Modulation of GABA release by 5-HT1B receptors: An interplay with AMPA-receptors and voltage-gated Ca2+ channels

Obesity has become a worldwide health challenge and commonly results from the intake of more calories than the body requires. The brain represents the master controller of food intake and as such has been the target of obesity medications. However, key mechanisms of druggable targets remain to be defined. Neurons within the arcuate nucleus of the hypothalamus co-expressing neuropeptide Y (NPY), agouti-related protein (AgRP) and GABA (NAG) are fundamental stimulators of hunger and food intake. NAG neurons also inhibit local satiety-promoting pro-opiomelanocortin (POMC) neurons. Agonists of the 1B subtype of metabotropic serotonin receptor (5-HT1BR) reduce food intake in part through the inhibition of hunger-promoting NAG neurons. We first confirmed that 5-HT1BR activation suppressed intake of a palatable Western diet in a mouse model of common dietary-induced obesity and genetically prone obesity. Next, we combined several electrophysiological approaches to analyse the effect of 5-HT1BRs in NAG neuron cell activity and GABA release. 5-HT1BR activation reduced NAG neuron action potential frequency and neurotransmitter release. We found that 5-HT1BR impact on GABA release from NAG neurons is mediated through voltage-gated Ca2+ channels with a critical input from glutamate receptors of AMPA subtype (AMPARs). As a fundamental outcome, this type of interplay provides an uncommon example of metabotropic action of AMPARs which regulates inhibitory signalling due to modulation of GABA release. As a translational outcome, our results provide a key mechanism through which 5-HT1BR drugs inhibit appetite-stimulating neurons within the brain to suppress food intake.


Introduction
Central nervous system (CNS)-focused pharmacological approaches to control food intake and treat obesity have dominated over approaches focusing on exclusively peripheral mechanisms (Sargent and Moore, 2009).Obesity medications augmenting brain serotonin (5-hydroxytryptamine; 5-HT) bioavailability (e.g.d-fenfluramine and sibutramine) improved human obesity worldwide, but were withdrawn from clinical use due to side effects (Heisler and Lam, 2017;McNeely and Goa, 1998;Sargent and Moore, 2009).Efforts to identify selective serotonin receptor agonists improving obesity focussed on the 5-HT 2C and 5-HT 1B subtypes (Clifton and Kennett, 2006;Heisler and Lam, 2017;Lam et al., 2010;McNeely and Goa, 1998;Vickers and Dourish, 2004).This led to 5-HT 2C receptor (R) agonist lorcaserin's development and subsequent use to treat human obesity from 2012 to 2020.5-HT 1B/1D R agonists are widely used to treat migraine, but 5-HT 1B R agonists have not yet been developed for obesity treatment.D-fenfluramine, sibutramine, 5-HT 2C R and 5-HT 1B R agonists utilise a common therapeutic mechanism to influence food intake, by acting at the brain melanocortin system (Burke et al., 2014;Burke et al., 2017;D'Agostino et al., 2018;Heisler et al., 2002;Heisler et al., 2006;Li et al., 2022).However, the specific mechanism through which 5-HT 1B R agonists influence melanocortin neurons has not been fully established, and well-defined mechanisms are essential to advance new druggable targets for obesity treatment.
The brain melanocortin system is comprised of two specific types of hypothalamic neurons located within the arcuate nucleus (Arc).One set of neurons potently stimulates food intake, whereas the other promotes satiety, and both compete for action at melanocortin receptors (Lam et al., 2010;Sweeney et al., 2023).The hunger-stimulating neurons produce orexigenic neuropeptides neuropeptide Y (NPY), agouti-related protein (AgRP) and γ-Aminobutyric acid (GABA).These cells are referred to as NAG neurons.NAG neurons send GABA-ergic inhibitory synapses to the other type of melanocortin neurons, satiety stimulating pro-opiomelancortin (POMC) neurons (Atasoy et al., 2012;Dicken et al., 2015).Therefore, when NAG neurons are active and stimulate appetite, they also inhibit POMC neurons to suppress the feeling of satiety.Hence, the molecular mechanisms controlling signalling in an NAG→POMC circuitry is of importance for CNS-focused pharmacological control over appetite and body weight.
Metabotropic 5-HT 1B Rs have been shown previously to be present in NAG neurons (Heisler et al., 2006;Li et al., 2022).They suppress NAG neuron action potential propagation (Heisler et al., 2006) and downregulate GABA release in other neuron types (Chadha et al., 2000).In the CNS, 5-HT 1B Rs are expressed as postsynaptic receptors, autoreceptors and also in non-serotoninergic terminals where they act as heteroreceptors inhibiting the release of neurotransmitters other than 5-HT (Barnes and Sharp, 1999).Therefore, the presence of 5-HT 1B Rs in NAG cells makes them an attractive target for the modulation of POMC neurons' disinhibition via the NAG →POMC GABA-ergic synapse(s).On the other hand, 5-HT 1B Rs appear to modulate the release of other neurotransmitters such as glutamate via the downregulation of Ca 2+ influx through the voltage-gated calcium channels (VGCCs) (Mizutani et al., 2006;Nishijo et al., 2022).Taking into account the central role of Ca 2+ microdomains control through presynaptic VGCCs in synaptic vesicle release (Jarvis and Zamponi, 2005) and, as a consequence, in interneuronal signalling, in this research we set out to clarify the mechanisms allowing the 5-HT 1B Rs-induced modulation of vesicle release from NAG cells.Such a modulation of vesicle release could potentially be translated through the regulation of VGCCs opening directly and/or through control over the AP propagation which, in turn, modulates VGCC activation.The dissection of 5-HT 1B R-connected molecular mechanisms is important in the future development of 5-HT 1B R agonists for obesity treatment.

