| Stable wakefulness and sleep states are fundamental homeostatic processes that serve physiological activities.The normal transition between wakefulness and sleep is under the control of multiple internal and environmental factors.The stability of sleep-wake cycle ensures the coordination of virous of physiological functions.The rapid eye movement sleep(REM sleep)aroused widely interests in many researchers for its unique character of electroencephalogram and electromyography and primary connection with dream.It has been determined that the sublaterodorsal tegmental nucleus(SLD)is necessary and sufficient in REM sleep generation.Specifically,the firing activities of the SLD are increased before and persisted through REM sleep,which process is thought to be the key of initiating and maintaining the REM sleep state.And the glutamatergic neurons in SLD are thought to provide ascending projections for corticohippocampal EEG activation and descending projections that trigger muscle tone decrease during REM sleep.Many neurotransmitter systems,including acetylcholine,γ-aminobutyric acid(GABA),glutamate and the neurotransmitter system in the hypothalamus can excite or inhibit the neural activity of SLD so that participate in the regulation of REM sleep.Notably,the hypothalamic orexinergic system play a crucial role in the modulation of sleep/wakefulness state.The deficiency of orexin system in human and animals is well acknowledged to cause a severe sleep/wakefulness-state instability disorder called narcolepsy-cataplexy.Besides the prominent deficiency in maintaining wakefulness state,narcoleptic patients also suffer from severe rapid eye movement(REM)sleep abnormalities,such as REM sleep disorder(RBD)and sleep paralysis.Consistently,the orexin neurons only cease firing when entering non-rapid eye movement(NREM)sleep,but the activities of orexin neurons are elevated again during REM sleep.Microdialysis investigations also reveal an elevated level of orexin release when entering REM sleep.In this circumstance,it’s has been reported that SLD is directly innervated by orexin fibers.While the orexin signaling in SLD and its corresponding influence on SLD neuronal network urges to be elucidated.In the present study,we first used a combination of morphological and electrophysiological methods to investigate the modulatory effects of orexin on the activity of SLD neurons.Interestingly,we found that orexin not only induces broad excitation but also promotes gap-junction mediated spikelet activities in SLD neurons.Thus,we further determined the underlying mechanisms of orexin on broad excitation and gap-junction activities to SLD neurons,combining neuropharmacological methods with patch clamp recordings.The main findings in the present study are as follows,1.The modulatory effects of orexin on SLD neurons(1)Hypothalamic orexin signaling provids inputs to glutamatergic neurons in the SLDWe firstly observed the distribution patterns of orexin-A containing fibers and orexin-1/2 receptor in the SLD(bregma:-9.2～-9.8 mm).High resolution confocal images revealed that the orexinergic fibers with an obviously varicose-like shape distributed around the ox-1/2 receptor positive SLD neurons.Further double immunostaining of vglut1 and GAD 67 revealed that most neurons in the SLD were proven to be glutamatergic(97%).Moreover,ox-1/2 receptor was largely co-localized with vglut1 in the soma of SLD neurons,while GAD 67 immunostaining was mostly in puncta shape around the the ox-1/2 receptor positive SLD neurons.These data suggested orexin signaling mainly target on glutamatergic neurons in the SLD.(2)Orexin elicits depolarization and elevates the percentage of gap-junction mediated spikelet activities in SLD neuronsWe next examined the modulations of orexin in the neuronal activity of SLD neurons,using whole-cell patch clamp recordings.In the presence of chemical synaptic trans mission blockers,when gradually holding down the membrane potential to around-60 m V,gap-junction mediated spikelet activities that were proved to be carbenoxolone(CBX)-sensitive were observed in 21 of 32(65.6%)tested neurons.Further immunostaining experiments also proved the existence of gap-junctions.In this condition,bath-application of orexin-A(100 nM)elicited a strong depolarization of 7.9±0.8 m V(n=32,P<0.01)in all 32 tested SLD neurons.With the onset of the depolarization,orexin-A(100 n M)also increased the percentage of SLD neurons exhibiting the spikelet activities from 65.6%(21of 32)to 84.4%(27 of 32).These results indicated that orexin not only affected the excitability of single SLD neurons but also elevated electrical coupling in SLD neuronal network.(3)Orexin induces inward currents with noisily rhythmic fluctuations in SLD neuronsUnder voltage-clamp configurations,30-300 n M orexin-A elicited inward currents in all 8 tested neurons.30,100,and 300 nM orexin-A elicited inward currents at 12.1±3.0,22.7±3.6,and 37.0±8.7 pA,respectively(n=8).In addition,the orexin-induced inward current exhibit noisily rhythmic fluctuations at the steady state,reflecting elevated spikelet activities.30,100,and 300 nM orexin-A increased the noise level of the inward currents by17.0±8.2%,64.1±18.6%,and 91.8±20.7%,respectively(n=8).2.The mechanisms underlying the modulatory effects of orexin on SLD neurons(1)Orexin-induced inward currents with noisily rhythmic fluctuations in SLD neurons are mediated by postsynaptic orexin-1 receptor and orexin-2 receptorThe orexin-induced inward current(n=7,P=0.528)and the noise increasement of the inward current(n=7,P=0.854)remained unaffected in the presence chemical synaptic blockers.While bath application of orexin-2 receptor antagonist TCS OX2 29(1μM)partially blocked the orexin-induced inward current(n=6,P<0.05),and the noise increasement(n=6,P<0.05).Further adding orexin-1 receptor antagonist SB 334867(1μM)totally abolished the remaining component of the orexin-induced inward current(n=6,P<0.01)and the noise increasement(n=6,P<0.05).These results demonstrated that the orexin-induced inward currents with noisily rhythmic fluctuations were dependent on both postsynaptic orexin-1 and orexin-2 receptors.