Sleep And Brain Development During Postnatal Days 7-12 Of Infant Rats

Sleep is an important physiological function in human and animal survival process,and plays an important role in promoting brain development in early life.The spontaneous firing of neurons in the cerebral cortex,which can be recorded on the surface of the scalp or cortex,is a bio-electricity signal that reflects general brain activity.Through telencephalon electrocorticogram(ECo G),electromyography(EMG)and other characteristic changes in different periods,the functional state of adult rat brain can be divided into three periods: wakefulness(wake),non-rapid eye movement sleep(NREM sleep)and rapid eye movement sleep(REM sleep).The rat is an altricial born in a far less mature condition than humans and its cortical maturity during the first postnatal week corresponds to that of the young premature human brain,so it is mostly used in sleep developmental research.Before postnatal days 11-12(P11-12),the brain development(especially the cerebral cortex development)is not mature,so ECo G is not typical,and it is difficult to use adult criteria to determine the real state of the brain.Therefore,the scientific question of this study is to look for brain regions other than the traditional ECo G to record and accurately analyze the sleep-wake phase development of infant rats <P12,and to clarify the relationship between sleep and brain development.Studies have shown that adult rats can experience characteristic waves in specific sleep states of the subcortical nucleus and brain region,such as the theta(4-8.5 Hz)wave in field CA1 of the hippocampus(CA1)and the ponto-geniculo-occipital like(PGO-like)wave of dorsal lateral geniculate nucleus(DLG)of thalamus and pons during REM sleep.The slow waves(0.5-4 Hz)in the piriform cortex(Pir)associated with NREM.The high-frequency electrical activity of locus coeruleus(LC)associated with wake.Therefore,this study aims to find the electrical activity pattern of subcortical nucleus and brain regions in adult rats during sleep and wake phases,and to find the electrical activity of brain regions with the most reference in each phase.Then we establish the stereotaxic brain atlases of P7-P13 rats to provide parameters for recording the subcortical nuclei and brain regions of rats,and to analyze the sleep development of P7-P12 rats.Furthermore,neural tissue staining was used to analyze the corresponding developmental changes of brain regions which was related to sleepwake in P7-P12 rats.Objective: To reveal the regularity of sleep and brain development at postnatal P7-P12 rats,and to find the corresponding relationship between sleep and brain development.Methods:(1)The local field potential(LFP)in the Pir,CA1,DLG and LC regions of the deep brain nuclei and brain regions of adult rats were collected by ECo G recording system,and its waveform,power spectrum and other specific characteristics were analyzed.It is found that it can represent a new criterion for ECo G to determine the sleep-wake phase,and solve the difficulty of analyzing the sleep-wake phase of P7-P12 rats by ECo G alone.(2)In order to accurately record the LFP of deep brain nuclei and brain regions of infant rats,the atlases of the infant rat brain at postnatal day 7-13 were made by Nissl staining.Common nuclei and brain structures were annotated,and the stereotaxic coordinates of each section were calculated,and verified by Di I staining and immunohistochemistry,so as to provide a reference for stereotaxic localization of each brain region and nucleus of P7-P13 rats.(3)The LFP of deep brain nuclei and brain regions,ECo G,EMG,twitch,electromyography(EOG),and activity behaviors were integrated to determine the sleep-wake phase an analyze the development pattern of P7-P12 rats with age.(4)Golgi-Cox staining was used to mark and statistically analyze the morphology of cortical pyramidal cells and hippocampal CA1(Field CA1 of the hippocampus)pyramidal cell neurons of P0,P5,P7,P9,P12,P14,P18,and P30 rats,to find the rules of early brain development of infant rats,and finally explain the relationship between sleep and brain development of P7-12 infant rats.Results:(1)The theta(4-8.5 Hz)wave recorded in hippocampal CA1 of adult rats during REM sleep has good synchronization with ECo G during REM sleep.The average accuracy of CA1 LFP determination method is: 94.06% ± 2.80%.The theta wave recorded in CA1 can more accurately represent REM sleep,which can be used to determine the early REM sleep of infant rats that cannot be determined by ECo G.