Modulatory Effects Of Adenosine On Activity Of Orexin Neurons And The Actions Of Orexin A On The Intracellular Calcium Of Neurons From Prefrontal Cortex

Orexins localized specifically in neurons within the lateral hypothalamus and perifornical area are the novel neuropeptides discovered in 1998 by two groups respectively. Orexin system includes two separate peptides orexin A (hypocretin 1) and B (hypocretin 2) proteolytically derived from the same precursor protein. The actions of orexins are mainly transduced by orexin receptor 1 and orexin receptor 2 belonging to G-protein coupled receptors superfamily. Many studies have shown that these peptides are importantly implicated in the regulation and promotion of wakefulness. And this role may be mainly fulfilled by the excitatory actions of orexins on multiple subcortical arousal systems and cerebral cortex. As shown by recent electrophysiological studies, the activities of orexin neurons are influenced by several neurontransmitters and neuromodulators, as well as metabolic signals. It is implied that modulation of the activity of orexin neurons by these neurotransmitters or metabolic cues are critical for the regulation of sleep and wakefulness.Among these neuromodulators, adenosine, widely distributed in the mammalian central nervous system, has been proposed as an endogenous homeostatic sleep-promoting factor that accumulates during waking. A growing body of evidence has shown that adenosine may modulate excitatory glutamatergic synaptic transmission, inhibitory GABAergic synaptic transmission or both. An earlier study has demonstrated that adenosine exerts an inhibitory effect on glutamatergic transmission on orexin neurons, which is mediated by A1 receptors located on presynaptic terminals. It is well known that GABAergic synaptic transmission is the major inhibitory input to orexin neurons; however, whether adenosine modulates the inhibitory GABAergic transmission on orexin neurons as well has not been investigated. Specially, whether endogenous adenosine released in lateral hypothalamus is involved in the regulation of synaptic transmission on orexin neurons is still unclear.In addition, our previous study has shown that a few of cultured cortical cells tested show intracellular calcium elevation in response to orexin A, which depend on extracellular Ca2+ entry. Also, our electrophysiological results have illuminated that orexin A can excite the freshly dissociated neurons from layers V and VI of prefrontal cortex (PFC). It is intriguing to test whether neurons in PFC show the same intracellular calcium increase in response to orexin A.In the present study, we have focused on a possible role for adenosine in regulating inhibitory synaptic transmission on orexin neurons by performing perforated patch clamp recording in hypothalamic slices from orexin-EGFP transgenic mouse under voltage-clamp conditions. And we also examined the activity-dependent release of endogenous adenosine in regulating the activity of synaptic transmission as well. The effect and mechanisms underlying cytosolic Ca2+ changes induced by orexin A was investigated by using calcium imaging in acutely dissociated neurons from layers V and VI of PFC.1. Adenosine suppresses evoked inhibitory synaptic transmission on orexin neuronsPerforated path clamp recordings of orexin neurons in hypothalamic slices from orexin-EGFP transgenic mice showed that evoked inhibitory postsynaptic currents (evIPSCs) was 51.4241% of control after superfusion with adenosine (100μM; n=14, P<0.05) and 87.1222% of control after its withdrawal. Moreover, adenosine reduced the evIPSCs amplitude in a concentration-dependent manner. The amplitude of evIPSCs was reduced, on average, to 93.0183% (n=6, P>0.05), 75.1114% (n=9, P<0.05) and 56.8569% (n=7, P<0.05) of control for 1, 10, 50μM adenosine, respectively.2. Presynaptic A1 adenosine receptor is responsible for the effect of adenosine on GABAergic synaptic transmissionApplication of A1 receptor antagonist CPT (200 nM) completely blocked the inhibitory effects of adenosine on evIPSCs (98.408% of control; n=9, P>0.05). During the course of the experiments it was noted that bath application of CPT alone did not affect evIPSC amplitude compared to control condition. Application of DMPX (10μM) did not alter the amplitude or the duration of evIPSCs (99.351 % of control; n=8, P>0.05), nor did it affect the inhibitory action of adenosine on the evIPSCs of orexin neurons (59.139 % of control; P<0.05). These results suggest that adenosine inhibits evoked inhibitory transmission in orexin neurons through activation of A1 receptors.In the presence of 50μM adenosine, the average