| Background For micro-organisms,plants and animals,circadian rhythm is a common phenomenon.Being able to maintain circadian rhythm is very important for human health.In the present fast-paced life,shift work or jet lag caused by trans-continental travel will lead to circadian rhythm disorders,and further disorders in gene expression,protein synthesis,energy metabolism etc.Chronic rhythm disorders can eventually induce tumor,heart vascular disease,sleep disorders and many serious illnesses,which will become a huge economic and spiritual burden to the society and the family.Therefore,studies in the generation and regulation mechanisms of circadian rhythm are urgently needed for improving human beings' health and quality of life.Suprachiasmatic nucleus(SCN),which is located in the hypothalamus,is the master center of mammalian circadian rhythm.It controls the circadian rhythm of mammal's daily physiological functions and behaviors by synchronizing the circadian rhythm with the 24-hour light/dark cycle.Anatomically,the SCN is divided into at least two regions,including the ventral lateral core region and dorsal shell region.It was found that a large number of neurons secrete vasoactive intestinal peptide(VIP)and?-aminobutyric acid(GABA)in the core region.In the dorsal shell region,a large number of neurons express vasopressin(AVP).Buijs et al.used co-immunostaining to label the terminal of VIP neurons and AVP neurons in the rat and found that both of them secreted GABA.For decades,investigators have diligently studied the role of these neuropeptides in the circadian rhythm system.With the VIP gene knockout mice,Colwell and his colleagues observed under constant darkness,the phase of VIP-deficient mice free-moving forward by 8 hours compared to that of those maintained under the bright/dark cycle.The periods of their free activities were also much shorter.In addition,Colwell et al.found that VIP can significant enhance the inhibitory transmission in wide-type and VIP-deficient mice.They confirmed the important role of VIP in regulating physiological functions and behaviors.At the same time,Evan et al.pointed out that VIP signal and GABA signal modulate the synchronization of SCN.Interestingly,most of the neurons in SCN are GABAergic,and almost all SCN neurons exhibit spontaneous inhibitory postsynaptic currents,indicating that GABA receptors exist in almost all of the SCN neurons.While Takahashi et al.concluded that the input and output of ?-aminobutyric acid between VIP neurons and other neurons is very sparse,it remains to be defined that whether the inhibitory synaptic transmission of VIP neurons are turely sparse as Takahashi described.We hypothesize that the electrophysiology results may depend upon the orientation of the SCN slice preparation.For instance,in the study of Takahashi et al.,they used coronal sections.Whether the properties of inhibitory synaptic of VIP neurons in brain slice in the sagittal plane and the coronal plane are the same,and the distribution of GABAnergic synapse is turely spare,need to be studied On the other hand,the most important inputs to SCN are originated from the intrinsically photoreceptive retinal ganglion cells(ip RGCs).From which light signal is transmitted by the retinohypothalamic tract(RHT).The synaptic terminals of ip RGCs co-release glutamate and adenylate cyclase activating peptide(PACAP),which mainly affect VIP neurons in SCN.Therefore,the properties of excitatory input onto VIP neurons need to be studied.In addition,in PACAP-/-mice,the rhythm of light-induced phase delay and advance were both reduced.However,the mechanism of PACAP's effect and modulation of the circadian rhythm remains unclear.Based on prior findings,the aim of the present study is to examine the excitatory and inhibitory synaptic properties of VIP neurons in SCN and the neuropeptides in SCN using a combination of electrophysiological patch-clamp recording and opti-genetic techniques.Objectives Spontaneous action potentials of VIP and non-VIP neurons were recorded during the day or night by use of electrophysiological patch-clamp whole-cell recordings from transgenic mice in which VIP-expressing neurons were labeled.Fresh brain slices containing SCN regions were obtained at different times during the day.The IPSCs,m IPSCs of VIP and non-VIP neurons were recorded in the sagittal plane or coronal plane,respectively.EPSCs and m EPSCs VIP and non-VIP neurons were recorded in coronal plane.In addition,in order to study the effect of PACAP on the circadian rhythm which is released from the ip RGCs terminals in SCN via RHT,we used transgenic mice that express channelrhodopsin in ip RGCs.Light stimulated the axon terminals of the ip RGCs and