(2)Orexin excites 2/3 orexin receptor positive SLD neurons and spreads the excitation to the whole SLD neuronal network via gap junctionsIn voltage-clamp configuration,CBX(100μM)largely attenuated the noise increasement of the orexin-induced inward current(n=15,P<0.05),and eliminated the rhythmic fluctuations of the inward current.Interestingly,CBX(100μM)also decreased the amplitude of the orexin-induced inward current(n=15,P<0.01)and that of 4 of the tested SLD neurons(4/15,26.7%)were totally abolished by the application of CBX(100μM).We used mefloquine(MEF),a more specific blocker of connexin-36(cx-36,the major gap-junction type in neurons)for confirmation.Combining biocytin based post-hoc analysis,we found that MEF(10μM)also decreased the orexin-induced inward current(n=25,P<0.05).Consistently,6 of 25(24%)tested neurons showed no response to orexin-A(100 nM)in the presence of MEF(10μM).Interesting,post-hoc immunostaining for ox-1/2 receptor in these electrophysiologically tested neurons revealed that the orex in non-responsive neurons(n=6)did not express ox-1/2 receptor,compared to those orexin responsive neurons(n=19).Based on above results,we further performed double immunostaining experiments of ox-1/2 receptor with Neu N.The results showed orexin-receptor negative neurons occupied 33.1%(45/136)of total SLD neurons,in similar percentage to the electrophysiological experiments.These data suggest that there only 2/3 SLD neurons express orexin receptors,but the orexin-elevated SLD electrical coupling enables the orexin excitatory input widely spread to the whole SLD networks via gap junctions.In addition,the orexin-induced inward current remains to be unaffected(n=8,P=0.059)by TTX(tetrodotoxin,1μM).While the application of TTX totally eliminated the orexin-induced rhythmic current fluctuations(n=8,P<0.05).Thus,it is the orexin-induced sub-threshold modulations on the SLD orexin receptor positive neurons that were spread through the gap junctions to form the orexin-induced global excitatory effects.(3)The orexin-induced excitation in 2/3 orexin receptor positive SLD neurons is mediated by the opening of the non-selective cationic conductanceIn order to identify the underlying ionic mechanisms of the direct excitation of the orexin receptor positive SLD neurons induced by orexin,we performed series experiments as follow.Firstly,we established the current-voltage plots(I-V curves)of the orexin-induced net current in the SLD neuros and found that the orexin-induced net current was reversed near-26.5±2.5mV,which is close to the equilibrium potential of the Na~+-permeable non-selective cationic conductance(NSCCs).Next,we reduced the Na~+concentration(from 152.25 to 70 mM)in the ACSF for further study.Indeed,changing the extracellular Na~+concentration decreased the amplitude of the orexin-induced inward currents(n=8,P<0.01).Apart from this,the reversal potential of the orexin-induced net current exhibited to a more negative value,-42.3±3.2mV,without a significant change of the Na~+/K~+permeability ratios(n=8,P=0.338).Moreover,with the application of the flufenamic acid(Flu,100μM),which is a selective blocker of the NSCCs,the orexin-induced inward currents were largely blocked in all the test SLD neurons(n=6,P<0.05).Through sets of experiments,we demonstrated that the orexin-induced inward currents in the SLD neurons were mediated by the opening of the NSCCs.(4)Orexin increases the conductance of gap junctions in SLD neuronal networkWe combined neuropharmacological methods with double-patch clamp recordings to investigate the direct modulatory effects of orexin on gap junctions.Firstly,we measured the coupling coefficient between electrically coupled SLD neurons and found that orexin-A(100 n M)bilaterally increased the coupling coefficient,from cell1 to cell2(n=7,P<0.05)and cell2 to cell1(n=7,P<0.05).Besides,we also calculated the coupling conductance of each pair of gap junction-coupled SLD neurons in and without the presence of orexin.Orexin also increased the coupling conductance,both from cell1 to cell2(n=7,P<0.05),and cell2 to cell1(n=7,P<0.05).While orexin did not influence the input resistance of cell1(n=7,P=0.676)or cell2(n=7,P=0.057).Together,these results demonstrated that orexin promotes electrical coupling via increasing the conductance of gap juncti ons in SLD.In summary,the present study revealed that orexin can not only excite the orexin receptor positive SLD neurons by postsynaptic excitation but also increase the conductance of gap junctions in the SLD.Above modulatory effects can result in the spreading of excitation to the whole SLD neuronal network.Thus,orexin can regulate the activities of the SLD neuronal network integrally,which led to the broadly and efficiently excitation of the SLD and promote the synchronized neuronal ensemble activity.Our study provides a new mechanism for REM sleep control.In addition,these finding also provide exciting new possibilities for the treatment of orexin deficiency related diseases. |
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