| The singing of songbirds is similar to the language learning of human.The male songbirds can sing complexly and regularly,while females only tweet simply.The structure and function of song control nuclei has obvious gender differences.RA is the premotor nucleus of songbirds,which directly controls the output of song.RA not only receives synaptic afferents fromHVC,the high vocal center in the vocal motor pathway,but also receives synaptic afferents fromLMAN in anterior cerebral pathway that controls vocal learning.HVC-RA and LMAN-RA afferent pathways are related to the vocal learning of songbirds.The former is responsible for transmitting the auditory signals and motor instructions fromHVC to RA,and the latter is responsible for transmitting signals fromthe forebrain through memory comparison feedback to RA.Then,RA integrates the signal to the vocal muscle control nucleus in turns,and produce sound at last.The central cholinergic systemis an important neurotransmitter systemin brain,which is involved in neural processes including sensorimotor regulation through acetylcholine binging its M-type and N-type receptors.In our previous studies,the excitability and intrinsic membrane properties of RA projection neurons were directly related to cholinergic signal,suggesting that the cholinergic systemcan regulate singing behavior,but the mechanismis unknown.It is necessary to elucidate the cholinergic regulation mechanisms of HVC-RA and LMAN-RA,two main afferent pathways of song premotor nucleus RA,which is of great significance in revealing the neural mechanismof song behavior.In this experiment,we further explore the receptor mechanisms of cholinergic modulation on excitatory synaptic transmission in RA projection neurons of adult zebra finches(a typical songbird for the study of song behavior and its neuromodulation mechanism),and the different regulation mechanisms of cholinergic signaling on the excitatory synaptic transmission of HVC-RA and LMAN-RA pathways in amphiprotic zebra finches by cholinergic signaling.The results show that:1.The frequency and amplitude of the mini-excitatory postsynaptic currents(mEPSCs)of the RA projection neurons in adult amphiprotic zebra finches were significantly decreased by non-selective acetylcholine receptor agonist carbachol(CAR),indicating that CAR inhibited the excitatory synaptic transmission received by RA projection neurons.The M-acetylcholine receptors(mAChRs)antagonist atropine(ATR)blocked the action of CAR,and the mAChRs agonist oxotremorine M(OM)could mimic the effect of CAR.The N-acetylcholine receptors(n ACh Rs)antagonist mecamylamine(MEC)could not block the effect of CAR,and the n ACh Rs agonist DMPP had no effect,indicating that the cholinergic signal through mAChRs reduced the excitatory synaptic transmission in RA projection neurons,but not n ACh Rs.2.As stimulating the HVC-RA projection fibers electrically,the amplitude of evoked excitatory post-synaptic current(e EPSC)was decreased by CAR in adult male zebra finches,indicating that CAR inhibited excitatory synaptic transmission of HVCRA pathway.ATR blocked the effect of CAR,and OM could mimic the effect of CAR;while MEC could not block the effect of CAR,and DMPP has no effect,which indicated that the cholinergic signaling on excitatory synaptic transmission of the HVCRA pathway was mediated by mAChRs,but not n ACh Rs.In addition,fluctuation of the paired-pulse ratio(PPR)was caused by adding CAR and OM alone,suggesting that the presynaptic mechanisminvolved in the regulation of cholinergic signaling on excitatory synaptic transmission in the HVC-RA pathway.On the other hand,CAR had no effect on the amplitude of e EPSC in RA projection neurons of adult female zebra finches,and OM and DMPP had no effect respectively,suggesting that excitatory synaptic transmission of the HVC-RA pathway in female birds is not regulated by cholinergic signaling.3.As stimulating the LMAN-RA projection fibers electrically,the amplitude of e EPSC in RA projection neurons in adult amphiprotic zebra finches was significantly decreased by CAR,indicating that CAR inhibited excitatory synaptic transmission of LMAN-RA pathway.In male birds,MEC blocked the effect of CAR,and DMPP could mimic the effect of CAR.Moreover,ATR could not block the effect of CAR,and OM had no effect,indicating that the effect of cholinergic signaling on excitatory synaptic transmission of the LMAN-RA pathway in adult male songbirds was mediated by n ACh Rs.Futhermore,PPR had changed after adding CAR and DMPP respectively,suggesting that the presynaptic mechanismis participate in the regulation of cholinergic signaling on excitatory synaptic transmission in LMAN-RA pathway in male songbirds.Unlike in male birds,the n ACh Rs antagonist MEC in females could not block the effect of CAR on excitatory synaptic transmission in LMAN-RA pathway in female birds,and DMPP could not mimic the effect of CAR.ATR can block the effect of CAR,and OM can also mimic the effect of CAR,indicating that the receptor mechanismof cholinergic signaling on the excitatory synaptic transmission of the LMAN-RA pathway in adult female songbirds is controlled by mAChRs instead of n ACh Rs.In addition,CAR and OM can cause changes in PPR,which suggested the presynaptic mechanismtake part in the regulation of cholinergic signaling on excitatory synaptic transmission of the LMAN-RA pathway in adult female songbirds.To sumup,cholinergic signaling is involved in the regulation of excitatory synaptic afferents of RA projection neurons in songbirds,and there are receptor mechanisms and gender differences between the regulation of two main afferent pathways,included HVC-RA and LMAN-RA excitatory synaptic transmission of RA projection neurons.Therefore,the diverse regulation of cholinergic signaling in two RA afferent pathways of songbirds may be an important mechanismto the generation of song learning behavior.This study provides important data for revealing the mechanismof central cholinergic system,which can regulate songbird song behavior. |
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