Animals
All animal procedures were conducted in accordance with the United Kingdom Home Office (Scientific Procedures) Act of 1986.Adult male and female Npy GFP mice (Jackson Labs strain number 008321), male C57BL/6 (Jackson Labs strain number 000664) and male obesity prone leptin deficient ob/ob mice (Jackson Labs strain number 008321) were maintained under standard housing conditions.All mice were 3-5 months of age and were of a C57BL/6 background.

Electrophysiology
Male and female Npy GFP mice were anesthetised by overdose of isoflurane and then rapidly decapitated.Coronal brain slices of the Arc were cut at 250 μm with a Leica VT1200S vibratome using neuroanatomical landmarks from − 1.23 mm to -2.91 mm from Bregma.Brain sectioning was performed in an ice-cold solution containing (in mM) 64 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 0.5 CaCl 2 , 7 MgCl 2 , 25 NaHCO 3 , 10 Dglucose and 120 sucrose, saturated with 95% O 2 and 5% CO 2 .Single channel openings to certain conductance were selected automatically by the threshold-detection algorithm of Clampfit 10 software with a minimum event time length of 0.2 ms.For the semi-quantitative assessment of amount of neurotransmitter agonists released from Npy GFP cells in response to high-frequency stimulation in a sniffer patch experiment, we used an open time fraction of single-channel patch recording.This was calculated as a ratio t o /t c , where t o is the full time of a given recording when ion channel(s) in a patch were in the open state and t c is the time when receptor(s) were in closed state.

Behavioural experiments
Male dietary-induced obese (DIO) C57BL/6 (n = 8; body weight 29-38 g) and genetically obesity prone leptin deficient ob/ob (n = 7; body weight 41-45 g) mice were maintained on a palatable 58% fat diet (Research Diets) post-weaning for 8 weeks.Food was removed and weighed 45 min before the onset of the dark cycle.Mice were then treated with either vehicle (saline) or CP-94253 (8 or 16 mg/kg, i.p.).Food was replaced at the onset of the dark cycle and subsequent food intake was manually measured 1, 3 and 6 h later.These time points were selected because CP-94253 reduces 6 h dark cycle cumulative ad libitum food intake.Each mouse was tested with drug and saline, administered in a counterbalanced order, with a minimum of two days between the treatments.Studies were performed in the home cage and mice were single housed during experimentation.

Immunohistochemistry
Under deep terminal anaesthesia, 0.9% saline followed by 10% neutral buffered formalin (Sigma) was transcardially perfused in male and female Npy GFP mice.Brains were removed, immersed in 10% formalin for 4 h, and then transferred to 20% sucrose in PBS for 36 h at • C. Brains were sectioned on a freezing sliding microtome at 25 μm.
Immunohistochemistry was performed using standard methods (Heisler et al., 2006;Wagner et al., 2023).Briefly, free floating tissue was incubated with a chicken anti-GFP primary antibody (Abcam, 1:1000) for 24 h at 4 • C and then incubated with a donkey anti-chicken IgG secondary antibody (Jackson IummnoResearch,1:500) for 2 h.Sections were mounted onto slides, air dried, and cover slipped with Vectashield antifading mounting medium (Vector Labs).Hypothalamic Npy GFP immunoreactivity was visualised using a Zeiss Axioskop 2 microscope equipped with an Axiocam HRc camera.The Arc was defined using neuroanatomical landmarks.

