| ObjectiveIdentificating the types of ventrolateral preoptic nucleus (VLPO) and investigate their action potential (AP) firing roles during day and light. To investigate the synaptic transmission manner of the projection from intrinsically photosensitive retinal ganglion cells (ipGGCs) to VLPO, and explore the micro connection among different types of neurons within VLPO.Methods1. Identification of cell types in VLPO and their firing rateThe fresh slices were made from WT mouse aged 4-6 weeks, and the neurons were identified by their shape, the response characteristics to noradrenaline (NA) and electrical characteristics. AP firings of neurons were recorded during 11:00-16:00 and 22:00-02:00 with patch-clamp recording. The firing rates of these neurons were analyzed using t-test;2. Investigating the trasmmison from ipRGCs to VLPOThe fresh slices were made from Rosa-lsl-ChR2-eYFP(Ai32) mouse aged 1-2 mooth,470 nm blue light was used to stimulate the terminals of iPRGCs in VLPO area, postsynaptic current or voltage was recored under voltage clamp or current clamp mode.3. The study of micro-circutry within VLPOThe fresh slice was made from Galanin-cre; Rosa-TDT aged 4-6 weeks. Patched two kind of neurons in VLPO area, interneurons and sleep-promoting neurons, stimulated the interneurons in the mode of current clamp, and the response of sleep-promoting neurons was recorded in the mode of voltage clamp.Results1. The neurons in VLPO were identified into two types. One type neurons with triangular and multipolar shape can be hyperpolarized by NA and have the electrical property of low-threshold spike (LTS). The other type neurons with fusiform and bipolar shape can be depolarized by NA and lack of the electrical property of LTS (non-LTS). Of 59 recorded neurons in VLPO,44 neurons have the character of LTS, and 15 neurons are non-LTS. Using t-test statistics, the AP firing rate of LTS neurons which were hyperpolarized by NA shows higher at day time than that at night (P<0.01), and the AP firing rate of Non-LTS neurons which were depolarized by NA shows the similar tendency, that AP firing rate at day time is higher than that at night (P<0.05).2. Of the 30 recored neurons in VLPO, only 2 neurons responsed to 470 nm blue light stimuli, and these neorons were sleep-promoting neurons in according to electrical characteristics and pharmacological test.3. Of the 25 pare neurons with patch clamp recording within VLPO, there did not find any pare neurons which had functional connections between them.Conclusion1. There are two types of neurons in VLPO. Both of them show high firing rate at day time and low firing rate at night time, which indicate they participate in regulating circadian rhythem.2. ipRGCs has direct projection to VLPO, which may excited sleep-promoting neurons in VLPO to regulate sleep and wake. This find will prove functional evidence for light remedy remedy in clinic. |
