The Effect And Mechanism Of Alcohol On Glutamatergic Neurons In The Parabrachial Nucleus Of Mice

Purpose:As a psychoactive substance,alcohol is widely used in life,and its intake at a certain concentration can affect human emotions,thinking,behavior and consciousness.Studies have shown that alcohol may affect the nerve nuclei that regulate sleep and wakefulness in the brain,and the parabrachial nucleus,as an important sleep and wakeful regulatory nucleus,is likely to be affected by alcohol to play its role in regulating sleep and wakefulness.Therefore,this experiment will explore the effect of alcohol on glutamatergic neurons in the parabrachial nucleus and its mechanism at the level of brain slices through the in vitro patch clamp technique.Methods:⑴Acute brain slice preparation:Using a vibrating microtome,the brain tissue of male C57BL/6J wild-type mice aged 6 to 8 weeks was sliced into a thickness of 280μm containing the lateral parabrachial nucleus(LPB)and parabrachial nucleus Acute brain slices of neurons in two subregions of the medial parabrachial nucleus(MPB).⑵The classification of glutamatergic neurons in the parabrachial nucleus:After clamping the target neuron,inject a negative current stimulation with a current intensity of 500 ms,and then classify the recorded neurons according to their electrophysiological characteristics.(3)In the whole-cell current clamp mode,record the spontaneous discharge and membrane potential changes of glutamatergic neurons in LPB and MPB with different concentrations of alcohol.⑷The effect of different concentrations of alcohol on the inhibitory transmitters afferent of glutamatergic neurons in LPB and MPB:First,D-AP5 and NBQX are added to the perfusion fluid,and the target neurons NMDA type and AMPA type glutamate are affected.In whole-cell voltage clamp mode,record spontaneous inhibitory postsynaptic currents(s IPSCs);add TTX to the above-mentioned perfusate to block Na~+channels,in whole-cell voltage clamp mode,To record miniature inhibitory postsynaptic currents(m ISPCs).⑸The influence of different concentrations of alcohol on the excitatory transmitter afferent of glutamatergic neurons in LPB and MPB:First,add PTX to the perfusate to block the GABAA receptor of the target neuron.In the whole-cell voltage clamp mode,Record spontaneous excitatory postsynaptic currents(spontaneous excitatory postsynaptic currents,s EPSCs);add TTX to the above-mentioned perfusate to block the Na~+channel,and record tiny excitatory postsynaptic currents in the whole-cell voltage clamp mode(miniature excitatory postsynaptic currents,mEPSCs).Results:⑴Typing results of glutamatergic neurons in the parabrachial nucleus:There are two types of glutamatergic neurons in the recorded LPB.The first is the neuron without Ih current,and the second is the neuron with Ih current.And the effects of alcohol on these two types of neurons are not different;Glutamatergic neurons in MPB are also divided into two types with Ih current and without Ih current,and there is no difference in the effects of alcohol on these two types of neurons.⑵36 mM alcohol has no significant effect on the firing frequency of action potentials on glutamatergic neuron in LPB,but it increases the firing frequency of action potentials on glutamatergic neuron in MPB;60 mM alcohol significantly reduced the firing frequency of action potentials on glutamatergic neuron in LPB and MPB;180 mM alcohol completely inhibited the firing frequency of action potentials on glutamatergic neuron in LPB and MPB,and significantly reduced membrane potential of glutamatergic neurons in LPB and MPB.(3)The effect of different concentrations of alcohol on the inhibitory transmitters afferent of glutamatergic neurons in LPB and MPB:36 mM alcohol has no significant effect on s IPSCs and m IPSCs of glutamatergic neurons in LPB,but reduces the firing frequency and amplitude of s IPSCs and m IPSCs of glutamatergic neurons in MPB;60 mM alcohol increased the firing frequency of s IPSCs and the firing frequency and amplitude of m IPSCs of glutamatergic neurons in LPB,and increased the firing frequency and amplitude of s IPSCs and m IPSCs of glutamatergic neurons in MPB;180mM alcohol increased the frequency and amplitude of s IPSCs and m IPSCs of glutamatergic neurons in LPB and MPB.⑷The influence of different concentrations of alcohol on the excitatory transmitter afferent of glutamatergic neurons in LPB and MPB:36 mM of alcohol has no significant effect on s EPSCs and mEPSCs of glutamatergic neurons in LPB,but it increases the firing frequency and amplitude of s EPSCs and mEPSCs of glutamatergic neurons in MPB;60 mM and 180 mM alcohol both reduced the firing frequency and amplitude of s EPSCs and mEPSCs of glutamatergic neurons in LPB and MPB.Conclusion:The excitatory effect of 36 mM alcohol on glutamatergic neurons in MPB may be achieved by inhibiting the inhibitory input and enhancing the excitatory input.By attenuating the release of pre-synaptic GABA transmitter and the opening of post-synaptic GABA_Areceptors,and enhancing the release of pre-synaptic glutamate transmitter and the opening of post-synaptic glutamate receptors;The inhibitory effect of 60 mM and 180 mM alcohol on glutamatergic neurons in LPB and MPB may be achieved by enhancing the inhibitory input of their neurons and inhibiting the excitatory input.By enhancing the release of pre-synaptic GABA transmitter and the opening of post-synaptic GABA_Areceptors,and attenuating the release of pre-synaptic glutamate transmitter and the opening of post-synaptic glutamate receptors.