Data analysis
All experimental data are provided as Mean ± standard error of the mean.Statistical comparisons were performed with repeated measures D. A. Najm Al-Halboosi et al. analysis of variance (ANOVA) followed by Dunnett's post hoc test, Student's paired t-test or unpaired t-test, as indicated.Statistical significance was defined as P < 0.05.

5-HT 1B R agonist modulation of NAG cell signalling and GABA release
We and others have reported that a subset of 5-HT 1B Rs is expressed in NAG neurons and that NAG neurons contribute to 5-HT 1B R agonist's effects on feeding (Heisler et al., 2006;Li et al., 2022).We therefore next focussed on the effect of 5-HT 1B R agonists on NAG cell activity and GABA release.We previously reported that both CP-94253 and CP-93129 hyperpolarize NAG neurons (Heisler et al., 2006).We next performed whole-cell current-clamp recording from Npy GFP cells simultaneously with a sniffer-patch recording with an outside-out patch excised from a nearby non-fluorescent cell and pre-positioned over the patched Npy GFP cell (Fig. 2B-G) (McLaughlin et al., 2019).The application of the 5-HT 1B R selective agonist 100 nM CP-93129 demonstrated that 5-HT 1B R activation significantly decreased the AP frequency in Npy GFP cells to 0.58 ± 0.08 of the control (t (4) = 5.53, P = 0.0058, n = 5 pairs, Student's paired t-test), whereas washout restored the AP frequency value to 0.93 ± 0.03 of that of control (t (4) = 2.32, P = 0.08, n = 5 pairs, Student's paired t-test; Fig. 2B and C).However, the impact of CP-93129 on cell membrane potential in tested cells did not reach statistical significance: 2.16 ± 1.34 mV from control (t (4) = 2.77, P = 0.08, n = 5 pairs, Student's paired t-test).These data indicate that 5-HT 1B R agonists inhibit NAG neuron activity.
Since GABA A R's opening probability is highly positively correlated with GABA binding to the receptor (Carter et al., 2010;Jembrek and Vlainic, 2015), an increase of GABA concentration in a close receptor's proximity immediately results in increased GABA A R opening frequency.We therefore used GABA A R opening as a measure of GABA release.CP-94253 lowered GABA A R opening frequency to 0.53 ± 0.09 of that of the control (t (4) = 5.37, P = 0.0058, n = 5 pairs, paired Student's t-test), and it was restored to 0.8 ± 0.12 of the control after the CP-93129 washout (t (4) = 1.7,P = 0.13, n = 5 pairs, paired Student's t-test): Fig. 2E and F. These results suggest that 5-HT 1B R agonist inhibit GABA efflux in the Arc.

