| The incidence of epilepsy has been gradually increasing over the past decade andaround50million people worldwide have been suffering from this disease. It has becomethe second most common nervous system disorder, after the cerebrovascular disease. Theincidence of epilepsy is on average0.7%worldwide. Nearly30%of patients cannotcontrol the disease by conventional drugs or through other treatments. Roughly half ofepilepsy-related deaths occur among those younger than the age of55. The disease notonly decreases the quality of life, but also causes a large number of psychological andeconomic burdens to family and society. Moreover, the above situation is exacerbated inChina and other developing countries. Many years of clinical and laboratory research hasfocused on pathogenesis and treatment of epilepsy, however the cellular and molecularmechanisms of the incidence of epilepsy remain unclear Traditional antiepileptic drug therapy often brings resistance and toxicity issues. Therefore, it is necessary tostudymechanisms of epilepsy in order to develop safer and more effective treatments.Bombesin is a biologically active polypeptide containing14amino acid residues. Manystudies found it exists widely in mammals as a brain-gut peptide and it playes an importantrole in nervous system tumors, ischemia-reperfusion injury, and learning and memorythrough BB and G protein-coupled receptors. Recent studies show a significant change inbombesin expression in the pathogenesis of degenerative neurological illness associatedwith Parkinson and Alzheimer’s diseases. Meanwhile, the entorhinal cortex (EC) is the agateway to the hippocampus plays a significant role in consolidation and recall ofmemories as well as pathological processes including Alzheimer’s disease, schizophreniaand temporal lobe epilepsy. This study explores the mechanism by which bombesininfluences inhibitory neuronal excitability and synaptic transmission in the EC. The resultthat bombesin reduces the excitability of principle glutamatergic neurons indicates thatbombesin-induced enhancement of GABA release contributes to its antiepileptic effects.Finally, it supports the proposition that the endogenous peptide bombsin can suppressepileptic activity. This study provides important suppport for bombesin-releated researchon pathogenesis of epilepsy as well as clinical antiepileptic pharmaceutical development.Experiment1: Bombesin Increases the Frequency and Amplitude of sIPSCsRecorded From the Principal Neurons in the ECObjective: To investigate the effects of bombesin on GABAA receptor-mediated sIPSCsrecorded from the principal neurons in the EC.Methods:(1) We used15to20days male SD rat brain slices and whole-cellvoltage-clamp to recording the frequency and amplitude changes of the spontaneousinhibitory postsynaptic currents (sIPSCs) of stellate and pyramidal neurons in the layer IIof entorhinal cortex after given the bombesin.(2) Dose-response relationship wasobserved at different concentrations (0.01/0.05/0.075/0.1/0.25/0.5μM) bombesinaccording the stellate neuron’s sIPSCs changes.(3) We observed stellate neuron’s sIPSCschanges after given two bombesin receptor antagonists (PD176252,10μM/D-Phe6, Leu-NHEt13, des-Met14,6μM).(4) We observed the stellate neuron’s sIPSCs changesafter given bombesin-like peptide NMB and GRP (0.25μM).Results:(1) We examined the effects of bombesin on GABAAreceptor-mediated sIPSCsrecorded from the principal neurons in each layer of the EC. Stellate and pyramidalneurons are the principal neurons in layer II whereas pyramidal neurons are the majorneuronal type in layer III and layer V. In layer II stellate neurons, application of bombesin(0.25μM) significantly increased the frequency (control:9.0±1.6Hz, bombesin:16.7±1.7Hz,204±26%of control, n=10, P=0.01) and amplitude (control:28.9±1.7pA, bombesin:37.3±3.0pA,128±6%of control, n=10, P=0.006) of sIPSCs.(2) The EC50value ofbombesin was measured to be42nM. Similarly, application of bombesin (0.25μM)significantly increased the frequency and amplitude of sIPSCs recorded from thepyramidal neurons in layer II (Freq:196±15%of control, n=10, P=0.002; Amp:126±6%of control, n=10, P=0.005), layer III (Freq:190±14%of control, n=10, P=0.002; Amp:134±6%of control, n=10, P=0.003), and layer V (Freq:200±13%of control, n=10,P=0.005; Amp:132±10%of control, n=10, P=0.03). Whereas these results indicate thatbombesin facilitates sIPSC in all the principal neurons in the EC, we used layer II stellateneurons as an example to determine the underlying cellular and molecular mechanisms forthe rest of the experiments. We applied bombesin at the concentration of0.25μM for therest of the experiments for better comparison.