electrophysiological patch clamp whole-cell recording were performed to study the effects of PACAP antagonists on postsynaptic currents in SCN neurons.Methods1.In vitro brain slice preparation Transgenic mice(10-12 weeks old)were anesthetized with isoflurane and the whole brain was quickly and gently removed,during night the whole procedure was performed under dim red light to avoid the effects of additional illumination on the animals.Brain slices containing SCN were obtained in sagittal plane or coronal plane of 300 ?m thickness in a 4 °C NMDG –cutting solution in a Lecia VT1200 S vibratory slicer,placed in a container containing artificial cerebrospinal fluid(ACSF)and incubated in water bath at 37 °C for 45 min with 95% oxygen(O2)and 5% carbon dioxide(CO2).Electrophysiological recording was carried out at room temperature(about 25 °C)after the incubation.2.Whole-cell patch-clamp electrophysiology Spontaneous action potential,IPSCs,m IPSCs,EPSCs,and m EPSCs of VIP and non-VIP neurons were recorded by whole-cell voltage-clamp or current-clamp recording.Whole-cell voltage–clamp configuration was used to record the postsynaptic currents induced by 470 nm wave length blue light in SCN neurons.3.RT-PCR Brain slice containing SCN in 10-12 weeks old OPN4-Td T mice was prepared.SCN tissue was isolated using glass electrode under microscope and the total RNA was extracted.The expression of PAC1,VPAC1,VPAC2 and NPY in SCN was examined by RT-PCR.4.Drug perfusion with Micro-circulation system The PACAP receptor antagonist,PACAP6-38,was administered at a final concentration of 500 n M for 2 or 5 min.Terminals of ip RGCs were stimulated with light at 470 nm wave length before and after the drug application.The effect of PACAP6-38 on the projection of ip RGCs to the SCN was recorded.5.Statistics All the electrophysiological data was analyzed with Clampfit10.6,Igor6.0 and Graph Pad Prism5.The results were expressed as`x ± SEM.Statistical significance was determined by student t test.P < 0.05 was considered statistically significant.Results1.Action potential: The firing rates of VIP neurons were significantly higher than that of non-VIP neurons both during day and night.2.IPSCs: The frequency of IPSCs of VIP neurons in coronal plane of brain slices was lower than that of in non-VIP neurons during day and night.The frequency of IPSCs of VIP neurons during the day was higher than that at night.On the sagittal plane of brain slices,the frequency of IPSCs in non-VIP neurons during daytime was significantly higher than that of in VIP neurons.Compared with the coronal plane of brain slices,the amplitude of IPSCs in the two types of neurons increased significantly during the day,and the frequency of the IPSCs in the VIP neurons were also significantly increased.3.m IPSCs: In the coronal plane,the amplitude and frequency of the m IPSCs in the VIP neurons during the day were higher than those in the night.The amplitude of m IPSCs in the non-VIP neurons was much higher during the daytime than that during the night.The amplitudes of the m IPSCs in the two types of neurons were significantly different in the daytime.In sagittal plane,the amplitude and frequency of m IPSCs were not different during day and night for both VIP and non-VIP neurons.The amplitude and frequency of m IPSCs were also not different between these two types of neurons.There was no difference in amplitude and frequency of m IPSCs between coronal and sagittal planes for both VIP and non-VIP neurons4.EPSCs: The frequency and amplitude of EPSCs in two types of neurons in the daytime and night showed no difference.5.m EPSCs: The frequency and amplitude of m EPSCs in VIP neurons in the daytime and night showed no difference.6.The strength of ip RGCs to SCN connection at night was significantly higher than that of daytime.7.PACAP antagonist can significantly inhibit the projection strength of ip RGCs to SCN during daytime.Conclusion1.Higher firing rate in VIP neurons during the daytime and night compared with non-VIP neurons may be critical for VIP neurons in regulating circadian rhythm.2.Significant differences in synaptic properties of VIP neurons between sagittal and coronal planes suggest a spatial bias of inhibitory signaling.3.The frequency of m IPSCs was much higher in VIP neurons during the day,suggesting light may regulate synaptic strength,but whether light affect pre-synaptic vesicle package or the number of post-synaptic receptor was not known.4.PACAP receptor antagonist significantly inhibited the signal input from ip RGCs in SCN,indicating that PACAP is an important daily rhythm modulator of glutamate signal in the retinal hypothalamic pathway. |
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