein Gs and PKC activity, but not on PKA activity, suggesting a regulation mechanism independent of adenylate cyclase. VPAC2‐mediated enhancement of GABA outflow through a PKC‐dependent mechanism might involve a PKC‐mediated recruitment of GAT‐1 to the membrane (Quick et al., 1997, 2004), allowing an enhancement of GABA outflow upon depolarization‐induced reversal of the carrier. It is possible that different PKC subtypes are involved in VPAC2‐mediated regulation of exocytotic and GAT‐1‐mediated GABA release. The finding that both PKA and PKC contribute synergistically to the enhancement of GABA release by VIP and the VPAC2 receptor agonist is in agreement with previous observations that stimulation of either PKA or PKC enhances inhibitory synaptic transmission through independent presynaptic mechanisms of action (Capogna et al., 1995).

VPAC1 receptor activation inhibited GABA release, as shown by the facilitation of VIP enhancement of GABA release in the presence of the selective VPAC1 receptor antagonist PG 97‐269 and by the inhibition of GABA release caused by the VPAC1 agonist [K15, R16, L27] VIP (1‐7)/GRF (8‐27). This VPAC1 receptor‐mediated inhibition of GABA release is dependent on activation of Gi proteins and VGCCs and partly dependent on PKC activity (see Figure 8). This implies that VPAC1‐mediated inhibition of GABA release involved two different mechanisms, one involving VGCCs and PKC and other dependent on VGCCs but not on PKC. The first mechanism may involve activation of PLCβ by the release of Gβγ subunits from Gi proteins (Clapham and Neer, 1997) and subsequent activation of PKC. Although it is currently established that PKC is a key modulator of the exocytotic machinery, the role of different PKC isoforms in this regulation is not known (Parker and Murray‐Rust, 2004). Furthermore, the consequences for neurotransmitter release of PKC‐mediated phosphorylation of proteins of the exocytotic machinery remain controversial (Morgan et al., 2005) as PKC mediates both enhancement and inhibition of exocytotic GABA release. The second mechanism for Gi‐dependent VPAC1 receptor‐mediated inhibition of GABA release may involve a direct inhibition of VGCCs by Gβγ subunits (Jarvis and Zamponi, 2001b) or an interaction of the Gβγ subunits with proteins of the exocytotic machinery, as has been described with SNAP‐25 and synapsin‐1 (Jarvis and Zamponi, 2001a; Gerachshenko et al., 2005). Any of these actions would cause a VGCC‐dependent inhibition of GABA release. The fact that the VPAC1 receptor is operating in a Gi‐dependent (instead of a Gs‐dependent) manner is not surprising as the ability of the VPAC1 receptor to couple to Gi proteins has previously been observed in CHO cells and hippocampal membranes (van Rampelbergh et al., 1997; Shreeve, 2002).

Figure 8.

Figure 8

Schematic representation of the main mechanisms involved in VPAC1‐mediated inhibition of GABA release from hippocampal nerve terminals. G,Gi protein α subunit; Giβγ,Gi protein β–γ dimer; VPAC1 R, VPAC1 receptor 1.