Quantification of NAG cells' contribution to neurotransmitter release in the Arc and dissection of 5-HT 1B R's role in this process
To establish whether 5-HT 1B R activation inhibits neurotransmitter release from NAG neurons, we next performed measurements of wholecell membrane capacitance fluctuations (C m ) in response to two different excitatory inputs in the Arc of Npy GFP mice: (i) series field stimulation (5 stimuli with 50 ms intervals) (Chen et al., 2020) and (ii) depolarization to 0 mV (Pekel et al., 1993) (Fig. 3).To quantify the effect of CP-93129, we used the area under the curve of the C m fluctuation (AUC).CP-93129 caused a significant reduction in AUC after the series stimulation, but not after the depolarization.The effect of CP-93129 after series stimulation was as follows: 0.47 ± 0.12 of the control (t (5) = 4.38, P = 0.007, n = 6 pairs, Student's paired t-test); after the depolarization episode: 0.81 ± 0.102 of the control (t (5) = 1.83,P = 0.13, n = 6 pairs, Student's paired t-test) (Fig. 3A-B).
To explain this difference, we hypothesized the involvement of other receptor types which were involved after series stimulation (due to an efflux of neurotransmitters from neighbouring cells), but not after the depolarization episode.To test this hypothesis, we performed the depolarization experiment with a parallel pharmacological activation of the GABA B receptors (with 1 μM SKF 97541), metabotropic glutamate receptors (with 1 μm CHPG, 1 μM (RS)-3,5-DHPG and 1 μM AMN 082) and acetylcholine receptors (with 100 nM 3-Bromocytisine), but without any significant effect of CP-93129 on AUC (data not shown).However, when we selectively activated the AMPA receptors (AMPARs) with μM AMPA 1 min before depolarization, CP-93129 again demonstrated a significant impact on AUC: decrease to 0.42 ± 0.11 of the control (t (5) = 5.7, P = 0.0001, n = 6 pairs, Student's paired t-test): Fig. 3B.Then, for the sake of clarity, we repeated the experiment with a series stimulation and step depolarization + AMPA, with a pharmacological block of AMPARs by 10 μM CNQX added to perfusion solution.This AMPAR antagonist prevented changes of AUC induced by CP-93129.For series stimulation + CNQX, the amplitude changed to 0.97 ± 0.07 of control (t (5) = 0.45, P = 0.67, n = 6 pairs, Student's paired t-test).For depolarization + AMPA, the amplitude changed to 0.96 ± 0.1 of control (t (5) = 0.35, P = 0.74; n = 6 pairs, Student's paired t-test): Fig. 3B.
As a control, we tested whether AMPARs in our preparation deliver their impact on the neurotransmitter vesicle release via the G i/o -protein- controlled metabotropic signalling chain.To achieve this, we repeated the experiments on AUC with 250 μM of a G i/o -protein inhibitor Nethylmaleimide (NEM) in a recording pipette.Here we found that NEM made the effects of both series stimulation and depolarization + AMPA on AUC non-significant (Fig. 3B): decrease to 0.96 ± 0.04 of the control, t (5) = 1.01,P = 0.35 for the difference from control for series stimulation; decrease to 0.94 ± 0.06 of the control, t (5) = 1, P = 0.36 for the difference from control for depolarization + AMPA, n = 6 pairs for both cases, Student's paired t-test.
To test the involvement of VGCCs with regard to these effects, we set out to repeat the experimental set of whole-cell recordings with VGCCsm in a perfusion solution.Here CP-93129 did not have any significant effect on AUC in all three cases -series stimulation, depolarization episode, depolarization episode with AMPA (Fig. 3B).These data indicate that selective 5-HT 1B R agonist inhibits GABA release from NAG neurons via action at VGCCs with a pivotal input from AMPARs.

AMPAR-VGCC interaction is a critical contributor to 5-HT 1B R initiated downregulation of VGCC
The data above demonstrated the specific role of interaction between AMPARs and VGCCs in the development of 5-HT 1B R effects.To quantify the AMPAR-VGCC interaction we next decided to test the AMPAR impact on the 5-HT 1B R control over the charge transfer through VGCCs.We therefore generated VGCC openings by voltage steps from − 70 mV to 0 mV (Pekel et al., 1993), followed by voltage steps after a series stimulation of neural tissue, and with a voltage step + AMPA added to the perfusion solution (Fig. 4).We found that the effect of 5-HT 1B R is significantly higher after series stimulation and after the depolarization step + AMPA compared with the depolarization step alone (Fig. 4B).For series stimulation: 0.58 ± 0.06 of control vs. 0.85 ± 0.05 for depolarization only (t (7) = 3, P = 0.02, n = 8 pairs, paired Student's t-test); for depolarization + AMPA: 0.55 ± 0.07 of control vs. 0.85 ± 0.05 for depolarization only, (t (7) = 3.33, P = 0.013, n = 8 pairs, Student's paired t-test; Fig. 4A).Again, as we did in our experiments on membrane capacitance, here we repeated the experiment with series stimulation and step depolarization + AMPA, applying a pharmacological block of AMPARs by CNQX.Similarly, CNQX prevented changes of the response amplitude induced by CP-93129 (Fig. 4B).For series stimulation: 0.94 ± 0.05 of control, t (5) = 1.14, P = 0.31; for depolarization step + AMPA: 0.95 ± 0.07 of control, t (5) = 0.69, P = 0.52, n = 6 pairs for both cases, Student's paired t-test.
Our whole-cell electrophysiological recordings demonstrated the involvement of AMPARs and the critical role of VGCCs in the development of 5-HT 1B R effects on neurotransmitter release from NAG neurons.However, one could question such a conclusion since the results obtained in living tissue could be due to contamination (especially after the stimulation series) resulting from the release of different neurotransmitters from a number of neighbouring cells.Hence, to generate 5-HT 1B R effects in a more controlled environment, we performed a rapid solution exchange experiment with nucleated patches pulled from NAG cells using similar methods to those previously reported (Sylantyev and Rusakov, 2013).Nucleated patches contain a part of intracellular milieu, thus preserving signalling pathways between metabotropic receptors and other membrane-localized effectors (O'Neill et al., 2018).Here we repeated the previous experiment involving the voltage steps, but in parallel we applied perfusion solution with different pharmacological agents (Fig. 5).We found that the application of both CP-93129 and CP-93129 + AMPA significantly decreases the amplitude of the VGCC response generated by voltage steps to 0 mV: to 0.82 ± 0.03 for CP-93129 (t = 5.69, P = 0.0012 for the difference from control, n = 7 pairs, Student's paired t-test) and 0.61 ± 0.04 for CP-93129 + AMPA (t (6) = 9.57, P = 0.000076 for the difference from control, n = 7 pairs, Student's paired t-test) -Fig.5B; the difference between CP-93129 and CP-93129 + AMPA effects also turned out to be highly significant: t ( 12  0.178, n = 7 pairs, Student's paired t-test; Fig. 5B) -see Fig. 4B.VGCC-sm, added to the perfusion solution in a control experiment, fully suppressed the response of VGCCs (Fig. 5A, rightmost trace).Our results demonstrate that a mechanism through which a 5-HT 1B R agonist inhibits GABA release from NAG neurons involves AMPAR activation (as a compulsory element) to downregulate VGCC.This then decreases calcium influx into the cell and suppresses GABA release from NAG cells.

