| Entorhinal cortex(EC)is the gateway for connections between hippocampus and neocortex.Anatomically,the EC includes medial entorhinal cortex(MEC)and lateral entorhinal cortex(LEC).The former can be divided into superficial layers of MEC(sMEC)and deep layers of MEC.The s MEC plays a key role in the encoding of spatial information.On one hand,the sMEC contains typical spatially tuned “grid cells” and “border cells”.On the other hand,the spatial information is persistently transmitted from the s MEC to hippocampus by synchronized theta and gamma oscillation,as well as the oscillation-related spiking.During spatial exploration in the two dimensional environment,the spiking of excitatory neurons in the sMEC is concentrated in a specific theta phase,which is called the “theta phase-locking” of neuronal spiking.Similarly,the gamma oscillatory power(40–100 Hz)is regularly maximized at specific theta phase,which is the so-called “theta-gamma coupling”.The function of spatial recognition relies on the regulation of sleep and wakefulness.The state of wakefulness is maintained by various arousal-promoting systems in the brain.The histaminergic system is one of the essential arousal-promoting systems.It includes a small group of neurons located in the tuberomammillary nucles(TMN),the wide spread histaminergic fibers and extensive distributed receptors in the brain.Melatonin is an important sleep-promoting factor that secreted by the pineal gland.The levels of melatonin in the brain are fluctuated with the circadian rhythm,with high concentrations in the dark phase and low concentrations in the light phase.Our previous studies in the brain slice showed that histamine specifically modulates the excitability and synaptic transmission of sMEC neurons by postsynaptic histamine receptor 1(H1Rs)and presynaptic histamine receptor 3(H3Rs).However,it remains largely unknown that how histaminergic system regulates the neuronal activity,information encoding in the s MEC and the relationship between histamine-modulated effects and spatial exploration behaviors.Here,we firstly investigated,by multi-channel recordings,neuropharmacological techniques and immunofluorescence methods,the effects of histamine and melatonin on the electrophysiology of sMEC principal neurons,and then explored the role of histamine in sMEC information encoding and spatial exploration.The main results are listed as follows:1.Histamine upregulates the neuronal excitability in the sMEC in vivoDuring freely moving test,administration of histamine at different concentrations(30μM,300μM,3m M)in the s MEC increased the firing rate of putative principal neurons(Paired t test,all P < 0.001,n30 μM = 190,n300 μM = 194,n3 mM = 153).The effect of histamine was dose-dependent(Chi-square test,P < 0.001).In order to clarify the receptor mechanisms for histamine-induced excitatory effects,we injected the antagonists for histamine receptors in the s MEC.We found that H1 R antagonist triprolidine(10 μM)decreased the firing rate of putative principal neurons(Paired t test,P < 0.001,n = 192)while infusion of H2 R antagonist ranitidine(100 μM)or H3 R antagonist clobenpropit(10 μM)did not influence the firing rate(Paired t test,all comparisons P > 0.05,nranitidine = 189,nclobenpropit = 196).Similarly,the histamine-elicited excitatory effects on putative principal neurons were diminished in the presence of triprolidine(10 μM,paired t test,P < 0.001,n = 65),but ranitidine(100 μM)or clobenpropit(10 μM)were failed to block the histamine-induced increase of firing rate in the s MEC neurons(Paired t test,all comparisons P > 0.05,nranitidine = 59,nclobenpropit = 54).Moreover,melatonin in the sMEC significantly decreased the firing rate of putative principal neurons(Paired t test,P < 0.01,n = 42).The antagonits for melatonin receptor 3 prazosin(0.5 mM)upregulated the firing of principal neurons while antagonists for receptor 1 and 2 luzindole(0.5 mM)has no effects on the excitability of these neurons(Paired t test,luzindole: P > 0.05,n = 46,prazosin: P < 0.001,n = 51).2.H1 Rs and H3 Rs mediate the histamine-induced elevation of theta and high gamma power in the s MECTheta and gamma oscillations are typical representations for information encoding in the EC-hippocampal circuits.Analyzing the LFP in different frequency bands(delta: 0.5–4 Hz;theta: 4–12 Hz;beta: 12–25 Hz;low gamma: 25–48 Hz;high gamma: 60–120 Hz)in the