(3) We next examined whether the effects ofbombesin on sIPSCs were mediated by activation of bombesin receptors. Application ofPD176252(10μM), a BB1and BB2receptor antagonist significantly reducedbombesin-induced increases in sIPSC frequency (136±12%of control, n=10, P=0.034vs.control without PD176252) and blocked the facilitatory effect of bombesin on theamplitude (122±13%of control, n=10, P=0.19vs. baseline). Application of the selectiveBB2antagonist,(D-Phe6, Leu-NHEt13, des-Met14)-bombesin (6-14) failed to significantlyalter the effects of bombesin (Frequency:180±21%of control, n=10, P=0.43vs. controlwithout the inhibitor; Amplitude:129±8%of control, n=10, P=0.98vs. control without theinhibitor).(4) Application of NMB (0.25μM), a selective BB1receptor agonist,significantly increased sIPSC frequency (188±26%of control, n=10, P=0.027) and amplitude (152±16%of control, n=10, P=0.036). Similarly, application of GRP (0.25μM),a selective BB2receptor agonist, also significantly increased sIPSC frequency (190±24%of control, n=10, P=0.02) and amplitude (129±4%of control, n=10, P=0.004). Theseresults together suggest that activation of either BB1or BB2receptors is capable ofincreasing sIPSC frequency and amplitude and the effects of bombesin might be mediatedby both BB1and BB2receptors.Experiment2: Bombesin Exerts no Effects on mIPSCs But Slightly Reduces theAmplitude of eIPSCsObjective: To investigate the synaptic mechanisms of bombesin promotes neuronal GABArelease.Methods:(1) We used15to20days male SD rat brain slices and whole-cellvoltage-clamp to recording the frequency and amplitude changes of the small inhibitorypostsynaptic currents (mIPSCs) of stellate neurons in the layer II of entorhinal cortex afterblocked the action potential and given the bombesin.(2) The stellate neuron’s sIPSCschanges were observed after administration of bombesin when removed extracellular Ca2+or used voltage-gated Ca2+channel blocker Cd2+/Ni2+(100μM).(3) The evoked IPSCs(eIPSCs) changes were observed after administration of bombesin by placing a stimulationelectrode in a location of200μm from the recorded neurons.(4) The changes of PPR wereobserved after given bombesin and synaptic responses were evoked by a paired pulse(50-ms interval) at0.1Hz by low-intensity stimulation (80-100μsec duration;10-40μAintensity) via a constant-current isolation unit.Results:(1) To determine whether bombesin-induced increases in sIPSCs might be relatedto alterations of the firing of inhibitory interneurons, we examined the effects of bombesinon mIPSCs recorded in the presence of TTX (0.5μM). Application of bombesin failed toalter mIPSC frequency (109±5%of control, n=10, P=0.12) and amplitude (96±2%ofcontrol, n=10, P=0.14). Together, these results suggest that bombesin facilitatespresynaptic GABA release with no effects on postsynaptic GABAAreceptors and theeffects of bombesin are AP-dependent.(2) We further tested the role of Ca2+in bombesin-induced increases in GABA release. In the extracellular solution containing0Ca2+and1mM EGTA, application of bombesin failed to increase either the frequency(117±14%of control, n=10, P=0.27vs. baseline) or the amplitude (95±3%of control,n=10, P=0.12) of sIPSCs suggesting that Ca2+influx is required for bombesin-inducedincreases in GABA release. We then examined the role of voltage-gated Ca2+channels inbombesin-mediated increases in GABA release. Inclusion of Cd2+(100μM) and Ni2+(100μM) in the extracellular solution to block voltage-gated Ca2+channels significantlyreduced sIPSC frequency (33±5%of control, n=10, P<0.001) and amplitude (71±2%ofcontrol, n=10, P<0.001) suggesting that voltage-gated Ca2+channels contributesignificantly to the generation of sIPSCs. Application of bombesin in the presence of Cd2+and Ni2+failed to further change sIPSC frequency (133±34%of control, n=10, P=0.56)and amplitude (96±2%of control, n=10, P=0.07). These results suggest thatbombesin-induced increases in GABA release require an influx of Ca2+via voltage-gatedCa2+channels.(3) Bath application of bombesin significantly reduced the amplitude ofeIPSCs evoked by the first stimulation (85±5%of control, n=5, P=0.04).(4)Bombesin-induced depression of eIPSC amplitude was presynaptic in origin becausebombesin significantly increased the PPR (n=5, P=0.012).Experiment3: Ionic Mechanisms Underlying Bombesin-Induced Depolarization ofGABAergic Interneurons in the ECObjective: To investigate the ionic mechanisms of bombesin depolarizes GABAergicinterneurons in the EC.Methods:(1)15to20days male SD rat brain slices and whole-cell current-clamp wereused to identify entorhinal cortex layer III interneuron types. The changes of restingmembrane potential (RMPs) and input resistance (Input Resistance,-50pA,500ms) wereobserved after administration of bombesin.(2) The changes of interneuron’s actionpotentials were observed using current clamp after given bombesin;(3) The holdingcurrent changes were observed at-60mV from interneurons using voltage clamp afteradministration of bombesin.(4) We used of Na+/Ca2+blockers and the changes of RMPs were observed after given bombesin from the interneuron.