Effects Of Acetylcholine On Adult Neurogenesis And Spatial Learning: With Analyses Of Underlying Mechanisms

Hippocampus is regarded to be the major subcortical cetral involvement of learning and memory.The dentate gyrus(DG)is one of the subunits of the hippocampus and the granule neurons in the granular cells layer(GCL)can extend dendrite to the molecular layer where they receive the innovations of medial perforant pathway(MPP)from the basal forebrain and also project axons terminals forming the mossy-fiber pathway and connect with pyramidal neurons in the CA3.The subgranular zone(SGZ)of the DG was one of the two regions in the central nervous system(CNS)where adult neurogenesis happens.Every day nearly 9,000 new neurons can be produced and grow to the glutamatergic neurons.Indeed,most of them will die and the survivals will mature and integrate into the existing circuits of physiological functions.The newborn immature neurons will migrate from subgranular zone to the molecular layer and extend the dendrite to form synaptic connection with other dendrite or axons.They also receive the innovations from the septum nuclei and involve the synaptic plasticity and learning and memory.The dentate gyrus receives innervations from both excitable glutamatergic and GABAergic fibers;it also receives innervations from cholinergic neurons.Acetylcholine(ACh)is a very important neurotransmitter which was regarded to be involved in learning and memory performance dysfunction of the AChergic synaptse in the major cause of the Alzheimer's disease (AD).Although studies demonestrated that both ACh and adult neurogenesis involved in the learning and memory,it is still unresolved about whether and how ACh affects adult neurogenesis and what the exact mechanism is underlying this process.The purposes of the present study,therefore,were as follows:(1)by using high frequency stimulation to study the effects and mechanisms of the newborn immature neurons and mature neurons in the DG on the long-term potentiation(LTP)in the adult rat;(2)by using Morris water maze and immunohistochemistry to observe the effects of ACh on adult neurogenesis and spatial memory;(3)by using field electrophysiological recording to investigate effects and mechanism of the ACh on LTP in the DG. PartⅠ:Effects of the Granule Neurons on the Long-term Potentiation in the Adult RatThe present study was aimed to examine the effects and the mechanisms of the newborn immature neurons and mature neurons in the DG on the long-term potentiation(LTP)in adult rats,electrophysiological recording method was used to record the field excitatory postsynaptic potential(fEPSP)in the medial perforant pathway(MPP)and LTP was induced by the pair-pulsed high-frequency stimulation(HFS),in order to investigate and understand the synaptic plasticity,learning and memory.Bicuculline(10μM)was used to block the inhibition of GABA on the mature granule neurons.The main results were as follows:1.There were two types of LTP that can be induced by HFS in the MPP of the DG. ACSF-LTP was contributed by newborn immature neurons and BIC-LTP was from mature neurons.The baseline synaptic transmission was induced by lower frequency stimulation (0.05Hz),LTP was induced by HFS and lasted at least for 1 hour.At the end of the 1h(during the 50-60mins after tetanic stimulation),the ACSF-LTP was 107.85±1.34%(N=7).We also added 10μM bicuculline into the perfusate 10 mins to block the GABAergic inhibition of slice and gave HFS,then washed out the bicuculline immediately,larger LTP(called BIC-LTP)was induced and lasted at least 1 hour.At the end of the 1h(during the 50-60mins after tetanic stimulation), the BIC-LTP was 164.67±2.40%(N=8).2.NR2B antagonist Ro25-6981 fully blocked the induction of the ACSF-LTP,but NR2A antagonist NVP-AMM077 did not affect it in the MPP of the DG.Ro25-6981(3μM)and NVP-AMM077(0.2μM)were added in the perfusate 10mins,after tetanic stimulation they were washed out immediately.The LTP at the end of the lh(during the 50-60mins after tetanic stimulation)was 100.75±2.75%(N=5)and 106.53±.36%(N=5)respectively.The