of paired-pulse ratio (PPR) was increasing from 1.095891 to 1.75679 (n=9, P<0.05). Application of adenosine did affect the cumulative inter-event interval distribution of the mIPSC, producing a rightward shift of the curve. The cumulative amplitude distribution of the mIPSC was not affected after application of adenosine. These observations indicate that adenosine inhibits evIPSCs in orexin neurons by a predominantly presynaptic mechanism (n=10, P<0.05).3. Activity-dependent release of adenosine inhibits excitatory synaptic transmission on orexin neuronsBath application of adenosine (100μM) induced a decline in the amplitude of evEPSCs to 56.413% of controls (n=11, P<0.05). Glutamatergic synaptic transmission after the application of CPT (200 nM) did not differ from the control condition when the stimulation rate was 0.033Hz and 1 Hz (n=7, P>0.05). The depression of evEPSCs elicited at 2Hz~5Hz was not abolished (P>0.05) by CPT (200nM), whereas when the frequency of stimuli was raised to 10 Hz, the depression of evEPSCs began to be partially blocked by CPT after 360s of stimuli (n=10, P<0.05). Application of DMPX (10μM), a A2 receptor antagonist, did not block the depression induced by 10 Hz stimulation (n=8, P>0.05). In the presence of CPT (200 nM), the depression of evEPSCs induced by 100Hz for 1s was also significantly partially inhibited during the course of stimulation (n=11, P<0.05).4. Activity-dependent release of adenosine inhibits inhibitory synaptic transmission on orexin neuronsNo change of the amplitude of evIPSCs was observed after application of CPT (200 nM) (n=9, P>0.05) in 1Hz stimulation. In the presence of CPT, the depression of evIPSCs elicited at 2Hz~5Hz was not abolished. When the frequency of stimuli reached to 10 Hz, the depression was then partially inhibited by CPT (200 nM) (n=9, P<0.05), while superfusion of DMPX (10μM) did not block the depression (n=6, P>0.05). When the frequency of stimulation increased up to 100Hz for 1s, the depression of evIPSCs was not inhibited by the CPT (n=6, P>0.05).5. A1 receptor antagonist enhances the induction of long term potentiation on excitatory synaptic transmission of orexin neuronsIn the perforated path clamp recordings, high frequency stimulation (HFS) or theta burst stimulation (TBS) can induce the long term potentiation (LTP) of excitatory synaptic transmission in orexin neurons, and the amplitude after induction was 119.836% (n=12) and 128% (n=12) of control, respectively. While in the presence of CPT during the induction by HFS, the amplitude of currents will increase up to 165.9375% after induction (n=12, P<0.05). The results indicate that endogenous adenosine may be involved in the induction of LTP in orexin neurons. 6. Extracellular calcium through L-type Ca2+ channels by activation of PLC-PKC pathway contribute to elevation of intracellular calcium of acutely dissociated neurons from PFCThe calcium imaging studies demonstrated that intracellular calcium in about of 20% (n=8, 8 of 40) of dissociated acutely PFC neurons was increased in a dose-dependent manner in response to orexin A (1μM). The increase in peak of calcium fluorescence intensity induced by orexin A was abolished in the presence of orexin receptor 1 antagonist SB 334867. Preincubation of PLC inhibitor D609 or PKC inhibitor BIS II, no elevation of intracellular calcium was observed in the presence of orexin A. Also, in extracellular calcium free condition, orexin A failed to increase the intracellular calcium, but the elevation was still observed when pretreatment of thapsigargin, an inhibitor of calcium store, was applied. Furthermore, the elevation induced by orexin A was not examined in the presence of L-type calcium channels inhibitor nifedipine. These findings illuminate that orexin A-induced increase of intracellular calcium in PFC neurons is mainly from extracelluar calcium influx through L-type calcium channels by activation of orexin receptor 1 and PLC-PKC signaling pathway.In conclusion, these observations provide evidence that the inhibitory effects of adenosine on inhibitory synaptic transmission in orexin neurons are mediated by presynaptic A1 receptors in a dose-dependent manner. Under strong and sustained synaptic transmission activities, endogenous adenosine will be released to extracellular space in hypothalamus, modulating the activity of synaptic transmission as well as the synaptic plasticity. Moreover, the increase in intracellular calcium induced by orexin A in freshly dissociated PFC neurons may be attributed to the extracellullar calcium influx through L-type calcium channels mediated by activation of orexin receptor 1 and PLC-PKC signaling pathway.