Growth hormone secretagogue receptor constitutive activity impairs voltage-gated calcium channel-dependent inhibitory neurotransmission in hippocampal neurons

Abstract

Key points: Presynaptic Ca_V 2 voltage-gated calcium channels link action potentials arriving at the presynaptic terminal to neurotransmitter release. Hence, their regulation is essential to fine tune brain circuitry. Ca_V 2 channels are highly sensitive to G protein-coupled receptor (GPCR) modulation. Our previous data indicated that growth hormone secretagogue receptor (GHSR) constitutive activity impairs Ca_V 2 channels by decreasing their surface density. We present compelling support for the impact of Ca_V 2.2 channel inhibition by agonist-independent GHSR activity exclusively on GABA release in hippocampal cultures. We found that this selectivity arises from a high reliance of GABA release on Ca_V 2.2 rather than on Ca_V 2.1 channels. Our data provide new information on the effects of the ghrelin-GHSR system on synaptic transmission, suggesting a putative physiological role of the constitutive signalling of a GPCR that is expressed at high levels in brain areas with restricted access to its natural agonist.

Abstract: Growth hormone secretagogue receptor (GHSR) displays high constitutive activity, independent of its endogenous ligand, ghrelin. Unlike ghrelin-induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic Ca_V 2 voltage-gated calcium channels. Here we postulate that GHSR constitutive activity-mediated modulation of Ca_V 2 channels could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole-cell patch-clamp in hippocampal primary cultures from E16- to E18-day-old C57BL6 wild-type and GHSR-deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs Ca_V 2.1 and Ca_V 2.2 native calcium currents and that Ca_V 2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher Ca_V 2.2 over Ca_V 2.1 contribution to GABA release (∼40% for Ca_V 2.2 in wild-type vs. ∼20% in wild-type GHSR-overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra-granule cell inhibitory postsynaptic current (IPSC) size ∼-67 pA in wild-type vs. ∼-100 pA in GHSR-deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a Ca_V 2-dependent mechanism without affecting glutamatergic transmission.

Keywords: GABA; GPCR; Synapse; brain slices; electrophysiology; ghrelin; inhibitory postsynaptic current; primary cultures.

Figure 1. GHSR constitutive activity affects native I _CaV levels in hippocampal cultured neurons

A, average I _Ba levels from 5–14 DIV wild‐type (WT) and GHSR‐deficient (def) hippocampal neurons transduced with lentiviral plasmids encoding GHSR (+GHSR) or GHSRA204E (+A204E) or not transduced. Statistical significance evaluated by Mann–Whitney test (n = 122, 3–12 per mean data point). B, representative traces and average I _Ba levels from mature (>14 DIV) hippocampal neurons in the same conditions as in A. Statistical significance evaluated by ANOVA and Tukey's post hoc test.

Figure 2. GHSR constitutive activity impairs Ca_V‐dependent GABAergic transmission while fails to modify glutamatergic transmission

EPSC (A) and IPSC (B) evoked by electrical stimuli (indicated by arrowheads). Representative traces and average values from mature (>14 DIV) wild‐type (WT) and GHSR‐deficient (def) hippocampal neurons transduced with lentiviral plasmids encoding GHSR (+GHSR) or GHSRA204E (+A204E) or not transduced. Statistical significance evaluated by Kruskal–Wallis ANOVA and Dunn's post hoc test.

Figure 3. Paired pulse ratio (PPR) is modified by GHSR expression levels only for GABAergic transmission

Average EPSC (A) and IPSC (B) PPR at different inter‐stimulus intervals (20–200 ms) from >14 DIV GHSR‐deficient (def) and wild‐type (WT) hippocampal neurons transduced with lentiviral plasmids encoding GHSR (WT+GHSR) or not (WT) and examples of paired pulses for WT overexpressing GHSR (WT+GHSR) and GHSR deficient (def) conditions evoked by 1 ms depolarizing stimuli at a 20 ms inter‐stimulus interval. Electrical stimuli are indicated by arrowheads. Statistical significance evaluated by Kruskal–Wallis ANOVA and Dunn's post hoc test (n = 283, 4–11 per mean data point).

