Effect Of Metabotropic Glutamate Receptors 2/3 Activity On Synaptic Transmission Of GABAergic Interneurons In The Anterior Cingulate Cortex

Background:Major depressive disorder(MDD)has become a severe mental illness worldwide.At present,clinical antidepressants act mainly through regulating the function of monoamine nervous system,but these drugs have many shortcomings such as low efficiency and slow onset,which greatly reduce the patient’s clinical compliance,thereby increasing the risk of suicide.Therefore,the development of antidepressant drugs with rapid onset and low toxic side effects has become a focus of research.Metabotropic glutamate receptors 2/3(mGluR2/3)antagonists have been shown to have rapid antidepressant effects in animal models and have entered clinical trials,but the mechanism is not clear.It was found that the expression of mGluR2/3 in the anterior cingulate cortex(ACC)of depression patients was increased,and the number of the γ-aminobutyric acid(GABA)interneurons containing somatostatin(SST)was reduced.In addition,antidepressant treatment increases the concentration of GABA and the activity of GABA receptors in the brain.Therefore,we hypothesize that mGluR2/3 antagonists exert antidepressant effects by affecting synaptic transmission in GABAergic neurons in the ACC region.In this study,we used polymerase chain reaction(PCR),immunofluorescence,and electrophysiology to study the effect of mGluR2/3 activity on the synaptic transmission in two subtypes of GABAergic interneurons,SST-expressing interneurons and parvalbumin(PV)-expressing interneurons.Our study would provide important information for our understanding of the mechanism of rapid antidepressants.Methods:1.SST-CRE and PV-CRE tool mice were used to breed with Ai9-RFP(Red Fluorescent Protein,RFP)tool mice to obtain SST-CRE/Ai9-RFP and PV-CRE/Ai9-RFP,which fluorescently labeled SST and PV neurons respectively.PCR and immunostaining methods were used to confirm the specific expression of RFP in SST and PV interneurons in the ACC region.2.Whole-cell patch-clamp recordings were performed in the ACC region of the brain slices to study the electrophysiological properties of SST and PV interneurons.The properties included resting membrane potential,input resistance,action potential(AP)threshold,AP half-width and amplitude,afterhyperpolarization,initial and steady spiking frequency,and spiking frequency adaptation.3.Whole-cell patch-clamp recordings were performed to record spontaneous excitatory postsynaptic currents(sEPSCs),miniature excitatory post synaptic currents(mEPSCs),miniature inhibitory postsynaptic current(mIPSCs)mGluR2/3 agonist DCGIV(4 μM)and antagonist LY341495(2 μM)were used to study the effect of mGluR2/3 activity on the synaptic transmission in SST and PV interneurons..Results:1.Immunofluorescent results showed that of RFP-expressing neurons in the ACC region of SST-CRE/Ai9-RFP transgenic mice,80% were immunoreactive to SST antibody,and of RFP-expressing neurons in the ACC region of PV-CRE/Ai9-RFP transgenic mice,81% were 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.Electrophysiological characteristics of SST interneurons(n=28)were: resting membrane potential-69.82±0.70 mV,input resistance 312 ±25.87 MΩ,AP threshold-35.49 ±1.17 mV,AP half-width 0.36±0.02 ms,AP amplitude 83.44±2.31 pA,afterhyperpolarization potential 20.63 ±1.02 mV,spiking initial frequency 111.30 ±8.69 Hz;AP steady frequency 39.88 ±4.30 Hz,AP frequency adaptation 3.66 ±0.61;.Electrophysiological properties of PV neurons(n=27)were: resting membrane potential-73.52 ±0.64 mV;input resistance 171 ±8.01 MΩ;AP threshold-26.15 ±0.91 mV;AP half width 0.25±0.02 ms,AP amplitude 72.52 ±1.55 pA;hyperpolarization potential 25.68 ±0.66 mV;spiking initial frequency 93.42±10.14 Hz;AP steady frequency 73.66 ±6.87 Hz and AP frequency adaptation 1.25±0.07.3.The frequency of sEPSCs in SST interneurons was significantly decreased after activation of mGluR2/3 by perfusing DCG-IV(p<0.05),but the amplitude was not significantly changed.Perfusion of LY341495 blocking the mGluR2/3 decreased the frequency of sEPSCs induced by DCG-IV(p<0.05)without affecting the amplitude;perfusion of mGluR2/3 antagonist LY341495 did not significantly affect the frequency and amplitude of sEPSCs and mEPSCs.DCG-IV did not significantly affect the frequency and amplitude of mIPSCs in SST interneurons.Perfusion of LY341495 alone significantly increased the frequency of mIPSCs(p < 0.05)but not affect the amplitude.4.The frequency of sEPSCs in PV interneurons was dereased significantly after activation of mGluR2/3 by perfusion of DCG-IV(p<0.05),but the change of amplitude was not significant.Perfusion of LY341495 blocking mGluR2/3 activity decreased the sEPSCs frequency induced by DCG-IV(p<0.001)without affecting the amplitude;perfusion of mGluR2/3 antagonist LY341495 did not significantly affect the frequency and amplitude of sEPSCs in PV interneurons,but increased frequency of mEPSCs(p < 0.01)Perfusion of only LY341495 did not affect the frequency and amplitude of mIPSCs in PV interneurons.Conclusion:1.Activation of mGluR2/3 attenuated excitatory synaptic transmission in SST and PV interneurons of the ACC regions.Antagonism of mGluR2/3 had no significant effect on excitatory synaptic transmission in SST interneurons,while enhanced excitatory synaptic transmission in PV interneurons.2.Activation of mGluR2/3 had no effect on inhibitory synaptic transmission in SST and PV interneurons of the ACC regions.Antagonism of mGluR2/3 enhanced inhibitory synaptic transmission in SST interneurons,without significant effect on inhibitory synaptic transmission in PV interneurons.3.Our results indicated that activity of mGluR2/3 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 mechanism of the rapid antidepressant action.