Discussion
The well-recognised impact of 5-HT obesity medications on appetite control has primarily been attributed to action at the 5-HT 2C Rs (Heisler et al., 2002;Xu et al., 2008).5-HT 1B Rs have been much less studied in this context, though agonists produce a significant effect on food intake and body weight (Halford and Blundell, 1996;Heisler et al., 2006;Lee and Simansky, 1997;Li et al., 2022).Given that 5-HT 1B/1D R agonists are in human use for migraine treatment, it is possible that targeting this receptor for obesity may be better tolerated than earlier 5-HT pharmacological approaches.Defining the 5-HT 1B R mechanism of action in food intake control is therefore important in considering this receptor as a druggable obesity target.
Previous reports indicate that 5-HT 1B R mediated (i) NAG neuron inhibition and (ii) POMC neuron disinhibition is a mediator of 5-HT 1B R agonists' effects on food intake (Heisler et al., 2006).Here we demonstrate that an important feature of 5-HT 1B R-mediated modulation of neurotransmitter release is regulation of Ca 2+ influx through VGCCs, even though 5-HT 1B R impact on NAG cell membrane potential did not reach statistical significance in our preparation.Such an interaction has been shown previously for different neural cell types (Mizutani et al., 2006;Nishijo et al., 2022).In turn, the suppression of VGCC activity to the level sufficient for a significant impact on neurotransmitter release requires the involvement of AMPA receptors.Such a complex action pattern involving metabotropic receptors, voltage-independent ionotropic receptors and voltage-dependent ion channels with all three elements being critical for the propagation of a significant impact, provides a relatively unusual multi-mechanism effect.
The importance of the hypothalamus in the central regulatory mechanisms of appetite control has been reported for decades (Grossman and Grossman, 1977;Hetherington and Ranson, 1940).The significance of the Arc in these effects was further established in later studies (Saper et al., 2002;Schwartz et al., 2000), with a central role played by NAG neurons in hunger being discovered (Aponte et al., 2011;Gropp et al., 2005;Krashes et al., 2013).The adipocyte derived hormone leptin inhibits NAG neurons.In common dietary induced obesity, leptin resistance can occur (Enriori et al., 2007).Here we show that NAG neurons are responsive to the inhibitory effect of 5-HT 1B R agonists in both common DIO and ob/ob mice.This finding supports the development of 5-HT 1B R agonists as an obesity medication target.
The orexigenic nature of signalling molecules AgRP and NPY is wellestablished (Engström Ruud et al., 2020;Ollmann et al., 1997).Nevertheless, apart from these peptides, GABA release from NAG neurons also plays a role in appetite regulation (Atasoy et al., 2012;Wu et al., 2009).The involvement of other neurotransmitters released from NAG neurons in POMC neuron disinhibition has also been suggested (Rau and Hentges, 2017).In terms of particular glutamate receptor type involvement, NMDA receptors were demonstrated to play a role in appetite regulation by acting at NAG cells (Liu et al., 2012).However, the regulation of NAG cell activity initiated by NMDA receptors is connected to control over cell excitation and to the AP generation probability.In contrast, our data suggest that a glutamatergic action in terms of suppression of neurotransmitter release could be delivered through G-protein-connected signalling mechanisms.This provides further evidence of an AMPAR functional profile, attributing unusual metabotropic function to this classical ionotropic receptor in accord with earlier studies (Satake et al., 2004;Takago et al., 2005).
In conclusion, 5-HT medications have been used to treat human obesity, but the neuropharmacological mechanisms of action have not been fully defined.Work of this nature is essential for the development of new, more selective and effective ligands.Here we focused on the less well characterised 5-HT 1B R subtype, agonists of which reduce food intake in obesity.We investigated the mechanism of action of a key target of 5-HT 1B R agonists in the regulation of appetite, hungerpromoting NAG neurons.We identify an unusual multi-mechanism effect though which 5-HT 1B R agonists inhibit NAG neurons involving metabotropic receptors, voltage-independent ionotropic receptors and voltage-dependent ion channels.This research provides an advance not only in 5-HT 1B R pharmacology but also in the understanding of fundamental neurocircuitry controlling appetite and body weight.

