Septo-hippocampal Cholinergic Projection Regulate Neural Activity Of Hippocampus

Acetylcholine plays a critical role for brain cognitive function such as learning and memory as a neuromodulator or neurotransmitter in central nervous system(CNS).Hippocampus,which is located under cerebral cortex and plays important roles in consolidation of information from short-term memory to long-term memory,and in spatial memory that enables navigation,receives cholinergic inputs mainly from medial septum.We combined multi-channel in-vivo recording technology with optogenetics,local brain micro-injection and electrochemistry detection technology,using ChATChR2(choline acetyltransferase-channelrhodopsin2)transgenic mice,to study how medial septal cholinergic neurons influence on hippocampal CA1 rhythmic neural activities.First of all,we combined multi-channel in-vivo recording technology and optogenetics to determine spontaneous firing pattern of medial septal cholinergic neurons.We recorded 10 neurons which fired following optical stimulations in medial septum altogether in 20 ChAT-ChR2 mice,and we make sure that these 10 neurons are cholinergic cells.We found that 7 of the 10 cholinergic cells showed a theta-dependent firing pattern,which was never reported.During the theta rhythm dominated AE state and REM state,the mean firing rate is 7.0 ± 1.3 Hz and 3.0 ± 0.9 Hz,significantly higher than 0.5 ± 0.8 Hz of sharp-wave ripples(SWRs)dominated SWS state.Moreover,the higher frequency theta oscillation became,the faster the 7 cholinergic cells fired.Further study indicates that although there exists strong coherence between medial septal cholinergic cells and hippocampal theta oscillation,no significant reciprocal Granger causality was presented between the two factors.Above these results,we concluded that medial septal cholinergic cells fired more actively during theta oscillation dominated behavioral states but they didn't participate in the generation of hippocampal theta oscillation.Besides,the other three cholinergic neurons fired much faster during rapid-eye movement(REM)than active exploration(AE)and slow-wave sleep(SWS)as previous study.And then,to study if septal cholinergic cells' firing activities directly influence the hippocampal acetylcholine concentration,we modified the electrode with acetylcholinesterase and Choline Oxidase and planted it into hippocampal CA1.Results indicated that the ACh concentration during AE were much higher than SWS.And then,10 Hz activation of septo-hippocampus cholinergic projection by optical fiber during SWS induce strongly increased ACh concentration in hippocampus and made hippocampal acetylcholine concentration keeping stable level in ChAT-ChR2 transgenic mice.Above these results,we concluded that septo-hippocampal cholinergic projection could directly regulate hippocampal acetylcholine content.After that,to explore the influence from septo-hippocampal cholinergic projection on neural network activities,we gave 10 Hz optical stimulation to MS during AE,REM and SWS respectively in 22 ChAT-ChR2 mice.According to the results,we found that activation of septo-hippocampal cholinergic projection could completely suppress SWRs of SWS but not alter frequency or power of theta and gamma oscillation of AE and REM.During SWS,activation of septal cholinergic cells could suppress ripple generation,the inhibition index was 88±63 %.Above these results,we understood that activation of septal cholinergic cells during SWS induced high acetylcholine content of hippocampus,which could completely suppress ripple generation so that hinder memory consolidation.To explore which acetylcholine receptor subtypes mediate the suppression,first we injected ChAT-ChR2 mice with atropine(muscarinic acetylcholine receptors antagonist)and mecamylamine(nicotinic acetylcholine receptors antagonist)intraperitoneally,then we found it was muscarinic receptors which mediated the suppression of ripple generation of hippocampus.Further experiments found that it was mAChRs subtype 2 and 4 but not other AChRs subtypes which mediated the ripple suppression by distinctive antagonist intraventriclly injection.Then we found that activation of septo-hippocampal cholinergic projection not only suppressed ripple generation,but also changed firing activities of hippocampal CA1 neurons: most hippocampal CA1 pyramidal cells were suppressed during optical activation of septo-hippocampal cholinergic projection;for interneurons,which were divided into two major groups: ripple-associated neurons and theta-associated neurons.42 of 46 SWRs-associated neurons were inhibited by activation of septo-hippocampal cholinergic projection,and this inhibition was eliminated by M4 antagonist;13 of 24 theta phase locked interneurons were inhibited,the activities of other 11 theta phase locked interneurons were activated.Above all results,we combined optogenetics and multi-channel in-vivo recording technology and successfully recorded real-time medial septal cholinergic neurons in freely behavioral ChAT-ChR2 transgenic mice.We revealed that medial septal cholinergic neurons' firing pattern was theta-dominated-behavioral-denpendent,showing high activation during AE and REM state while low activation during SWS state.Furthermore,we found that high acetylcholine content in hippocampus during SWS state completely suppressed hippocampal ripple oscillation generation,and M2/M4 acetylcholine receptors mediated the suppression effect.Moreover,septohippocampal cholinergic projection could regulate hippocampal CA1 pyramidal cells and interneurons.A series research of our experiments is a powerful demonstration that septo-hippocampal cholinergic projection influences learning and memory by regulating hippocampal neural activities.