Hypocretin Potentiates NMDA Receptor-mediated Somatic Noradrenaline Secretion From Locus Coeruleus Neurons

Hypocretins(orexins) have been implicated in feeding,neuroendocrine homeostasis, autonomic functions,drug addiction,and sleep/wakefulness.They act on two G-protein-coupled receptors,hypocretin receptor 1 and 2(Hcrtr 1 and 2),which are differentially expressed throughout the central nervous system.Locus coeruleus(LC) neurons are densely innervated by hypocretinergic-immunoreactive fibers and mainly express Hcrtr 1 but less Hcrtr 2.Hpocretins increases LC activity and induces a direct excitatory effect on LC neurons.Furthermore,hypocretins evoke noradrenaline(NA) release from the axonal terminals of LC neurons in the dentate gyrus and cerebral cortex.Generally,vesicles are not only released from axon terminals but also from cell bodies in various cell types.We recently observed NA secretion from the somata of LC neurons.These results,combined with the findings that the main typical responses to hypocretin receptor activation include elevation of intracellular calcium concentration([Ca²⁺]_i),an event essential for somatic release,prompted us to investigate whether hypocretins modulate somatic secretion in LC neurons.Excitatory amino acids,including N-methyl-D-aspartate receptor(NMDAR)-mediated neurotransmission,provide the major excitatory input to LC neurons and are involved in many of their physiological functions.Interactions between hypocretins and glutamate/NMDA have been repeatedly reported.Our earlier data showed that hypocretin-1 (HCRT) and glutamate have a synergistic effect on the excitability of prefrontal cortical neurons.Importantly,HCRT induces potentiation of NMDAR-mediated transmission in the ventral tegmental area,which is critical for synaptic plasticity.Nonetheless,little is known about HCRT-NMDA interactions in LC neurons.In the present study,using amperometry, whole-cell patch-clamp recordings,and calcium imaging in rat brain slices,we first determined whether HCRT or NMDA would induce somatic secretion from LC neurons. Subsequently,we investigated whether HCRT would affect NMDAR-mediated somatic secretion. 1.Morphological and electrophysiological properties of LC neuronsLC neurons are easily distinguished by their typical morphological features and electrophysiological properties.LC was identified as an aggregate of neurons that located in a region adjacent to the medial side of the mesencephalic trigeminal nucleus and to the lateral side of the fourth ventricle at the levels of the cerebellar peduncle.The dissociated LC neuron had a large and oval shaped soma(diameter:20～50μm) with a few short robust dendrites. Almost all large neurons are tyrosine hydroxylase positive neurons,suggesting they are noradrenergic neurons.Whole-cell recordings were obtained from a total of 21 LC neurons in brain slices.The mean resting membrane potential was -55.9±0.9 mV,and the mean input resistance was 179.8±0.5 MΩ.The majority of LC neurons were producing spontaneous firing having the amplitudes from 0.5 to 5 Hz.LC neurons fire in two distint acitivity modes: tonic and phasic.Aslo,we obtained whole-cell sodium,potassium and calcium currents respectively.In addition,NA had a significant inhibitory effect on the excitability of LC neurons.2.HCRT induces somatic release from LC neuronsPuff application of 100 nM HCRT did not induce NA secretion,while a higher concentration(1μM) elicited a significant response.Similarly,whole-cell recordings showed that the excitatory action of HCRT on the firing rate was also concentration-dependent.In addition,HCRT-induced secretion was dramatically inhibited by the selective Hcrtr 1 antagonist SB 334867(1μM),confirming the role of Hcrtr 1.When we used a glass pipette to clean off extracellular materials overlying the somata before recording,individual amperometric spikes induced by 1μM HCRT were detected.Furthermore,in the presence of