Declaration of competing interest
The authors declare that they don't have any conflict of interests.

Fig. 2 .
Fig. 2. Activation of 5-HT 1B Rs suppresses AP frequency in NAG cells and neurotransmitter release.A: experimental schematic and images of NAG cells in hypothalamus labelled with GFP fluorescence.Scale bar in left image: 100 μm.Scale bar in right image: 20 μm.B: Example trace of AP recording.Grey horizontal bar denotes time interval of 100 nM CP-93129 application.C: Statistical summary for AP recordings without block of VGCCs.D: Statistical summary for AP recordings when VGCCs were blocked.E: Sniffer-patch recordings, example traces.From top to bottom: individual traces with single-channel openings recorded at control, nM CP-93129 and washout experimental intervals; time points of the recordings are marked by red dotted lines in B. Scale bars apply to all three traces.F: Statistical summary for sniffer-patch recordings without block of VGCCs.G: Statistical summary for sniffer-patch recordings when VGCCs were blocked.Asterisks denote significance of difference from unity.** -P < 0.01, *** -P < 0.001, n.s.-non-significant; for AP recordings: paired Student's t-test, for single-channel recordings: unpaired Student's t-test.

Fig. 3 .
Fig. 3. Activation of 5-HT 1B Rs inhibits neurotransmitter vesicle release from NAG neurons.A: Example traces.The effect of 100 nM CP-93129 (bottom) on membrane capacitance fluctuations compared to control (top).Capacitance fluctuations were generated by (from left to right) series of electrical stimuli delivered to neural tissue, 0.2 s membrane depolarization episode, and depolarization episode + 10 μM AMPA.Grey shadows: individual traces; red lines: averaged curves.B: comparison of the reduction of the area under averaged curve.Asterisks denote the significance of difference from control (unity).** -P < 0.01, *** -P < 0.001, n.s.non-significant, Student's paired t-test.
Fig. 4. 5-HT 1B R activation suppresses the amplitude of the whole-cell VGCCs response in NAG neurons.A: Example traces: the impact of 100 nM CP-93129 on electrical charge transfer through VGCCs generated by (from left to right) series of electrical stimuli, 0.2 s membrane depolarization episode, and depolarization episode + 10 μM AMPA.Blue traces: control.Red traces: effect of CP-93129.Dashed lines show the stable response interval used for comparison of the response amplitudes.Solid black line denotes depolarization protocol: step from − 60 mV (holding voltage) to +40 mV.B: Statistical summary.Asterisks denote the significance of difference from "Depolarization" value.* -P < 0.05, n.s.-non-significant, Student's paired t-test.

Fig. 5 .
Fig. 5. AMPAR-VGCC interaction via G i/o signalling is a critical contributor to 5-HT 1B R initiated downregulation of VGCC.A: Example traces showing the effect of CP-93129 on VGCC response generated by the membrane depolarization episode under different pharmacological interventions.Blue trace: control.Red trace: CP-93129 100 nM.Solid black line denotes depolarization protocol: step from − 60 mV (holding voltage) to +40 mV.B: Statistical summary of A: activation of AMPARs enhances the effect of CP-93129 which is critically dependent from G-protein signalling.Asterisks denote the significance of difference from control (unity).** -P < 0.01, *** -P < 0.001, n.s.-non-significant, Student's paired t-test.