The Mechanisms Of Hypocretin In Regulating The Medial Entorhinal Cortex And Its Role In Learning And Memory

Medial entorhinal cortex?MEC?is essential for learning and memory.The stellate neurons and pyramidal neurons are two major types of excitatory principal neurons in the superficial layer of MEC.Inhibitory interneurons include parvalbumin positive?PV⁺?,somatostatin positive?SOM⁺?and serotonin receptor type 3A positive(5-HT_3AR⁺)neurons.One of the prominent characteristics of neuronal networks in the superficial layer of MEC is that rare direct connections are formed between principal neurons,of which form numerous bidirectional projections with interneurons.The activities of neurons are manifested as rhythmic oscillations at local filed potential?LFP?levels.Theta?4-12 Hz?and gamma?25-120 Hz?oscillations are important features of spatial information processing and transmission between MEC and hippocampus.High levels of wakefulness are the premise for the completion of learning and memory tasks.The maintaining of wakefulness depends on various arousal-promoting system in the brain.Hypocretin/orexin?Hcrt/orexin?system is one of the key arousal-promoting systems in the brain.Currently,it is still unknown about whether the Hcrt system modulates neural activities in the MEC,as well as MEC-related spatial learning and memory.Here,we investigated,for the first time,the neuronal modulatory role of Hcrt system in the MEC and spatial learning and memory.By applying patch clamp,virus-assisted neural tracing,optogenetics and chemogenetics,and manipulating the Hcrt fibers in the superficial layer of MEC during spatial memory tasks,we systemically explored the effects of arousal-promoting Hcrt system on the MEC neural activities and spatial learning and memory.The main results are summarized as follows:1.Hcrt1 increases the excitability of interneurons in the superficial layer of MEC through activation of HcrtR1?1?Hcrt1 increases the firing rate of interneurons in the superficial layer of MEC by activation of Hcrt/orexin receptor type 1?HcrtR1/OX₁R?.Under the current clamp mode,we found that infusion of Hcrt1?500 nM?elicited significant increase in firing rate of PV⁺,SOM⁺and 5-HT_3AR⁺neurons(PV⁺interneurons:P<0.05,n=8;SOM⁺interneurons:P<0.01,n=9;5-HT_3AR⁺interneurons:P<0.01,n=9).In addition,in the presence of HcrtR1antagonist SB-334867?2?M?,the Hcrt1-induced increase in firing rate was diminished?n=7?.There results indicated that Hcrt1 increased the firing of interneurons through HcrtR1in the superficial layer of MEC.?2?Hcrt1 increases the excitatory input to the interneurons in the superficial layer of MEC.The excitability of neurons is affected by the synaptic input.We found that inhibitors of glutamatergic AMPA receptors?CNQX,10?M?and NMDA receptors?AP-5,50?M?completely blocked the effect of Hcrt1 for increasing the firing rate of interneurons?n=9?.Under voltage clamp,Hcrt1?500 nM?significantly increased the frequency of spontaneous excitatory postsynaptic currents?sEPSCs?of the interneurons?P<0.05,n=8?,but the amplitude of sEPSCs was not affected?n=8?.Thus,Hcrt1 enhances the excitatory input to interneurons by HcrtR1,thereby elevating their excitability.2.Hcrt1 enhances the excitatory-inhibitory neuronal connections in the superficial layer of MEC.?1?Hcrt enhances the excitatory synaptic transmission between local excitatory-inhibitory neurons in the superficial layer of MEC.Under double-patch recording,Hcrt1?500 nM?significantly increased the response reliability of unitary EPSCs?uEPSCs?in the PV⁺interneurons,SOM⁺interneurons and 5-HT_3AR⁺interneurons in response to the firing of principal neurons with direct connections(principal neuron-PV⁺interneuron:P<0.01,n=5;principal neuron-SOM⁺interneuron:P<0.05,n=3;principal neuron-5-HT_3AR⁺interneuron:P<0.001,n=4).These results indicated that Hcrt1 enhances the excitatory synaptic transmission efficiency between principal neurons and interneurons in the superficial layer of MEC.?2?HcrtR1 is expressed in the presynaptic glutamatergic terminals of interneurons in the superficial layer of MEC.Immunohistochemical and immune electron microscopical results showed that HcrtR1 was expressed in the excitatory presynaptic terminals of the GABA interneurons.These results further indicated that Hcrt1 increased the excitatory input to the interneurons through activation of HcrtR1 that located in the axonal terminals of excitatory neurons