Figure 4. Increased IPSC size due to the absence of GHSR is conserved at different Ca²⁺ concentrations

EPSC (A) and IPSC (B) average values at different Ca²⁺ concentrations (logarithmic scale) and linear regression plot from >14 DIV wild‐type (WT, open circles) and GHSR deficient (def, open squares) hippocampal neurons. Linear regression fitting parameters: EPSC slope test: F = 1.22978, degree of freedom for numerator (DFn) = 1, degree of freedom for denominator (DFd) = 75, P = 0.271, pooled slope = 0.576134 r ² (WT) = 0.7167, r ² (def) = 0.5260; intercepts test: F = 0.0203306, DFn = 1, DFd = 76, P = 0.887, pooled intercept = 2.77994. (n = 79, ranging between 3–12 per mean data point.) IPSC slope test: F = 0.699603, DFn = 1, DFd = 81, P = 0.4054, pooled slope = 0.501558, r ² (WT) = 0.5170, r ² (def) = 0.5888; intercepts test F = 29.3249, DFn = 1, DFd = 82, P < 0.0001, intercept (WT) = −0.50 ± 0.05 nA, intercept (def) = −0.82 ± 0.06 nA. (n = 85, ranging between 3–13 per mean data point.)

Figure 5. Ca_V‐independent mEPSCs and mIPSC are unaffected by GHSR constitutive activity

mEPSC (A) and mIPSC (B) average amplitude and frequency values and representative traces from mature (>14 DIV) wild‐type (WT) and GHSR‐deficient (def) hippocampal neurons transduced with lentiviral plasmids encoding GHSR (+GHSR) and GHSRA204E (+A204E) or not transduced. Statistical significance evaluated by Kruskal–Wallis ANOVA and Dunn's post hoc test. Both mEPSC and mIPSC were recorded in the presence of 1 μm TTX and the Ca_V blocker 100 μm Cd²⁺.

Figure 6. Hyperosmotic sucrose response is unaffected by GHSR expression

Representative traces and average mobilized charge values from mature (>14 DIV) wild‐type (WT) and GHSR deficient (def) hippocampal neurons transduced with lentiviral plasmids encoding GHSR (+GHSR) and GHSRA204E (+A204E) or not transduced (non‐transduced) in response to 0.5 m sucrose stimulation in the presence of 1 μm TTX and 100 μm Cd²⁺. Statistical significance evaluated by Kruskal–Wallis ANOVA and Dunn's post hoc test.

Figure 7. GHSR expression reduces the contribution of Ca_V2.2 to total Ca_V currents

I _Ba time courses of application of 1 μm ω‐conotoxin‐GVIA (+cono) and 0.2 μm ω‐agatoxin‐IVA (+aga) (A), averaged basal I _Ba levels (B) and average percentage of I _Ba inhibition (C) from >12 DIV GHSR‐deficient (def), wild‐type (WT) or wild‐type transduced with lentiviral plasmids encoding GHSR (WT+GHSR) hippocampal neurons. Statistical significance evaluated by Kruskal–Wallis ANOVA and Dunn's post hoc test.

Figure 8. GHSR expression inhibition of GABAergic transmission is related to a higher reliance on Ca_V2.2

A, IPSC representative traces before (control) and after successive application of 1 μm ω‐conotoxin‐GVIA (+cono) and 0.2 μm ω‐agatoxin‐IVA, B and C, average basal levels (B) and percentage of inhibition (C) from >12 DIV wild‐type (WT) hippocampal neurons and wild‐type hippocampal neurons transduced with lentiviral plasmids encoding GHSR (+GHSR). Statistical significance evaluated by Student's t test.

Figure 9. GHSR constitutive activity modulates synaptic transmission in hippocampal slices

A, diagram of the DG‐CA3 circuit showing positions of extracellular stimulating and recording electrodes on the granule cell layer of the dentate gyrus (GC layer of DG) and pyramidal cell layer of CA3 (PYR layer of CA3). B, representative traces and average intra‐granule cell layer IPSCs (GC‐IPSCs) from wild‐type (WT) and GHSR‐deficient (def) mice. C, representative traces and average EPSCs from WT and GHSR‐deficient (def) pyramidal cell layer (PYR‐EPSCs) mice. Statistical significance evaluated by Mann–Whitney test.