results showed that Ro25-6981 completely blocked the ACSF-LTP(P＜0.05)but NVP-AMM077 remained unaffect it(P＞0.05).3.Ro25-6981(3μM)and NVP-AMM077(0.2μM)were used together with bicuculline (10μM)and the LTP was recorded for at least 1h.The LTP at the end of 1h(during the 50-60mins after tetanic stimulation)were 147.56±6.63%(N=6)and 109.04±1.93%(N=6) respectively compared with BIC-LTP(164.67±2.405,N=8).There was significant difference between Ro25-6981&BIC group and BIC group(p＜0.05),so did it between NVP&BIC group and BIC group(p＜0.001).This indicated that BIC-LTP induced from mature neurons was mainly depended on the NR2A receptor.4.We also compared LTP of the NVP&BIC group(109.04±1.93%,N=6)with ACSF-LTP (107.85±1.34%,N=7)and found that there was no significant difference between the two groups (T=0.519,P=0.614).These results indicated that in the DG of the hippocampus,new born immature granule neurons without being inhibited,participate the physiological function and facilitate the synaptic plasticity.They can produce ACSF-LTP which depended on the activation of NR2B subunit. The mature granule neurons can be induced to form LTP under the GABAergic antagonists which mainly depended on NR2A receptor.PartⅡ:Aeetyleholine Regulates Adult Hippoeampal Neurogenesis And Learning in the RatThe hippocampus also receives innervations from cholinergic neurons from septum nuclei. In present study,acetylcholine chloride(ACh)was injected stereotaxically into the lateral ventricles and effects were observed on adult neurogenesis and learning and spatial memory. Sham group and control group were set to compare with the ACh treated group.In sham group ACh was replaced by 0.9%HC1,and in control group nothing was given except anaesthetized. The main results were as follows:1.In the Morris water maze ACh treated group need less time to complete the experimental trial than control and sham groups in the place navigation task(PNT).After being trained several days the time on escaping to the platform in treated group were 8.074±1.26s(3^rdday),6.75±1.16s (4^thday)6.06±0.85s(5^thday)compared with sham group(12.16±1.54s in 3^rdday),12.13±1.10s in 4^thday),13.15±2.73s in 5^thday)and control group(14.52±1.45s in 3^rdday),11.38±1.24s in 4^th day),11.77±2.00s in 5^thday),there were statistical significant difference in ACh treated group compared with sham group and the control group respectively(P＜0.05).There was no statistical significantly difference between sham group and the control group(P＞0.05).2.In the Morris water maze,swimming speeds of the rats were observed and there was no significant difference within the three groups.That showed ACh did not affect the animal's swimming velocity.3.In the spatial probe tesk(SPT),the crossing times of the ACh treated group was higher (7.4±0.51,N=5)than the sham group(4.2±0.37,N=5)and the control group(3.6±0.51,N=5), there were significantly difference between treated group and sham group(P＜0.05)or between treated group and control group(P＜0.05).There was no statistical significantly difference between sham group and the control group(P＞0.05).4.In the visible platform test(VPT),the time for three groups(ACh treated group,sham group,control group)to find the visional platform were 8.97±1.17s,8.25±1.06s,7.30±1.07s respectively.There were no significant difference with each other(P＜0.05).5.In the immunohistochemistry experiments,the numbers of the BrdU+ cells of the ACh treated group was higher(637.00±39.50 per hippocampi,N=3,n=36)than the sham group (339.00±17.62 per hippocampi,N=3,n=36)and control group(336.00±49.05,per hippocampi, N=3,n=36),there were significantly difference between treated group and sham group(P＜0.05) or between treated group and control group(P＜0.05).There was no statistical significantly difference between sham group and the control group(P＞0.05).The numbers of the BrdU+/CaBP+ cells of the ACh treated group was higher (491.00±47.29 per hippocampi,N=3,n=36)than the sham group(305.00±17.62 per hippocampi, N=3,n=36)and control group(304.00±30 per hippocampi,N=3,n=36).There were significantly