Studying The Indirect Pathway Of Noradrenaline Acting On The Ventrolateral Preoptic Region Neurons

Ventrolateral preoptic area(VLPO)is a sleep related center,which mainly contains two types of neurons: sleep-promoting neurons and interneurons.Previous studies have shown that noradrenaline(NA)can affect the excitability of two types of neurons in VLPO,but the mechanism is not clear whether the effect is through the indirect pathway of presynaptic neurons or the direct pathway of postsynaptic neurons.In this study,the whole cell patch clamp technique was used to study the mechanism of excitatory effect of norepinephrine on VLPO neurons in C57 mice brain slices.Our results show that NMDA receptor antagonist(AP5)can block the excitatory effect of NA on action potential firing frequency of interneurons,but not the inhibitory effect of NA on sleep promoting neurons.At the same time,neither AMPA receptor antagonist nor GABA receptor antagonist can affect the excitatory effect of Na on two types of neurons in VLPO.The results suggest that the stimulation of Na on the excitability of interneurons in VLPO is mediated by NMDA receptor indirectly.Therefore,RT-PCR and immunofluorescence were used to verify that glutamatergic neurons or their terminals exist in the brain region of VLPO to release glutamate,thus participating in the indirect effect.Objective:To explore the indirect pathway of noradrenaline on VLPO neurons.Materials and methods:1.Brain slice culture: 2-3 weeks old C57 mice were removed from the ice,and the brain slices containing VLPO were cut out with a vibration slicer,with a thickness of 300 ?m,and then transferred to the extracellular fluid,oxygenated,and incubated at 25°C for one hour.2.Whole cell patch clamp recording of brain slices: the incubated brain slices were absorbed by the prepared pipette and transferred to the recording bath,and the extracellular fluid was heated to 30°C for whole cell patch clamp recording.During the recording process,the peristaltic pump maintained the perfusion rate of 2 ml / min and oxygenated.After the stable recording,the corresponding drugs and blockers were given to record the changes of action potential firing frequency.3.RT-PCR: after inhaling isoflurane anesthesia,mice were decapitated and their brains were sliced with a vibration slicer,and then the tissues containing VLPO were collected at the location of VLPO according to the brain atlas.RNA was extracted first and then amplified by PCR.Finally,the expression of GAPDH,Glu N1 R,Glu N2 ARand Glu N2 BR was detected by 1% agarose gel electrophoresis.4.Immunofluorescence: after perfusion,the brain of mice was taken and dehydrated for three days.The brain slices containing VLPO were cut with frozen section agent,and the thickness was 30 ?m.Then,according to the principle of antigen and antibody binding,the corresponding antibody was incubated.After a series of steps such as PBS rinsing and sealing,the expression of Glu N1 R Glu N2 BR and VGLUT2 R were detected respectively.One group was the blank control,and the other was the first antibody.Results:1.Identification of two types of VLPO neurons: under the low power microscope,we found the VLPO brain area.Then switching 40 X high power microscope,we identified the two types of neurons according to their shape characteristics.The sleep-promoting neurons were multi-level triangles,and the intermediate neurons were bipolar fusiform.Select a neuron to clamp,break the membrane and record stably,then observe and record the change of action potential firing frequency of neurons through the administration of noradrenaline.If the frequency becomes slower,it is identified as sleep-promoting neuron,if the frequency becomes faster,it is interneuron.2.The effect of NA on the firing of VLPO neurons in the presence of GABA blocker: by microscopic observation,the clamp cells were selected in VLPO for electrophysiological recording.First,the type of neurons was identified by the effect of neurons on NA,and then NA was given in the presence of flumazenil to observe the change of action potential firing frequency of neurons.The results were obtained that in the presence of flumazenil,the firing frequency of action potential in the presence of flumazenil was significantly lower than that in the presence of Ctl + flumazenil(P < 0.05;n = 7).For interneurons,the firing frequency of action potential in the presence of flumazenil was significantly higher than that in the presence of Ctl+ flumazenil(P < 0.05;n = 7).The results were obtained that in the presence of flumazenil,the excitatory and inhibitory effects of NA on the two types of neurons still existed.3.Effect of NA on the firing of VLPO neurons in the presence of AMPA blocker: for sleeppromoting neurons,the firing frequency of action potential after NA administration was significantly lower than that of action potential after Ctl + CNQX administration in the presence of CNQX(P < 0.001;n = 7).For interneurons,the firing frequency of action potential in the presence of CNQX was significantly higher than that in the presence of Ctl + CNQX(P < 0.01;n = 8;).The results showed that in the presence of flumazenil,the excitatory and inhibitory effects of NA on the two types of neurons still existed.4.Effect of NA on the firing of VLPO neurons in the presence of NMDA blocker: for sleep-promoting neurons,the firing frequency of action potential in the presence of AP5 was significantly lower than that in the presence of Ctl + AP5(P < 0.001;n = 8).For interneurons,in the presence of AP5,between NA and Ctl + AP5(n = 8),there was no significant difference in the firing frequency of action potential.The results showed that in the presence of AP5,the inhibitory effect of NA on interneurons still existed,while the excitatory effect of NA on interneurons disappeared.5.Expression of glutamate receptors in VLPO: the expression of glutamate receptors(Glu N1 and Glu N2B)and VGLUT2 R in VLPO was detected by RT-PCR and immunofluorescence.Conclusion:1.These findings revealed that the excitatory effects of NA on VLPO interneurons is an indirect effects caused by glutamatergic neurons releasing glutamate,which is mediated by NMDA receptor of VLPO interneurons.2.In this study,the expression of glutamate receptor was verified by immunofluorescence.