in the superficial layer of MEC.3.Hcrt1 enhances the inhibitory input to principal neurons,which depends on excitatory synaptic transmission in the superficial layer of MEC.?1?Hcrt1 induces membrane hyperpolarization and an outward current in principal neurons in the superficial layer of MEC.Under voltage clamp,Hcrt1?500 nM?induced significant membrane hyperpolarization in both stellate neurons and pyramidal neurons?stellate neurons:P<0.001,n=23;pyramidal neurons:P<0.01,n=8?.Consistently,Hcrt1?500 nM?elicited an outward current in the principal neurons?P<0.001,n=16?.?2?Hcrt1 enhances the inhibitory GABAergic input to the principal neurons in the superficial layer of MEC.In the presence of GABA_a receptor antagonist picrotoxin?PIC,100?M?and GABA_b receptor antagonist CGP-55845?2?M?,Hcrt1-elicited hyperpolarization was completely blocked?n=8?,indicating a role of GABAergic synaptic transmission in the Hcrt1-induced hyperpolarization.Interestingly,Hcrt1?500 nM?did not affect the frequency?n=7?and amplitude?n=7?of spontaneous IPSCs?sIPSCs?.Then,we clamped the principal neurons at+10 mV to block the glutamatergic receptors on the recorded neuron and simultaneously maintained the intact excitatory glutamatergic transmission in the superficial layer of MEC.Surprisingly,Hcrt1?500 nM?significantly increased both the frequency?P<0.001,n=7?and amplitude?P<0.05,n=7?of sIPSCs on the principal neurons.Thus,Hcrt1 enhances the inhibitory input to principal neurons,which depends on excitatory synaptic transmission in the superficial layer of MEC.4.Endogenous Hcrt in the superficial layer of MEC modulates neuronal activities and enhances local gamma oscillations.?1?Endogenous Hcrt increases the firing of interneurons through activation of HcrtR1in the superficial layer of MEC.We injected the AAV-EF1?-DIO-mCherry in the LH of Hcrt-cre mice for anterograde neuronal tracing,and the hypocretinergic fibers were detected in the superficial layer of MEC.In addition,injection of retrograde transporting AAV_retro-EF1?-EYFP in the superficial layer of MEC resulted in positive expression of viruses in the soma of Hcrt neurons in the LH.Thus,the Hcrt neurons directly project to the superficial layer of MEC.The AAV-EF1?-DIO-ChR2-mCherry was injected into the LH of Hcrt-cre mice to specifically express the light-sensitive channel proteins ChR2 in the hypocretinergic fibers in the superficial layer of MEC.Optogenetic activation?473 nm,20Hz?of hypocretinergic fibers to stimulate the release of endogenous Hcrt significantly increased the firing rate of PV⁺interneurons in the superficial layer of MEC?P<0.05,n=7?.The antagonist of HcrtR1,SB-334967,completely inhibited the optogenetic activation-induced increase in firing rate of interneurons?n=5?.Thus,endogenous Hcrt increases the excitability of interneurons by activation of HcrtR1.?2?Activation of endogenous Hcrt increases the excitatory input to the interneurons in the superficial layer of MEC.Consistent with the results from in vitro patch clamp,light activation of endogenous Hcrt induced significant elevation of frequency of sEPSCs?P<0.05,n=6?,but the amplitude was not affected?n=6?.Therefore,endogenous Hcrt enhances the excitatory input to the interneurons,thereby increasing their firing rates.?3?Activation of endogenous Hcrt in vivo increases the firing of interneurons but inhibits the firing of principal neurons in the superficial layer of MEC.To further clarify the modulatory role of endogenous Hcrt in the superficial layer of MEC in vivo,we used t he multi-channel recording to investigate the effect of endogenous Hcrt on the activity of principal neurons and interneurons.We found that optogenetic activation of hypocretinergic fibers in the superficial layer of MEC elicited a significant increase in firing rate of interneurons?P<0.001,n=18?and a significant decrease in firing rate of principal neurons?P<0.001,n=43?.?4?Activation of endogenous Hcrt in the superficial layer of MEC in vivo enhances the gamma oscillations.In the analysis and comparison of LFP oscillations in different bands,we found that light activation of endogenous Hcrt in the superficial layer of MEC did not affect the local theta oscillations?n=27?,while the power of low-gamma?P<0.001,n=27?and high-gamma oscillations?P<0.001,n=27?were significantly elevated.Thus,the endogenous Hcrt is probably involved in the regulation of information processing in the MEC-related circuits in vivo by modulating the neuronal activities and oscillations.5.Manipulation of LH_Hcrt-MEC pathway affects spatial learning and memory behaviors?1?Endogenous Hcrt in the superficial layer of MEC regulates spatial exploration behaviorChemogenetic inhibition of endogenous Hcrt in the superficial layer of MEC impairs spatial exploration behavior and reduces gamma oscillations.We injected the AAV-EF1?