difference between treated group and sham group(P＜0.05)or between treated group and control group(P＜0.05).There was no statistical significantly difference between sham group and the control group(P＞0.05).These results indicated that ACh can promote the adult neurogenesis in the DG and suggested that the cholinergic functions in the DG may partly contribute to learning and spatial memory through increasing the newborn hippocampal neurons. PartⅢ:The Mechanism Underlying the Acetylcholine Potentiation of LTP in the Dentate GyrusThe partⅡexperiment had shown that acetylcholine(ACh)can affect the learning and spatial memory in the Morris water maze through improving the baseline of the adult neurogenesis in the DG.But the exact relationship between the ACh and adult neurogenesis is still not clearly.Long-term potentiation(LTP)is a widely used experimental model of frequency dependent synaptic plasticity in the central nervous system,and has been considered to be a candidate mechanism for the cellular processes underlying learning and memory.So electrophysiological recording and cholinergic receptor agonist carbachol(CCh)were used to investigate the effects of cholinergic receptor activation on LTP in the in vitro DG to understand the mechanisms of the ACh on LTP.The results showed as follows:1.CCh had a dose dependent of depressant effects on fEPSP in the slice of the DG.Three different concentrations of CCh(0.1μM,1μM and 10μM)were given on the hippocampal slice and depressant effects were observed after given carbachol 3 to 4 minutes,and at the 7-10 minutes after given carbachol,the slope of the fEPSPs were separately depressed 8.60±0.73% (0.1μM,N=3),18.17±0.58%(1μM,N=3),34.31±1.04%(10μM,N=3)compared with baseline (100%).10 minutes later CCh were washed out and the slope of the fEPSP began to recovery. Nearly after 25-30 minutes later after washout,the slope of the fEPSP were very close to the baseline,but later they depressed a little.2.CCh had a depression effects on ACSF-LTP.CCh(1μM)were given 10mins before tetanus,the slope of the fEPSP during the 50-60 minutes after tetanic stimulation was 99.83±2.14%(N=5)compared with ACSF-LTP(107.85±1.34,N=7).There was a statistic significant difference between the two groups(p＜0.05).3.CCh enhanced the BIC-LTP in the DG.GABAergic antagonist bicuculline(10μM)was used to block the GABAergic receptor.We found that during the 50-60 minutes the MPP-LTP in being given CCh(1μM)and bicuculline(10μM)group was 182.59±5.85%(N=8)compared with only bicuculline(10μM)164.67±2.40%(N=8).There was a statistic significantly difference between the two groups(p＜0.05).Muscarinic cholinergic antagonist—atropine(10μM)was given to block the muscarinic cholinergic receptor and the facilitation effects of CCh on MPP-LTP was observed to be blocked (151.66±5.76%,N=4,p＜0.05),that means the effects of CCh on MPP-LTP was dependence on the muscarinic cholinergic receptors.In short,our present study indicated that the effects of cholinergic receptor agonist CCh facilitating LTP was depended on the mature granule neurons but not on the new immature neurons in the DG,the facilitating effects could be blocked by muscarinic receptor antagonists.In conclusion,by using field potential recording,Morris water maze and immunohisto -chemistry technique,we have investigated the effects and the mechanisms of adult neurogenesis and ACh on the synaptic plasticity and spatial memory.The results have demonstrated that: 1.there was two types of LTP induced by immature neurons and matre neurons respectively in the DG.2.The mechanisms of the two types LTP were different.ACSF-LTP formed by immature neurons was NR2R dependend,LTP formed by mature neurons was NR2A dependend. 3.ACh can promote the adult neurogenesis in the DG and suggested that the cholinergic functions in the DG may partly contribute to learning and spatial memory through increasing the newborn hippocampal neurons.4.cholinergic receptor agonist CCh facilitated LTP formed by mature neurons through muscarinic cholinergic receptor,however,it inhibited the LTP formed by immature neurons.