Effect Of Muscarinic Cholinergic Receptor Activity On Synaptic Transmission Of GABAergic Interneurons In The Anterior Cingulate Cortex

Background:Major depressive disorder(MDD)is a recurring,debilitating illness characterized by depressed mood,diminished self-esteem,decreased motivation,and inability to experience pleasure.Moreover,MDD is one of the leading causes of morbidity and mortality worldwide leading to a significant socioeconomic impact on health care systems.used Antidepressants treat always by regulating the monoamine neurotransmitter systems,but these drugs have many disadvantages,including slow onset,low efficacy,and tolerability,thus,the development of new antidepressants that can overcome these limitations has been a priority.Previous studies have found that the anterior cingulate cortex(ACC)function of depressed patients is weakened,and gamma-aminobutyric acid(GABA)interfering neurons containing somatostatin(SST)In the ACC region,the expression of anti-depressant treatment can increase the concentration of GABA in the brain and increase the activity of GABA receptors.In the study of antidepressants,GABAergic interneurons containing Parvalbumin(PV)also be found.may mediates the antidepressant effect.These findings suggested that the activity of GABAergic interneurons in the ACC region may be an important target for the study of antidepressant mechanisms.Clinical and animal studies have found that scopolamine,a non-selective muscarinic cholinergic receptor(mAChR)antagonist,has a rapid and significant antidepressant effect,but its mechanism needs to be further clarified.Further revealing its specific mechanism of action can provide an important theoretical basis for the development of antidepressants.Therefore,we want to know whether the mAChR activity affects the synaptic transmission function in GABAergic interneurons in the ACC region.In this study,polymerase chain reaction(PCR),immunohistochemistry and patch-clamp whole-cell recording techniques were used to answer the questions and explore the activation and antagonism of mAChR activity in the middle of GAC energy in the ACC region.The effects of neuronal synaptic transmission provide theoretical support for revealing the specific mechanism of action of rapid antidepressants.Method:1.SST-Cre and PV-Cre transgenic mice were mated with Ai9-RFP(Red Fluorescent Protein)transgenic mice to obtain SST-Cre: Ai9-RFP and PV-Cre: Ai9-RFP mouse.The specificity of RFP expression by SST interneurons and PV interneurons in two types of mouse ACC brain regions was determined by immunohistochemistry.2.Using the Whole-cell Patch-calmp recording technique,the RFP-expressing neurons were recorded in the ACC brain regions of SST-Cre:Ai9-RFP and PV-Cre:Ai9-RFP mice.Electrophysiological properties,including Resting Membrane Potential(RMP),Input Resistance,Action Potential(AP)Threshold,Half-width,and Amplitude(Amplitude),after-polarization(AHP),initial and steady frequency(Steady),and Spike frequency adaptation,and observed the morphological characteristics of neurons by biotin staining.3.Spontaneous excitatory postsynaptic currents(sEPSCs),miniature excitatory post synaptic current(mEPSCs),and miniature inhibitory postsynaptic current(mIPSCs)were recorded in RFP expressing SST and PV interneuron to study the effect of mAChR activity on them.Result:1.Immunofluorescent results showed that of RFP-expressing neurons in the ACC region of SST-Cre:Ai9-RFP transgenic mice,81% were also immunoreactive to SST antibody,and of RFP-expressing neurons in the ACC region of PV-Cre:Ai9-RFP transgenic mice,83% were also immunoreactive to PV antibody.These results indicated that RFP-expressing neurons in SST-Cre:Ai9-RFP and PV-Cre:Ai9-RFP mice are largely SST and PV interneurons,respectively.2.SST-Cre: Electrophysiological characteristics of neurons expressing RFP in Ai9-RFP mice(n=35): resting membrane potential-69.54±0.7 mV,input impedance 319.0±22.69 M?,AP threshold-35.66±0.97 mV,The median width is 0.38±0.02 ms,the amplitude is 83.36±1.96 pA,the post-hyperpolarization potential is 20.22±0.84 mV,the initial frequency is 111.4 ±7.02 Hz,the stable frequency is 40.44 ±3.33 Hz,and the discharge frequency adaptability is 3.43±0.48.PV-Cre: Neurophysiological characteristics of neurons expressing RFP in Ai9-RFP mice(n=35): resting membrane potential-73.14±0.52 mV;input impedance 180.7 ±9.68 M?;AP occurrence threshold-27.06 ±0.81 mV;The median width is 0.26±0.01 ms,the amplitude is 73.31±1.35pA;the potential after hyperpolarization is 25.62±0.54mV;the initial frequency is111.9 ±9.60Hz;the stable frequency is 80.91 ±5.74Hz;the discharge frequency adaptability is 1.35±0.06.The neurons expressing RFP in SST-Cre:Ai9-RFP mice are mostly fusiform and have bipolar or tripolar morphology;PV-Cre:Ai9-RFP mice express RFP-like neurons mostly in a basket shape with multipolar form.3.After perfusion of Muscarine to activate mAChR,the frequency of sEPSCs in SST interneurons increased significantly(p<0.01)and the amplitude changes were not significant.This effect could be blocked by Scopolamine(p<0.01);perfusion of Scopolamine was not significant Affects the frequency and amplitude of sEPSCs and mEPSCs;Muscarine can significantly reduce the frequency of mSTSCs in SST interneurons(p<0.05)without significantly affecting the amplitude;this effect can be blocked by Scopolamine(p<0.05);When Scopolamine was perfused alone,the frequency of mIPSCs was significantly increased(p<0.05)without affecting its amplitude.4.The frequency of sEPSCs in PV interneurons was increased significantly after perfusion of muscarine(p<0.05)but the amplitude was unchanged.Scopolamine could block the effect induced by muscarine.Perfusion of Scopolamine alone did not significantly affect the frequency and amplitude of sEPSCs and mEPSCs;Muscarine had no significant effect on the frequency and amplitude of mIPSCs,but perfusion of Scopolamine increased the frequency of mIPSCs significantly(p<0.05).Conclusion:1.Combined with immunohistochemistry and neurophysiological and morphological characteristics of neurons,we basically determined that the neurons expressing red fluorescent protein in the ACC brain region we recorded were SST interneurons and PV interneurons.2.Activation of mAChR can enhance the excitatory synaptic transmission function of SST interneurons and PV interneurons in ACC region,while antagonism of mAChR has no significant effect on excitatory synaptic transmission.3.Activation of mAChR attenuates the inhibitory synaptic transmission of SST interneurons in the ACC region,but does not affect the inhibitory synaptic transmission of PV interneurons;antagonism of mAChR enhances the inhibitory synaptic transmission of SST interneurons,but not Inhibition of synaptic transmission of PV interneurons.4.Our results indicated that activity of mAChR differently affect the synaptic transmission in SST and PV interneurons of the ACC region.Further study is needed to understand the possible role of these different effects in the mechanisms underlying the rapid antidepressant effect of scopolamine.