(2)We established a stereotaxic atlas of the infant rat brain at postnatal day 7-13.The atlases contain a series of 530 coronal layers from the olfactory bulb to the end of the brainstem spaced about 0.5 mm apart,and provide stereotaxic coordinates parameters such as the distance between anteroposterior(AP),mediolateral(ML)and dorsal and ventral(DV)of each important nucleus at each day of age.This study provides a reference for electrode implantation and manipulation of nuclear and brain regions,and makes up for the lack of entire stereotaxic brain atlas in infant rats of P7-P13.(3)Theta waves were recorded in the hippocampus during REM sleep of P7 infant rats,which advancing the age at which complete sleep records of infant rats could be recognized to P7,and combining ECo G,EOG,EMG,twitch and activity behavior,we objectively distinguished the Wake,NREM and REM sleep phases of P7-P12 rats,and found the developmental rule: with the increase of age,the ratio of REM sleep decreased from 83.24 ± 1.2% in P7 to 43.12 ± 1.62% in P12,the ratio of NREM sleep increased from 0% in P7 to 23.27 ± 1.54% in P12,and the ratio of Wake increased gradually from 16.76 ± 1.22% in P7 to 33.60 ± 1.73% in P12(P<0.001),although there was no significant difference in daily increase between P8-P12(P>0.05).The high ratio of REM sleep at P7 was due to the fact that the average episode duration of REM sleep was 50.43 s ± 5.24 s,which was significantly higher than that at other days of age,while there was no significant difference in the duration of REM sleep at P8-P12(about 32-35 s).The episodes number of REM sleep per hour decreased from 75.97 ± 4.52 in P8 to 43.94 ± 1.94 in P12(P<0.01).The typical slow wave of NREM sleep could not be recorded in P7-P8 rats,while a gradual increase in NREM sleep with the increase of slow wave activity in P9-P12 rats,the episodes number of NREM sleep per hour decreased from 19.42 ± 2.61 in P9 to 30.88 ± 2.39 in P12(P<0.001),and the average episode duration of NREM sleep increased from10.69 s ± 1.18 s in P9 to 28.62 s ± 3.45 s in P12(P<0.05).There was no significant difference in Wake ratio in P8-P12 because the number of Wake episodes per hour decreased from 75.82 ± 4.54 in P8 to 58.56 ± 3.10 in P12,but there was no significant difference(P>0.05).The average episode duration of Wake increased from 14.32 s ±1.65 s in P8 to 21.32 s ± 1.42 s in P12(P<0.01).Both Wake and NREM sleep became continuous,and small Wake episodes and discontinuous NREM sleep were significantly reduced.In addition,during REM sleep,theta(4-8.5 Hz)waves in hippocampal CA1 in P7 changed from discontinuous to continuous in P10,and slow waves in NREM sleep(0.5-4 Hz)began to be recorded in P9,and become continuous in P11.(4)The total length of dendrites in P0-P30 cortical pyramidal neurons(126.94μm ± 5.77 μm vs.1885.34 μm ± 51.30 μm,P<0.001)and the number of branches(3.08 ± 0.22 vs.32.03 ± 1.19,P<0.001)increased with age,but there was no significant difference in the total number,and the number of branches of apical dendrites and basal dendrites of P12-P18 rats(P>0.05).The total length of dendrites of CA1 pyramidal neurons in P0-P30 was 214.29 μm ± 10.19 μm vs.2667.58 μm ±95.90 μm,P<0.001),and the total number of dendrites was 6.16 ± 0.34 vs.40.94 ±1.08,respectively.P<0.001)increased with age,but there were no significant differences in the basal dendrite length,total number of branches,basal dendrite and apical dendrite of P9-P14 rats(P>0.05).Conclusion: The clear theta waves during REM sleep could be recorded in CA1 of P7 rats.Combined with ECo G,EOG,EMG,twitch and activity behaviors,the ratio of REM sleep from P7 to P12 gradually decreased with the increase of age,while the ratio of NREM sleep and wake increased.The theta wave recorded in CA1 during REM sleep changed from discontinuous in P7 to continuous in P10,and the slow wave in NREM sleep appeared in P9 and began to be continuous in P11 and P12.The transition of theta wave from discontinuous to continuous in hippocampal CA1 may be related to the onset of P7 CA1 pyramidal neuron development and the gradual maturation of CA1 pyramidal neuron at P9.The occurrence of continuous slow waves may be related to the gradual development and maturation of P12 cortical pyramidal neurons.