Circadian Variation Of Electrical Activity Of VIP Neurons In SCN And Retrograde Tracing Of Brain Areas Projection To SCN

ObjectiveNeurons in the suprachiasmatic nucleus?SCN?exhibit circadian variation in their neuronal activities.Here,we show that their input synapses undergo daily regulation.The presence of light is the most important factor that affects the circadian rhythm of organisms,and glutamate is the major neurotransmitter secreted by synaptic terminals on the retino-hypothalamic tract?RHT?.We discovered daily variation in the frequencies,but not the peak amplitudes,of miniature postsynaptic currents?mPSCs?for both glutamatergic and GABAergic synapses.In addition,vasoactive intestinal polypeptide?VIP?neurons play an important role in the circadian rhythm in the SCN brain regions,which can directly receive input of glutamatergic information expressed in the RHT terminal.Therefore,we wanted to explore how VIP neurons play a role in the circadian variation of neuronal activity in the SCN.Methods1.In vitro brain slice preparationC57/B6 mice and VIP-ires-Cre::Rosa-GCaMP5 transgenic mice?2-3 months old?were anesthetized with 4%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 VT1200S 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?.2.Whole-cell patch-clamp electrophysiologyGABAergic and glutamatergic synaptic mPSCs were recorded with electrophysiological recordings in SCN brain regions at room temperature?using different bath solutions?.Transgenic mice brain slices were stimulated with blue light at a wavelength of 470 nm.VIP neurons in the SCN could be seen under a fluorescence microscope.3.StatisticsFor detection of miniature events,recording traces were further low-pass filtered with a Gaussian filter to 1 kHz in Clampfit 10.7?Axon CNS?.An event template was generated by averaging 20 visually detected events,and then an event detection routine provided by Clampfit went through the whole trace and detected all the events that statistically matched the kinetics of the template.Following this automatic detection,we applied human visual inspection to evaluate the quality of the detection,and if the total number of false-positive and true-negative events exceeded 10%of the number of detected events,the entire process would be repeated with a new template.The peak amplitude of each detected event was measured as the local minimum of the peak from the baseline.The frequency of the miniature events was calculated by dividing the number of events by the total time.Data were presented as mean±SEM.The n values indicated in the text were the number of cells recorded.The mean frequency of mPSCs was the average of miniature event frequencies from each cell.The mean mPSC amplitude was the average of the mean peak amplitudes of each cell.P<0.05 was considered statistically significant.Results1.The frequency of glutamatergic mPSCs was significantly higher in the dark phase?0.57±0.12 Hz,n=27?than in the light phase?0.24±0.05 Hz,n=24?.Their peak amplitudes were not significantly different between the light?7.29±0.47 pA,n=24?and dark phases?6.93±0.51 pA,n=27?;2.The frequency of GABAergic mPSCs was significantly reduced during the dark phase?1.14±0.14 Hz,n=29?compared with the light phase?3.56±0.98 Hz,n=13?,and their peak amplitudes were not significantly different between the light?42.43±6.38 pA,n=13?and dark phases?35.10±3.34 pA,n=29?;3.During the mouse's adaptation to the jetleg?time shift 4h?,on the third day of adaptation to the new dark environment,there was no statistical difference in the frequency of GABAergic?Daytime:0.24±0.05Hz,n=24;Daytime?Dark?:0.11±0.02Hz,n=13?and glutamatergic mPSC?Daytime:3.56±0.98Hz,n=13;Daytime?Dark?:2.85±0.58Hz,n=12?in the SCN neurons;4.During the photoconversion to dark phase,recordings of glutamatergic mPSCs?Night:0.40±0.14Hz,n=15;Night?Light?:0.18±0.03Hz,n=15?showed an decreased frequency,but recordings of GABAergic mPSCs?Night:1.46±0.27Hz,n=15;Night?Light?:2.12±0.38Hz,n=14?showed a increased frequency.5.The transgenic VIP-cre:GCaMP5 mice were used to identify VIP neurons and the VIP neurons and non-VIP neurons in the ventral SCN were recorded.Under the LD cycle,VIP neurons exhibited a higher frequency of glutamatergic mPSCs and lower frequency of GABAergic mPSCs in the light phase compared with the dark phase?glutamatergic mPSCs:0.42±0.14 Hz,n=14[L]vs.0.85±0.28 pA,n=11[D];GABAergic mPSCs:1.01±0.20 Hz,n=11[L]vs.0.50±0.10 Hz,n=11[D]?.ConclusionsFirstly,we verified that the external dark environment is an important factor affecting the activity of SCN neurons.The plasticity of the quantal events could affect the output of SCN neurons over the daily cycle,and we speculated that the circadian variation in the electrical activity of VIP neurons mediates the difference in day and night electrical activity of SCN neurons.Objective In the study of neural circuit tracing,retrograde is an efficient method to recongnize the upstreams of target neuron.Viral vectors are powerful gene delivery vehicles for the nervous system,but most available tools suffer from inefficient retrograde transport or limited clinical potential.Therefore,we apply a newly evolved variant,r AAV2-retro,permits robust retrograde access to suprachiasmatic nucleus with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.In this study,we observe and count upstreams of SCN.Methods 1.Virus injectionHealthy adult Ai9 transgenic mice were anesthetized by 1% pentobarbital sodium according with body weight.After narcotized,mice were fixed on an adapter.Then the mouse bregma point and lambda point were fully exposed.Then we corrected the horizontal line of the mouse brain according to the mouse brain alas and drilled.We tilted the syringe by 10° according to the given coordinates.2.Frozen brain slices After three weeks of virus injection,mice were perfused with 4% paraformaldehyde.Take out the whole brain,perfusion fixation about 20 hours.Then 4% paraformaldehyde were replaced with 30% sucrose solution.Avoid light as much as possible.After dehydration completed,the coronal brain slices were cut by a freezing microtome at a thickness of 50 ?m/piece,washed three times with PBS,covered slip and sealed it.3.Virus observation and filmingObserved brain slices under a fluorescent microscope according to mice brain map.Then used a confocal microscope to film.Results and Conclusions The upstream nuclei of SCN we observed are: VLPO,ACo,Me AD,IGL,VMH,Hb,PVP,ZI,Arc LP+Arc MP,PLCo,AHi AL+Me PV,BMP+BLP,Ect+Te A,EW,PMD,PH,etc.