Acetylcholine Regulates Theta Rhythm In Hippocampus

Theta rhythms(4-12Hz)represent the “on-line” state of the hippocampus,also assumed to be carriers of mnemonic processes.Studies have showed that lesion or inactivation of medial septal(MS)neurons abolishes theta waves in the hippocampus,thus MS has been regarded as the ultimate rhythm generator of theta.Furthermore,MS is mainly composed of interneurons containing inhibitory transmitter(GABA),excitatory glutamatergic and cholinergic neurons,and the number of cholinergic neurons in the hippocampus is low,thus hippocampal acetylcholine mainly came from septo-hippocampal cholinergic projections.Do the cholinergic neurons of MS participate in the regulation of theta rhythms or not? How to regulate? We combined transgenic technology,multi-channel in vivo recording technique,and optogenetics to manipulate the cholinergic neurons of MS to observe their effects on theta rhythms in the hippocampal CA1 region,then clarify the relationship and mechanism between MS cholinergic system and theta rhythms in hippocampal CA1 area.Optical fiber was embedded in MS of the choline acetyltransferase-channelrhodopsin2(ChAT-ChR2)mice and multi-channel in vivo recording was performed in the hippocampal CA1 region.In mice active exploration and rapid eye movement sleep,20 Hz blue light activates the cholinergic neurons of MS,and theta rhythms in the hippocampal CA1 area are not changed,as shown in the theta score no difference compared with the light before.In slow wave sleep,theta rhythms in the hippocampal CA1 area disappear,and typical ripple rhythms(100-250 Hz)appear.At this time,20 Hz blue light stimulus MS,and fails to induce theta rhythms in the CA1 area of the hippocampus,instead,it causes the inhibition of ripple rhythms.The ivermectin-gated silencing receptor(IVMR)mice was crossed with choline acetyltransferase-cre mice(ChAT-Cre)to construct a ChAT-IVMR(choline acetyltransferaseivermectin gated silencing receptor)transgenic mice that the release of acetylcholine by cholinergic neurons specifically will be inhibited after the injection of IVM.This result shows that the microinjection of IVM into MS effective inhibits the release of acetylcholine into the hippocampal during active exploration,which significantly reduce the amplitude of the theta waves in the hippocampal CA1 brain region,and the decreased percentage of amplitude of theta-waves is linearly related to the dose of IVM.Specifically,when 0.05 nmol of IVM was injected into the MS,3 hours later,theta score decreased from 0.0135±0.0119 to 0.0077±0.0114(P<0.01,T-TEST).After 24 hours,the amplitude of theta rhythms could completely recover to normal level.However,there is no influence on the frequency.To further investigate the effect of decreased acetylcholine levels on neurons in hippocampal CA1 region,according to the characteristics of neuron firing rate and the correlation between neuron and theta rhythm,we divide the hippocampal interneurons into two categories.Type I interneurons active have very high coherence with theta rhythm,while type II interneurons active has no coherence with theta rhythm.After microinjection of IVM into the brain area of MS in ChAT-IVMR mice,the coherence between type I interneurons and theta rhythm decreased,while the coherence between type II interneurons and theta rhythm increases.As for hippocampal pyramidal neurons,when the level of acetylcholine in the hippocampus falls,partial pyramidal neurons change their locked phase with theta rhythm.These findings suggest although the damage of MS caused the hippocampus theta rhythms disappear,the effect of MS cholinergic neurons on theta rhythms is limited.The decrease of acetylcholine in the hippocampus does not affect the frequency of the theta rhythms,but partially affects the amplitude of the theta rhythms.On the other hand,activating the MS acetylcholine neurons and increasing acetylcholine in the hippocampus has no effect on the theta rhythms already existed in the hippocampus,neither induce theta rhythms when theta rhythm absence in the hippocampus.In addition,changes in acetylcholine levels also affect active of some interneurons and pyramidal cells in the hippocampal CA1 region.These findings are important to understand the regulation and mechanism of acetylcholine in the hippocampal theta rhythm.