tetrodotoxin(TTX,1μM) to block all dendritic and axon terminal secretion,HCRT-evoked secretion was inhibited only by～20%.These results demonstrate that HCRT-evoked catecholamine release is mainly from the neuron somata in LC slices.3.NMDA induces somatic release from LC neuronsApplication of 100μM NMDA significantly induced secretion in LC slices.In contrast, LC neurons did not show a secretory response to normal ACSF.In addition,both 20μM and 1 μM NMDA had weaker effects on amperometric responses than that of 100μM,suggesting a dose-dependent response.Furthermore,NMDA-induced secretion was completely blocked by pretreatment with the NMDA receptor antagonist APV(50μM),confirming the specific action of NMDA receptor activation.These results reveal that NMDA evokes catecholamine secretion within LC somatodendritic region in a dose-dependent manner.4.HCRT potentiates NMDAR-mediated somatic release from LC neuronsApplication of 100μM NMDA evoked a pronounced secretion.This response to NMDA was subsequently enhanced by treatment with 100 nM HCRT for 5 min.As a control, application of ACSF did not affect the NMDA-induced amperometric response.In addition, SB 334867(1μM) completely abolished HCRT-enhanced NMDA-evoked secretion, confirming the prerequisite role for Hcrtr 1.Moreover,even in the presence of TTX(1μM), 100 nM HCRT still enhanced NMDA-induced amperometric response,indicating that this effect is TTX-resistant and thus HCRT modulates NMDAR-mediated somatic secretion. Further,in the presence of the PKC inhibitor BISâ…¡(1μM),the potentiation of NMDA-induced secretory response by 100 nM HCRT was completely blocked.5 min treatment with 1μM phorbol 12-myristate 13-acetate(PMA),a membrane-permeable agonist for PKC,significantly enhanced NMDA-evoked amperometric response,thereby mimicking the effect of HCRT.Finally,as a control,100 nM HCRT had no effect on the high-K⁺-evoked release.These results confirm that HCRT potentiates NMDAR-mediated somatic secretion through a PKC-dependent pathway in LC neurons.5.HCRT increases NMDAR-mediated[Ca²⁺]_i rises in LC neuronsWhen we added Ca²⁺-free ACSF containing 1 mM EGTA and 200μM Cd²⁺ to the bath solution for 30 min,neither 100μM NMDA nor 1μM HCRT induced any amperometric signals,confirming that NMDA- or HCRT-evoked somatic secretion is Ca²⁺ dependent in LC neurons.HCRT at the lower concentration(100 nM) caused barely detectable changes in[Ca²⁺]_i. As a positive control,high-K solution dramatically evoked somatic[Ca²⁺]_i elevation. Similarly,application of 100μM NMDA produced a significant[Ca²⁺]_i increase.After adding 100 nM HCRT to the bath solution for 5 min,the second puff of the same NMDA solution resulted in a larger[Ca²⁺]_i response,suggesting that HCRT potentiates NMDAR-mediated [Ca²⁺]_i rises.SB 334867(1μM) or BISâ…¡(1μM) blocked the HCRT potentiation of NMDAR-mediated[Ca²⁺]_i increase,indicating that activation of the Hcrtr 1 receptor and PKC plays a role.These results are consistent with the idea that the HCRT potentiation of NMDAR-mediated[Ca²⁺]_i elevation contributes to the enhanced somatic secretion in LC neurons.In summary,Either HCRT or NMDA applied alone dose-dependently induced somatic secretion.Application of HCRT notably potentiated NMDA receptor-mediated secretion in the absence and presence of TTX,suggesting that the somatic secretion can be modulated.This potentiation was blocked by SB 334867 or BISâ…¡,indicating the involvement of Hcrtr 1 and PKC.Consistent with this,phorbol 12-myristate 13-acetate(PMA),a PKC activator,could mimic the HCRT-induced potentiation.Furthermore,HCRT enhanced NMDA-induced intracellular Ca²⁺ elevation via activation of Hcrtr 1 and PKC,which may contribute to the HCRT-potentiated somatic secretion.These results suggest that HCRT modulates LC activity not only by regulating noradrenergic input to targets of the LC but also by affecting noradrenergic communication within the somatodendritic regions.