sMEC,we found that histamine at 30 μM increased the high gamma power but decreased the delta power(Paired t test,Pdelta < 0.01,Ptheta > 0.05,Plow gamma > 0.05,Phigh gamma < 0.001,n = 11).Furthermore,histamine at 300 μM or 3mM elicited the elevation of theta and high gamma power,with reduction of delta power in the sMEC(Paired t-test,300 μM: Pdelta < 0.001,Ptheta < 0.01,Plow gamma > 0.05,Phigh gamma < 0.001,n = 11;3 mM: Pdelta < 0.001,Ptheta < 0.001,Phigh gamma < 0.001,n = 11).It is believed that LFP oscillation is the result the membrane oscillation and activity of synaptic potentials.To further illustrate the histamine receptor involved in regulation of histamine-mediated LFP oscillations,we administrated the antagonists for histamine receptors in the s MEC.Interestingly,both H1 R antagonist triprolidine(10 μM)and H3 R antagonist clobenpropit(10 μM)significantly reduced the theta and high gamma power,accompanied by increase of delta power(Paired t-test,triprolidine,Pdelta < 0.001,Ptheta < 0.001,Pbeta < 0.01,Plow gamma < 0.05,Phigh gamma < 0.001,n = 11;Clobenpropit,Pdelta < 0.001,Ptheta < 0.001,Phigh gamma < 0.001,n = 11).H2 R antagonist ranitidine(100 μM)has no effects on the sMEC LFP oscillatory activity(Paired t-test,all comparisons P > 0.05,n = 11).3.Histamine does not affect the encoding of grid cells and border cellsAfter clarifying the effects of histaminergic system on neuronal activity in the sMEC,we wonder whether histamine modulates encoding of the two typical spatial tuning neurons,which are grid cell and border cell,in the s MEC.For the recorded grid cells,we calculated the gridness score,average rate,peak rate,spatial information rate,sparsity and mean field size.Administration of histamine(300 μM,3 mM)did not influence the ongoing encoding of grid cells(Paired t-test,all comparisons P > 0.05,n300 μM = 13,n3 mM = 17).Also,injection of H1 R antagonist triprolidine(10 μM),H2 R antagonist ranitidine(100 μM)or H3 R antagonist clobenptopit(10 μM)has no effects on the encoding pattern of grid cells(Paired t-test,all comparisons P > 0.05,ntriprolidine = 14,nranitidine = 16,nclobenpropit = 15).In terms of the border cell encoding,we did not observe significant change of borderness score after injection of histamine(300 μM)or H1 R antagonist triprolidine(10 μM,paired t-test,P > 0.05,nhistamine = 14,ntriprolidine = 13).4.Histamine enhances the theta phase-locking of neuronal spiking and theta-high gamma coupling in the sMECSince histamine has no effects on encoding of grid cells and border cells,we explored whether it is involved in theta-modulated spiking of putative principal neurons and gamma oscillations during spatial exploration.Comparing with the vehicle group,histamine(3 mM)significantly increased the strength of theta phase-locking of putative principal neurons in the sMEC(Paired t-test,P < 0.001,n = 112),with no effects on the preferred phase(Paired t-test,P > 0.05,n = 112).Infusion of H1 R antagonist triprolidine(10 μM),the locking strength was reduced(Paired t-test,P < 0.001,n = 119),but the preferred theta locking phase remained constant(Paired t-test,P > 0.05,n = 119),indicating that H1 Rs played a role in histamine-mediated enhancement of theta phase-locking of sMEC excitatory neurons.The antagonists for H2 Rs or H3 Rs did not change the theta phase-locking of putative principal neurons(Paired t-test,all comparisons P > 0.05,nranitidine = 136,nclobenpropit = 142).To further confirm the role of histamine receptors,we observed the effects of histamine in the presence of receptor antagonists.The results showed that H1 R antagonist triprolidine(10 μM)successfully inhibited the histamine-elicited increased of theta phase-locking strength(Paired t-test,P > 0.05,n = 129),while the H2 R antagonist ranitidine(100 μM)or H3 R antagonist clobenpropit(10 μM)was inefficient to block the effects of histamine(Paired t-test,all comparisons P < 0.001,nranitidine+histamine = 101,nclobenpropit+histamine = 99).In the s MEC,the gamma is also modulated by theta oscillations.We speculate that histamine plays a role in regulating the theta-gamma coupling.Interestingly,histamine enhanced the modulation of high gamma amplitude to theta phase(Paired t-test,P < 0.001,n = 11)but did not influence the modulation index for theta and low gamma(Paired t-test,P > 0.05,n = 11).Administration of H1 R antagonist triprolidine(10 μM)or H3 R antagonist clobenpropit(10 