(5) Perusing (μM)100Cd2+/0.5TTX/100Ni2+/10DNQX/50APV/10Bicuculline and recording Ramp voltage-currentcurve (-120~-60mV,0.1mV/ms) after given bombesin.(6) The changes of RMPs andsIPSCs frequency were recorded when using inward rectifier potassium channel (inwardrectifier K+channels, Kirs) blocker Ba2+(0.5mM) and bombesin from the entorhinalcortex layer II stellate neuron.Results:(1) After having electro-physiologically identified the interneurons, we recordedthe RMPs by washing in TTX (0.5μM) in the extracellular solution. The data recordedfrom Type I and II interneurons were pooled. Application of bombesin depolarized theinterneurons recorded at the RMPs (Control:-63.7±0.8mV, bombesin:-61.6±0.9mV, n=8,P<0.001) and significantly increased the input resistance of the interneurons (Control:315±36M, bombesin:362±46M, n=8, P=0.026).(2) We also probed the roles ofbombesin in AP firing by including in the extracellular solution10μM DNQX,50μMDL-APV,10μM bicuculline and2μM CGP55845without TTX. Application of bombesinsignificantly increased the firing frequency of APs (416±54%of control, n=6, P<0.001),but slightly decreased the amplitude of AP (92±1%of control, n=6, P<0.001).Bombesin-induced depression of AP amplitude might be due to its depolarizing effectresulting in inactivation of Na+channels.(3) Application of bombesin induced a smallinward HC recorded at-60mV from interneurons (-8.7±1.4pA, n=6, P=0.001).(4) Weinitially replaced the extracellular NaCl with the same concentration of NMDG-Cl. In thiscondition, application of bombesin still induced a comparable depolarization (2.5±0.3mV,n=7, P<0.001). Furthermore, replacement of extracellular Ca2+with Mg2+and inclusion ofEGTA (1mM) in the extracellular solution failed to change significantlybombesin-induced depolarization (3.2±1.6mV, n=7, P=0.7vs. bombesin in controlcondition). These results suggest that bombesin-induced depolarization of interneurons isindependent of extracellular Na+and Ca2+.(5) We used a ramp protocol (from-120mV to-60mV) to construct the voltage-current curve before and during the application ofbombesin. In this condition, bombesin induced a current, which had a reversal potential(-89.8±4.2mV, n=6) close to the calculated K+reversal potential (-85.4mV).(6) The bombesin-induced current showed an inward rectification suggesting that the involved K+channels are inward rectifier K+channels (Kirs). We further confirmed the involvement ofKirs by applying the Kir blocker, Ba2+. In the presence of Ba2+(0.5mM), applicationbombesin failed to induce significant depolarization (0.5±0.4mV, n=7, P=0.25). Moreover,bath application of Ba2+alone significantly increased sIPSC frequency (228±45%ofcontrol, n=8, P=0.026) and blocked bombesin-induced enhancement of sIPSC frequency(n=8, P=0.33). These data together demonstrate that bombesin increases GABA releasevia inhibition of Kirs.Experiment4: Bombesin and Bombesin-Like Peptides Inhibit Mg2+-Free-InducedEpileptiform Activity in EntorhinalObjective: To test the inhibitory effect of the activation of bombesin receptors in theentorhinal.Methods: Extracellular field recordings with a glass electrode containing2M NaCl wereconducted in layer III of the EC from the horizontal brain slices.15to20days male SD ratbrain slices were incubated in nominally Mg2+-free extracellular solution bubbled with95%O2and5%CO2for1-3h. The slice was then placed in the recording chamber andperfused with the Mg2+-free extracellular solution to record stable basal epileptiformactivity for15min. We applied bombesin, NMB or GRP at0.25μM in the Mg2+-freeextracellular solution for15min and continued to record the epileptiform activity in theabsence of bombesin or the bombesin-like peptides for another30min.Results: Omission of extracellular Mg2+induced epileptiform activity. After the inductionof epileptiform activity, bath application of bombesin significantly and reversibly reducedthe frequency of the epileptiform activity (control:4.38±1.12events/min, bombesin:1.07±0.43events/min, n=6, P=0.034). Similarly, the frequency of Mg2+-free-inducedepileptiform activity was significantly reduced by application of NMB (0.25μM, control:10.30±1.29events/min, NMB:2.04±0.88events/min, n=5, P=0.014) or GRP (0.25μM,control:5.93±1.67events/min, GRP:0.93±0.35events/min, n=5, P=0.027). These resultsdemonstrate that activation of BB1and BB2exerts antiepileptic activity in the EC. Conclusions：1. Bombesin increases presynaptic GABA release without affecting postsynapticGABAAreceptors.2. Bombesin inhibits presynaptic Kirs to depolarize GABAergic interneuronsresulting in increases in AP firing frequency and subsequent activation ofvoltage-gated Ca2+channels to increase GABA release.3. The facilitatory effects of bombesin on GABA release may contribute to itsanti-epileptic effect in the EC. |
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