The present study provides functional evidence for the presence of VPAC1 and VPAC2 receptors in hippocampal GABAergic nerve terminals. The opposing actions of VPAC1 or VPAC2 receptor activation on hippocampal GABA release may be related to the different hippocampal distribution of VPAC1 and VPAC2 receptors and to a differential target selectivity of terminals containing each of the receptors. VPAC1 and VPAC2 receptor mapping in the hippocampus by in situ hybridization, autoradiography or immunohistochemistry (Vertongen et al., 1997b; Joo et al., 2004; White et al., 2010) demonstrates a preferential location of VPAC1 receptors in the stratum oriens and radiatum and VPAC2 receptors in the stratum piramidale of the Ammon's horn. This distribution overlaps with the differential location of synapses of VIP‐containing interneurones targeting other interneurones (stratum oriens and radiatum) and VIP‐containing basket cells targeting pyramidal cell bodies (Acsády et al., 1996b) respectively. Thus, it is possible that VPAC1 and VPAC2 receptors are differently involved in the modulation of GABA release to interneurones and pyramidal cells. An unequivocal demonstration of this hypothesis would require a finer localization the immunocytochemical signal at nerve terminals from the different interneurone populations. This, together with either paired recordings or channel rhodopsin‐assisted circuit mapping would be required to elucidate the dual effect of VIP on GABA release. This approach has been used recently to discriminate the role of different VIP‐expressing cortical interneurones in a push–pull inhibitory circuit (Garcia‐Junco‐Clemente et al., 2017). A differential modulation of GABA release to interneurones and pyramidal cells by VIP is consistent with the current knowledge that GABAergic transmission to interneurones and to pyramidal cells is differently regulated (Jonas et al., 2004) and with previous evidence (Cunha‐Reis et al., 2005, 2006) that VPAC1 receptor activation is mainly associated with VIP modulation of synaptic transmission to pyramidal cell dendrites, whereas VPAC2 receptor activation is preferentially associated with modulation of transmission to pyramidal cell bodies. The present observation that upon VPAC1 receptor blockade with PG 97‐269 there is an enhancement of the facilitation caused by VIP on GABA release (mediated by VPAC2 receptor activation) suggests that a concomitant regulation of GABA release by VPAC1 and VPAC2 receptors, which might be present at the same nerve terminal, could occur, provided that the superfusion rates used in this study were sufficient to eliminate interactions between different terminals (Collard, 1996).

Neuropeptides, such as VIP, are stored in ‘special’ vesicles known as large dense‐core vesicles that are released from synaptic sites different from the active zone, where the release of classic transmitters like GABA or glutamate occurs. Neuropeptide release is also coupled to different VGCCs, and it requires strong and repetitive electrical activity patterns (see Ghijsen and Leenders, 2005), like tetanic stimulation. As such, the physiologically relevant actions of VIP in the control GABA release are likely in situations eliciting synaptic plasticity phenomena or in pathological conditions like epilepsy. VIP was shown to modulate long‐term depression and de‐potentiation induced by low‐frequency stimulation (Cunha‐Reis et al., 2014). In fact, a marked increase in VIP receptor binding sites was reported in the hippocampus of humans with temporal lobe epilepsy with multiple sclerosis (de Lanerolle et al., 1995), a disease that is also associated with marked changes in the nature and distribution of inhibitory circuits (Cossart et al., 2001; Sloviter, 2005).

In summary, this study clearly demonstrates that VIP modulates GABA release from hippocampal nerve terminals through the activation of both VPAC1 and VPAC2 receptors, which recruit and interact with multiple intracellular pathways. VPAC1 receptors inhibit GABA release through Gi/o coupling. Conversely, VPAC2 receptors enhance GABA release by coupling to Gs proteins. Differences in VIP modulation of distinct populations of nerve terminals may occur and may play an important role in the differential modulation by VIP of GABA release to interneurones, pyramidal cell dendrites or pyramidal cell bodies. This suggests that further studies in hippocampal slices could provide important insights into the effects of presynaptic activation of VIP VPAC1 and VPAC2 receptors on hippocampal microcircuits.

Author contributions

D.C.‐R. participated in the study conception and design, performed the GABA release experiments, analysed the data and wrote the paper. J.A.R. contributed to the study design and manuscript writing. R.F.A. contributed to the statistical analysis and critically revised the paper. A.M.S. participated in the study conception, design and statistical analysis and manuscript writing. All authors read and approved the final version of the paper.

Conflict of Interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

We acknowledge the Institute of Physiology, FMUL, for animal housing facilities. This work was supported by Fundação para a Ciência e a Tecnologia: research grants: PTDC/SAU‐NEU/103639/2008 and UID/MULTI/00612/2013; fellowships: SFRH/BPD/34661/2007 and SFRH/BPD/81358/2011.

Cunha‐Reis, D. , Ribeiro, J. A. , de Almeida, R. F. M. , and Sebastião, A. M. (2017) VPAC1 and VPAC2 receptor activation on GABA release from hippocampal nerve terminals involve several different signalling pathways. British Journal of Pharmacology, 174: 4725–4737. doi: 10.1111/bph.14051.

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