Somatodendritic Release of Cholecystokinin Potentiates GABAergic Synapses Onto Ventral Tegmental Area Dopamine Cells.

BACKGROUND: Neuropeptides are contained in nearly every neuron in the central nervous system and can be released not only from nerve terminals but also from somatodendritic sites. Cholecystokinin (CCK), among the most abundant neuropeptides in the brain, is expressed in the majority of midbrain dopamine neurons. Despite this high expression, CCK function within the ventral tegmental area (VTA) is not well understood. METHODS: We confirmed CCK expression in VTA dopamine neurons through immunohistochemistry and in situ hybridization and detected optogenetically induced CCK release using an enzyme-linked immunosorbent assay. To investigate whether CCK modulates VTA circuit activity, we used whole-cell patch clamp recordings in mouse brain slices. We infused CCK locally in vivo and tested food intake and locomotion in fasted mice. We also used in vivo fiber photometry to measure Ca2+ transients in dopamine neurons during feeding. RESULTS: Here we report that VTA dopamine neurons release CCK from somatodendritic regions, where it triggers long-term potentiation of GABAergic (gamma-aminobutyric acidergic) synapses. The somatodendritic release occurs during trains of optogenetic stimuli or prolonged but modest depolarization and is dependent on synaptotagmin-7 and T-type Ca2+ channels. Depolarization-induced long-term potentiation is blocked by a CCK2 receptor antagonist and mimicked by exogenous CCK. Local infusion of CCK in vivo inhibits food consumption and decreases distance traveled in an open field test. Furthermore, intra-VTA-infused CCK reduced dopamine cell Ca2+ signals during food consumption after an overnight fast and was correlated with reduced food intake. CONCLUSIONS: Our experiments introduce somatodendritic neuropeptide release as a previously unknown feedback regulator of VTA dopamine cell excitability and dopamine-related behaviors.

Periaqueductal Gray and Rostromedial Tegmental Inhibitory Afferents to VTA Have Distinct Synaptic Plasticity and Opiate Sensitivity.

The ventral tegmental area (VTA) is a major target of addictive drugs and receives multiple GABAergic projections originating outside the VTA. We describe differences in synaptic plasticity and behavior when optogenetically driving two opiate-sensitive GABAergic inputs to the VTA, the rostromedial tegmental nucleus (RMTg), and the periaqueductal gray (PAG). Activation of GABAergic RMTg terminals in the VTA in vivo is aversive, and low-frequency stimulation induces long-term depression in vitro. Low-frequency stimulation of PAG afferents in vitro unexpectedly causes long-term potentiation. Opioid receptor activation profoundly depresses PAG and RMTg inhibitory synapses but prevents synaptic plasticity only at PAG synapses. Activation of the GABAergic PAG terminals in the VTA promotes immobility, and optogenetically-driven immobility is blocked by morphine. Our data reveal the PAG as a source of highly opioid-sensitive GABAergic afferents and support the idea that different GABAergic pathways to the VTA control distinct behaviors.

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

KEY POINTS: Presynaptic CaV 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. CaV 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 CaV 2 channels by decreasing their surface density. We present compelling support for the impact of CaV 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 CaV 2.2 rather than on CaV 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 CaV 2 voltage-gated calcium channels. Here we postulate that GHSR constitutive activity-mediated modulation of CaV 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 CaV 2.1 and CaV 2.2 native calcium currents and that CaV 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 CaV 2.2 over CaV 2.1 contribution to GABA release (∼40% for CaV 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 CaV 2-dependent mechanism without affecting glutamatergic transmission.

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVß-dependent manner.

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range of Ca2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCR-dependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several CaV subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of CaV channels depends on the presence of a CaVß subunit. CaVß subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.

Ghrelin binding to serum albumin and its biological impact.

Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone.