The Electrophysiological Properties Of Dopaminergic Neurons In Brain Slice Model Of Epilepsy And Levetiracetam Condition

Backgroud:Psychosis of epilepsy(POE)is a term applied to a group of psychosis disorders in epilepsy patients,such as impatient,depression,irritability,hallucination and so on.But it is differernt from Schizophrenia.The prevalence of POE in epilepsy patients is higher than that in the general population,therefore it has raised more attention in recent years.POE is related to the hypersensitivity of DA neurons in midbrain according to the previous studies.Ventral tegmental area(VTA)is one of the brain regions where DA neuons congretated.It palys a central role in reward and aversion,drugs of abuse and addiction as well as some psychosis disorders.Several animal models of epilepsy demonstrate that the spontaneous firing rate of DA neurons is increased in epileptic condition when recorded in vivo.It was regulated by a Ventral subicular-nucleus accumbens-ventral pallidal-VTA pathway,but the detailed mechanism remaines unclear.POE can be induced by many factors apart from epilepsy.Antiepileptic drugs(AEDs)can lead to psychosis disorders as well.Levitiracetam(LEV)is a novel anti-epileptic drug in the treatment of patial and generalized seizure.It is known to specifically bind to synaptic vesicle protein 2A(SV2A).Unfortunately,LEV can induce psychotropic side effects in some patients.It may be linked to the dopaminergic activity.However,it is rarely reported about the effects of LEV on the electrophysiological properties of DA neurons.Objective:Utilizing brain slice model of epilepsy,we investigated the spontaneous firing rate,the intrinsic excitability and synaptic drive of DA neurons in epileptic and LEV condition by patch-clamp recordings.We sought to explore the electrophysiological changes of DA neurons,which may provide a new target for the therapy of POE.Methods:1.Preparation of neocortex-VTA brain slices was obtained from TH-GFP mice.We utilized magnesium-free artificial cerebrospinal fluid(ACSF)to induce epileptiform discharges for the prepatation of epileptic model(EP-model).2.We examined the spontaneous firing and the evoked firing pattern of neurons in VTA.Besidies,we distinguished VTA neurons by Avidin and immunohistochemical staining.We classified the DA neurons firing patterns according to the ramp.3.We examined the relationship between spontaneous firing rates and the intrinsic excitability of DA neruons.We compared the spontaneous firing rates,membrane resistance,membrane capacitance,half-width of action potentials,the afterhyperpolarization,rheobase,d V/dt(maximum),d V/dt(minimum)and the frequency-injected currents(F-I)curves in control and EP-model conditions.4.We examined the relationship between spontaneous firing rates and the extrinsic synaptic drive of DA neruons.We recorded the spontaneous excitatory postsynaptic currents(s EPSC)and spontaneous inhibitory postsynaptic currents(s IPSC)using internal solution containing Cs+ and QX-314 in normal and EP-model conditions.The inter-event interval(IEI),frequency and ampitude were analyzed.Furthermore,we investigated the evoked excitatory postsynaptic currents(e EPSC)and evoked inhibitory postsynaptic currents(e IPSC)by a concentric tungsten electrode and a stimulator.We analyzed the paired-pulse-ratio(PPR)to examine changes in presynaptic release probability.5.We investigated the contribution of extrinsic excitabilties to the spontaneous firing rates.We compared the spontaneous firing rates in normal,EP-model and the EP-model conditions in the presence of glutamate receptor blockers CNQX(AMPA receptor antagonist)and AP5(NMDA receptor antagonist).6.We examined the effects of LEV on the extrinsic synaptic drive to DA neurons.We investigated the frequency and amplitude of s EPSC and s IPSC in normal and LEV-treated conditions(with different concerntration).To record AMPA receptor mediated miniature excitatory postsynaptic currents(m EPSC),tetrodotoxin(TTX,the blocker of voltage-dependent sodium channel),PTX(GABAA receptor antagnoist)and AP5 were added to the bath solution.The same as to the minature inhibitory postsynaptic currents(m IPSC)except that the PTX was replaced by CNQX.The IEI,ampitude and frequency were then analyzed.7.We examined the effects of LEV on the intrinsic excitability of DA neurons.Similar to the method 3,we compared the related paremeters in normal and LEV-treated conditions.8.We probed the effects of LEV on the spontaneous firing rates of DA neurons.We analyzed the spontaneous firing rates of DA neurons in normal and LEV conditions using cell-attached patch clamp recordings.Results:1.According to the firing patterns and immunohistochemical stainings,the neurons in VTA can be mainly classified into two kinds,DAergic and GABAergic neurons.DA neurons are characterized by pacemaker firing pattern,with relatively broader width and slower firing rates than GABA neruons.The evoked firing patterns can be classified into three types,type I can accout for 37.5%,type II 47.9% and type III 14.6%.2.Cell-attached patch clamp recordings revealed an increase in the rate of spontaneous firing in DA neurons in EP-model,accompanied by a decrease in the half-width of action potentials.Similar results were obtained with whole-cell patch clamp recordings.3.The intrinsic excitability of DA neruons was significantly increased in EP-model.The F-I curve showed a skew toward higher evoked firing rates,significant increase was observed with current injection of 0-280 p A.The half-width of action potentials became narrower,and the d V/dt(minimum)and area were decreased.Whereas,the membrane resistance and capacitance did not show much statistical changes.4.The cumulative probability plot of s EPSC IEI shifted to the left accompanied by an increase in the mean frequency,while the ampitude was almost identical to control condition.In contrast,the frequency and ampitude of s IPSC did not change too much.No significant difference in e EPSC and e IPSC PPR was detected. antagonists CNQX and AP5 were added to the bath solution in EP-model,lying between the normal and EP-model conditions.5.The spontaneous firing rates could be reduced when glutamate receptors antagonists CNQX and AP5 were added to the bath solution in EP-model,lying between the normal and EP-model conditions.6.The cumulative probability plot of s EPSC IEI shifted to the left in 200 ?M LEV and 400 ?M LEV condition and showed statistically significant in 400 ?M LEV. But the mean frequency was not significantly affected by LEV.The parameters of s IPSC,m EPSC and m IPSC were not clearly changed.7.The intrinsic excitability of DA neruons was significantly reduced in 200 ?M LEV condition.The F-I curve showed a skew toward lower evoked firing rates and significant differences were detected with current injection of 40-100 p A and 140-160 p A.The membrane capacitance became significantly larger while the membrane resistance became smaller.8.The spontaneous firing rates of DA neruons were not significantly affected by 200 ?M or 400 ?M LEV.Conclusion:1.The brain slice model of epilepsy can be prepared by magnesium free ACSF after being incubated for 30 minutes.2.The DA neruons in VTA can be identified by their firing pattern and width of action potentials in TH-GFP mice,which is above 1 ms generally.The evoked firing patterns of DA neurons can be classified into three types according to the initial ramp in response to step current injection.3.The spontaneous firing rates of DA neurons increase in EP-model.It may result from the increased intrinsic excitability and extrinsic excitatory synaptic drive.The half-width of action potentials in DA neruons is shortened in EP-model.The frequency of s EPSC increases,while the paremeters of e EPSC and e IPSC did not show significant change.4.LEV exerts no significant effect on spontaneous firing.However,LEV inhibits DA neuron excitability by reducing the number of action potentials evoked by step current injections.