μM)reduced the modulation index of theta-high gamma(Paired t-test,Ptriprolidine < 0.001,ntriprolidine = 11;Pclobenpropit < 0.05,ntriprolidine = 11),indicating both H1 Rs and H3 Rs were involved in histamine-modulated theta-high gamma coupling in the s MEC during spatial exploration.H2 R antagonist ranitidine(100 μM)did not change the coupling between theta and high gamma(Paired t-test,P > 0.05,n = 11).5.Histamine in the s MEC regulates spatial recognitionSince histamine modulates the information encoding in the sMEC during spatial exploration,we,in the next step,investigated its role in sMEC-related spatial recognition behaviors.We applied two similar behavioral paradigms(one trial recognition task and object recognition task)to evaluate the performance of rats during spatial recognition.We found that H1 R antagonist triprolidine(10 μM),but not the H2 R antagonist ranitidine(100 μM)or H3 R antagonist clobenpropit(10 μM),impaired the performance of spatial recognition,with a decreased re-exploration score(Object recognition task: two-way repeated-measures of ANOVA,group effect,P < 0.001,n=6;One trial recognition task: one way ANOVA,P < 0.05,n=6).Interestingly,inhibition of hsitaminergic system in the s MEC did not affect the recognition for novel object(One way ANOVA,all comparisons P > 0.05,n=6).To confirm the role of s MEC neurons in histaminergic modulated spatial recognition,we detected the expression of c-fos protein after behavioral training.After the trial of exploration for changed spatial configuration,c-fos expression in the sMEC was signigicantly increased.Pre-administration of H1 R antagonist triprolidine decreased the expression of c-fos,and inhibited the elevation of c-fos expression after exploration for changed spatial configuration.H2 R antagonist ranitidine or H3 R antagonist clobenpropit were failed to inhibit block the increase of c-fos expression after spatial recognition training(One way ANOVA,P < 0.001,n = 9).6.Histamine-modulated spatial recognition is closely linked to theta phase-locking and theta-high gamma couplingAs mentioned above,histamine increases the theta phase-locking of putative principal neurons and theta-high gamma coupling during spatial exploration,and plays a role in regulating the spatial recognition.The relationship between histamine-modulated neural activity and behavior should be further clarified.We analyzed the electrophysiological activities in the s MEC during spatial recognition.Interestingly,the locking strength to theta phase of putative principal neurons in the s MEC was significantly increased during the trial when rats exploring the objects with changed spatial configuration(Paired t-test,all comparisions P < 0.001,nobject exploration task = 62;none trial recognition task = 59).H1 R antagonist triprolidine(10 μM)inhibited the theta locking strength during the baseline trial,as well as the elevation of locking strength during spatial recognition(Paired t-test,two comparisons P > 0.05,nobject exploration task = 85;none trial recognition task = 53).Similarly,theta-modulated local high gamma oscillation in the sMEC was also increased during the trial of exploring the changed spatial configuration(Two-way repeated measures of ANOVA,object exploration task,P < 0.001;one-trial recognition task,P < 0.05,n = 6).H1 R antagonist triprolidine decreased the theta-high gamma coupling and the modulation index did not increase when rats perform the spatial recognition trial(Two-way repeated measures of ANOVA,P > 0.05,n = 6).It is worth noting that H3 R antagonist clobenpropit(10 μM)was failed to block the increase of theta-high gamma coupling during spatial recognition(Two-way repeated measures of ANOVA,P < 0.001,n = 6)though it reduced the modulation index during the baseline.In summary,we found that histamine in the s MEC up-regulates the excitability of putative principal neurons through activation of H1 Rs in vivo.H1 Rs and H3 Rs mediate the histamine-induced elevation of theta and high gamma power in the sMEC.During spatial exploration,histamine does not affect the encoding of grid cells and border cells,but significantly enhances the theta phase-locking strength of neuronal spiking and theta-high gamma coupling.Histamine-mediated enhancement of theta-modulated spiking and high gamma oscillations correlates with spatial exploration. |
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