Effects Of Ghrelin On The Nigral Dopaminergic Neurons And Its Physiological Significance

Parkinson’s disease (PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons originating in the substantia nigra (SN) and the subsequent dopamine (DA) depletion in the striatum. This disorder is characterized symptomatically by resting tremor, rigidity and bradykinesia. The incidence of PD is close to 1.7% in the people over 65 in China. The number of PD patients is about 10 million in China and this number will be rising in the next 20 years with the ageing of our society. Therefore, PD becomes an overwhelming problem which could affect our health and life quality and hinder the economic sustainable development. Since the exact pathogenesis of PD is largely unknown and there are no cures, natural and efficient bioactive materials that could be neuroprotective in PD will have incalculable social and economic benefits.Ghrelin, a 28-amino-acid peptide, has been identified as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Ghrelin exerts its biological effects via its fully functional receptor, GHS-Rla. GHS-Rla is a number of G-protein coupled receptor with seven transmembrane-spanning domains. Several lines of evidence suggest that the effects of ghrelin are not only restricted to feeding behavior and energy homeostasis, but also concern neuroprotective effects on some brain regions, such as cortex, hippocampus and substantia nigra (SN). In our previous studies, we observed the neuroprotective effects of ghrelin on dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced neurotoxicity, mediated by inhibition of the apoptosis and attenuation of the oxidative stress. Similar studies showed that ghrelin also exerted its neuroprotective effects by enhancing the UCP2-dependent mitochondrial function. However, the biological effects and physiological significance of ghrelin on nigral dopaminergic neurons are largely unknown. In the present study, using whole cell patch clamp recording, electrochemical detection and animal behavior test both in vivo and in vitro, we study the electrophysiological effects of ghrelin on nigral dopaminergic neurons and its underlying mechanisms. The results are as follows:1. The nigral dopaminergic neurons could be divided into two types according to their firing mode in rat brain slices:tonic firing neurons and silent neurons. Application of 10 nmol/L ghrelin significantly depolarized the membrane potential and increased the firing rate in tonic firing neurons. In some silent neurons, application of 10 nmol/L ghrelin could also cause the depolarization of the membrane potential, resulting in a chain of action potentials. 2.0.1 and 1 nmol/L ghrelin had no effects on the firing rate of nigral dopaminergic neurons, whereas 100 and 1000 nmol/L increased the neuronal firing rate in a dose-dependent manner. Ghrelin could also modify the discharge pattern of some dopaminergic neurons from tonic firing to a burst firing pattern.3. GHS-Rla blocker D-[Lys3]-GHRP-6 (50μmol/L), Phospholipase C (PLC) blocker U73122 (10μmol/L) or Protein kinase C (PKC) blocker GF109203X (5μmol/L) could block the effects of ghrelin on the firing rate of dopaminergic neurons respectively. Application of these blockers alone had no effects on the neuronal firing rate.4. The presence of L-type calcium channel antagonist nimodipine (10μmol/L) did not alter the membrane depolarization induced by ghrelin. However, the effects of ghrelin could be fully blocked by voltage-gated potassium channels blocker tetraethylammonium (TEA). Twenty millimole of TEA alone increased the firing rate of dopaminergic neurons. XE991 (1μmol/L), one of the KCNQ channel blocker, could completely block the effects of ghrelin on the neuronal firing rate, and its opener retigabine (5μmol/L) could partially inhibit the effects of ghrelin. Application of XE991 or retigabine alone increased or decreased the firing rate of nigral dopaminergic neurons respectively.5. Ghrelin (300 nmol/L) inhibited 58.32%±8% of KCNQ current in rat DRG neurons and increased the neuronal firing. XE991 (30μmol/L) completely blocked the effects of ghrelin on DRG neurons and increased the neuronal firing.6. Ghrelin had no effects on the frequency, half decay time and amplitude of the glutamate receptor mediated mEPSCs. However, ghrelin decreased the frequency and half decay time of the GABA-A receptor mediated mIPSCs, but no effects on the amplitude.7. Intracerebroventricular injection of ghrelin (100 and 1000 nmol/L) increased the contents of DOPAC and HVA in the striatum, and the DA turnover rate. However, no effects of ghrelin (10 nmol/L) were observed. Intracerebroventricular injection of ghrelin (1000 nmol/L) could also increase the DA release electrical stimulated by medial forebrain bundle (MFB) in the rat striatum.8. Unilateral microinjection of ghrelin (1000 nmol/L) into the SNc induced a significant contralateral dystonic posturing in the haloperidol-induced catalepsy rats and attenuated the descent latency. One micromole XE991 could completely block the effects of ghrelin on the rat behavior, and 5μmol/L retigabine partially inhibited the effects of ghrelin.In conclusion, ghrelin enhances the excitability of nigral dopaminergic neurons in rat brain slices. This excitatory effect of ghrelin is mainly dependent on the inhibition of KCNQ potassium channels through activation of GHS-Rla-PLC/PKC pathway. Ghrelin could also inhibit the presynaptic inhibitory GABAergic inputs. Ghrelin-induced excitation of dopaminergic neurons results in increased DA release which attenuated haloperidol-induced catalepsy. Overall, these results suggest that ghrelin is capable of regulating dopaminergic neuronal excitability and the motor function of nigrostriatal system. Our data fully elucidate the physiological significance of ghrelin on the nigral dopaminergic neurons and the nigrostriatal system, which provide new evidence for the usage of ghrelin to treat PD.