-DIO-hM4D-mCherry in the LH of Hcrt-cre mice and implanted drug cannulae in the superficial layer of MEC.After chemogenetic inhibition of endogenous Hcrt by in vivo local infusion of clozapine N-oxide?CNO?into the superficial layer MEC,the latency of mice for finding the correct place of food in the spatial environment was significantly increased(P<0.001,n_mCherry=8,n_hM4Di=8),and the error counts were significantly higher than that of the control group(P<0.01,n_mCherry=8,n_hM4Di=8),indicating the impairments of spatial learning and memory.In addition,the low-gamma and high-gamma oscillations in the superficial layer of MEC during spatial exploration were significantly decreased compared with those in the control group(Low-gamma:P_trial4,8,20<0.05,P_{trial 16}<0.01,n_mCherry=56,n_hM4D=49;High gamma:P_{trial 12,20,24}<0.05,P_{trial 16}<0.01,n_mCherry=56,n_hM4D=49).It was suggested that Hcrt may play a role in the spatial exploration by regulating the gamma oscillations in the superficial layer of MEC.Opotogenetic activation of endogenous Hcrt in the superficial layer of MEC enhances spatial exploration behavior.AAV-EF1?-DIO-ChR2-mCherry was injected into the LH of Hcrt-cre mice,light activation of endogenous Hcrt in the superficial layer of MEC during spatial exploration task resulted in significant shortened latency of mice for finding the food in the early trials(compared with control group,Block 1:P_mCherry×_ChR2<0.001,n _mCherry=6,n _ChR2=5).Therefore,optogenetic activation of endogenous Hcrt enhances spatial exploration at behavioral levels.?2?Endogenous Hcrt in the superficial layer of MEC regulates spatial working memoryChemogenetic inhibition of endogenous Hcrt in the superficial layer of MEC damages spatial working memory.After local infusion of CNO in the superficial layer of MEC,the correct rate of mice both in normal and interference T-maze were significantly decreased(normal T-maze:group hM4D:P_saline×_CNO<0.05,n_saline=6,n _CNO=6;interference T-maze:group hM4D:P_saline×_CNO<0.05,n_saline=6,n _CNO=6),indicating impairments of spatial working memory.Optogenentic activation of endogenous Hcrt in the superficial layer of MEC in the execution trial of spatial working memory task enhances behavioral performance.We activated the endogenous Hcrt using optogenetics in the superficial layer of MEC in the sample trial,delay period and execution trial,respectively,and investigated the changes in behaviors of mice.The results showed that in the normal T-maze task,the correct rate of mice cannot be further improved by optogenetic activation regardless in the sample trial,delay period or execution trial(n _ChR2=6,n_mCherry=7).However,for the interference T-maze task,the correct rate of mice was significantly increased when optogenetic activation of endogenous Hcrt in the execution trial(P_{light off×execution}<0.05,n_ChR2=7,n_mCherry=4).Optogenetic activation in the sample trial or the delay period had no effect on the behavioral performance of mice(n _ChR2=7,n_mCherry=4).These results indicated that endogenous Hcrt in the superficial layer of MEC also played an essential role in anti-interference during spatial learning and memory.Conclusions:the above results showed that LH Hcrt neurons directly project to the MEC.Hcrt1 enhances the excitatory-inhibitory neuronal synaptic transmission and forms feedback inhibition through activation of HcrtR1 on the axonal terminals of excitatory neurons,thereby playing a role in modulating the excitatory-inhibitory neuronal circuits.Through regulating these neuronal circuits,Hcrt enhances the local gamma oscillations in the superficial layer of MEC,and LH_Hcrt-MEC pathway thus plays an important role in spatial learning and memory behaviors.Our results provide new solid evidence for deepening our understanding of the relationship between Hcrt system and neural networks in the superficial layer of MEC,as well as the working model of arousal-promoting system for regulating the functions of brain regions related to learning and memory.