Medial Prefrontal Cortex Dominates The Pure Tone Frequency Discriminative Learning

In some fear behavioral experiments,the freezing response of animals induced by auditory fear conditioning is independent of the pure tone frequency of the conditioned auditory stimulus(CS).But in discriminative fear conditioning experiments,the frequency of the CS is considered specific,and the freezing response induced by the CS is higher than that by the unrelated frequency(UF)pure tones.After auditory fear conditioning,how to elicit the pure tone frequency discrimination and which nuclei and pathways are involved in it remain unclear.Firstly,we trained mice with CS,and tested them with different frequencies of pure tones(CS or UF)separately.The results showed that there was no correlation between the freezing response and the frequency of the CS in each group,which was inconsistent with the results of discriminative fear conditioning experiments.We speculated that the different results may be attributed to the different conditions of sound stimulation in the testing,so we used the same training procedure,while multiple frequencies,including the CS and UFs,to perform the tests in the following behavioral experiments.The results showed that the mice could distinguish between the CS and UFs in different freezing responses in the latter stage of the testing.We also observed that the fear extinction of the CS was inhibited,which may contribute to the discrimination.Therefore,we set up a extinction group on the basis of two frequencies pure tones tests on the second day.Extinction group was divided into two groups,exposed to CS or UF,respectively,to perform fear extinction.Extinction of conditioned fear was evident in both groups,and the degree of fear maintenance(DFM)in the discrimination group tested with CS(D_C)was significantly higher,suggesting that there may be an emergent discriminative learning.The mPFC is involved in the regulation of conditioned fear learning,however,the frequency response properties of mPFC neurons to pure tones and the connection between their responses and any hypothetical emergent discriminative learning,remain unclear.We performed electrophysiological recording in mice,anesthetized them with sodium pentobarbital during the recording,and found that mPFC neurons only in awake mice responded to noise,and only some neurons responded to pure tones at high intensities.We performed loose-patch recordings in vivo in untrained and trained mice,and found that the response of mPFC neurons to the CS in trained mice was relatively greater,which was consistency with behavioral response.To explore whether mPFC was involved in the above discriminative learning,we silenced mPFC with muscimol before the two frequencies pure tones tests.The results showed that the discriminative learning in mice was blocked.When we inactivated the ipsilateral primary auditory cortex(Al)with lidocaine in electrophysiological recordings,the pure tone response of mPFC neurons was inhibited.When silencing both sides of A1 with muscimol,the discriminative learning in mice was also blocked.Based on the above experimental results,we draw the following conclusions:(1)Delay fear conditioning elicited fear generalization in mice.(2)Multifrequency testing after auditory fear conditioning could gradually reveal an emergent discriminative learning.(3)Some mPFC neurons in awake mice responded to pure tones at high intensities.(4)The response of mPFC neurons to the CS was enhanced relatively after auditory fear conditioning,which may be related to the discriminative learning.(5)Pure tone information ascending to mPFC neurons may mainly come from Al.These findings provide new insights for further studies of fear discriminative learning in the mPFC.