| Associative learning is a high-order brain function,which is vital for defense and survival.Deficits in associative learning have often been implicated in numerous neuropsychiatric disorders,such as aging,Alzheimer's disease and schizophrenia.The hippocampus is a key brain structure involved in associative learning.The crucial role and functional signature of learning-related activity of excitatory hippocampal pyramidal cells(PYRs)in associative learning have been extensively investigated.Although the inhibitory interneurons precisely modulate activity of pyramidal cells in the hippocampal network,their role and underlying mechanisms in such form of learning remain unclear.Notably,the hippocampal interneurons show a greater cellular diversity than pyramidal cells.At least 21distinct types of interneurons have been identified in the hippocampal CA1area,which innervate distinct subcellular compartments of the pyramidal cells and act in discrete time windows by unique firing patterns.Among these,a specific cell type,the parvalbumin positive interneuron(PVIN),has recently attracted more attention.Although the existing studies have implied a possible relationship between hippocampal PVIN and behavioral performance of associative learning,the detailed mechanisms of PVIN's participation in associative learning is required to be revealed.In this study,classical trace eyeblink conditioning(tEBC)was utilized as an experimental model system of associative learning.A combination of multichannel recording,optogentics and immunohistochemistry enabled cell type-specific tagging and modulation of neurons in the dorsal hippocampal CA1(dCA1).We first observed in vivo activity and functional role of dCA1 interneurons of freely-moving mice ongoing tEBC training.We further evaluated the involvement of dorsal hippocampal CA1 parvalbumin positive interneurons(dCA1-PVINs)during the acquisition of tEBC.We finally explored the potential working mechanisms underlying the participation of dCA1-PVINs in associative learning.Results:1.dCA1 interneurons show conditioned stimulus(CS)-evoked sustained activity during tEBC1)Strong CS-evoked firing activity was observed in the dCA1 interneurons,and it did not cease until sustaining into the trace interval period.2)In early stage of learning,the CS-evoked sustained activity of dCA1 interneurons in paired-training mice was greater than in unpaired-training mice(Independent t tests,Averaged Z score:PDay1=0.045,PDay2=0.039;Maximum Z score:PDay1=0.048,PDay2=0.023).In late stage of learning,no significant differences in the CS-evoked sustained activity of dCA1interneurons were observed between the two groups.(Independent t tests,Averaged Z score:PDay3=0.703,PDay4=0.194;Maximum Z score:PDay3=0.075,PDay4=0.194).2.Sustained activity of dCA1 interneurons is coupled with conditioned response(CR)in early stage of learningIn early stage of learning,sustained activity of dCA1interneurons in the CR trials was significantly higher than that in the no-CR trials(Paired t test,Averaged Z score:P<0.001;Maximum Z score:P<0.001;n=53).In late stage of learning,no significant bias was observed in the sustained activity of dCA1interneurons between the CR and no-CR states(Paired t test,Averaged Z score:P=0.083;Maximum Z score:P=0.0097;n=51).3.Optogenetic suppression of dCA1 interneurons impairs the acquisition of tEBC1)Suppressing sustained activity of dCA1 interneurons by 400ms green laser(520nm)after the CS onset resulted in an obvious impairment of CR acquisition(Two-way ANONAs with repeated measures,Main effects:PCR incidence=0.003,PCR amplitude=0.049;Interaction:PCR incidence=0.393,PCR amplitude=0.513).2)Suppressing sustained activity of dCA1 interneurons caused no significant change in the incidence of well-learnedCRs(Paired t test,P CR incidence=0.965;n=6).4.CS-evoked activities of PVINs and non-PVINs are distinct1)The recorded dCA1 interneurons were further identified as either putative PVINs or putative non-PVINs.2)In early stage of learning,the CS-evoked sustained activity of dCA1-PVINs in paired-training mice was greater than in unpaired-training mice(Wilcoxon rank sum tests,Averaged Z score:P=0.0166;Maximum Z score:P=0.0055).In late stage of learning,no significant differences in the CS-evoked sustained activity of CA1-PVINs were observed between the paired and unpaired training mice(Wilcoxon rank sum tests,Averaged Z score:P=0.3666,Maximum Z score:P=0.5478).3)In both early and late stages of learning,no significant differences in the CS-evoked increased activity of non-PVINs were observed(Paired t test,Averaged Z score:P=0.0889;n=43).5.Sustained activity of dCA1-PVINs is coupled with CR occurrence1)In early stage of learning,sustained activity of dCA1-PVINs in the CR trials was significantly higher than that in the no-CR trials(Paired t tests,Averaged Z score:P=0.0037;Maximum Z score:P=0.0167;n=23).In late stage of learning,the significant bias in the sustained activity of dCA1-PVINs between the two states was also observed.(Paired t tests,Averaged Z score:P=0.0135;Maximum Z score:P=0.3464;n=22).2)In both early and late stages of learning,no significant bias was observed in the CS-evoked activity of non-PVINs between the CR and no-CR states(Paired t test,Averaged Z score:PDay1-2=0.0889;n=43;PDay4-5=0.2861;n=23).6.Optogenetic suppression of dCA1-PVINs impairs the acquisition of tEBCSuppressing sustained activity of dCA1 interneurons by 400ms green laser(520 nm)after the CS onset resulted in an obvious impairment of CR acquisition.(Two-way ANONAs with repeated measures,Main effects:PCR incidence=0.001;Interaction:PCR incidence=0.780).7.PYRs and PVINs are functionally connected during learning1)CS-evoked increased activity andCS-evoked decreased activity were observed in the dCA1 pyramidal cells,and these responses to CS persisted across both the CS and trace interval periods.According to these two different responses,we classified pyramidal cells into increased firing and decreased firing subtypes.2)Fast excitation from increased firing PYRs to PVINs in paired-training mice was greater than that in unpaired-training mice(Independent t test,P=0.0350).No significant differences in fast excitation from increased firing PYRs to non-PVINs was observed between two group(Independent t test,P=0.8409).3)Fast excitation from decreased firing PYRs to PVINs in paired-training mice was greater than in unpaired-training mice(Independent t test,P=0.0012).Feedback inhibition of decreased firing PYRs by PVINs was indicated because the firing rate of decreased firing PYRs during 10-50ms after PVINs firing in paired-training mice was lower than in unpaired-training mice(Independent t test,P=9.8837×10-6).8.Optogenetic suppression of fast excitation from PYRs to PVINs impairs the acquisition of tEBC.1)In the second training phase of training day2 and day 5,suppressing sustained activity of dCA1-PVINs by 400ms green laser(520 nm)after the CS onset severely disrupted the fast excitation from PYRs to PVINs(Independent t test,P=1.0266×10-5).2)Suppressing sustained activity of dCA1-PVINs by 400 ms green laser induced significantly decreasedCR occurrence in both early and late stages of learning.(Paired t test,PDay2=0.003;PDay5=0.014;n=6).Conclusion:1.Associative learning requires CS-evoked sustained activity of hippocampal interneurons.2.Among hippocampal interneurons,PVINs play a critical role in associative learning.3.PVINs may correlate distinct functional ensembles of pyramidal cells through CS-evoked sustained activity,so as to precisely sculpt the outputs of the hippocampus and facilitate the associative learning processing.Taken together,the current study elucidated functional signature of firing activity of hippocampal interneurons which is essential for acquisition of associative learning.Furtherly,with deepening insight into the critical role of PVIN in this cognition processing,our results provided mechanistic understanding of the properties of hippocampal interneuron activity,which is required by successful associative learning. |
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