| 1.Imbalance of EPSC and IPSC at threshold level in auditory cortexNeurons in layers ?-? of the auditory cortex assemble auditory information from thalamocortical inputs[1][2][3].As with other excitatory neural circuitry,thalamocortical excitation is coupled with inhibition,both of which are essential for cortical function involving neural computation and plasticity[4][5][6].Studies of visual,auditory and somatosensory cortices have demonstrated that excitation and inhibition are often coupled in single cortical neurons[7][52][53][54][55].The degree of coupling describes the balance between excitation and inhibition in cortical information processing.In the auditory cortex,the neuronal receptive field constructed on excitatory postsynaptic conductance(EPSC)is largely mirrored by the neuronal receptive field constructed on inhibitory postsynaptic conductance(IPSC)[7][8][9][10].Studies in the visual cortex recently showed that the ratio of inhibition and excitation is mostly consistent across individual neurons at the thalamocortical recipient layer[56][57].These findings suggest that the excitatory and inhibitory feedforward microcircuitry is a fundamental unit of the thalamocortical system[58][59][50][60].The inhibition in this feedforward circuitry shapes the output,ie.,firing and receptive field of the recipient neurons in layers ?/? of the auditory cortex[7][8].Of note,previous studies that examined the balance of cortical excitation and inhibition have focused on neuronal responses to optimal stimulation.The dynamics of this feedforward inhibition appears to occur in a linear manner;the degree of inhibition is largely correlated to the increase or decrease in excitation following the changes in stimulation[7][53].However,the ratio of inhibition and excitation can largely decrease in response to higher sound levels in non-monotonic neurons.This suggests a level-dependent dynamics of thalamocortical feedforward excitation and inhibition[61][5].It remains unclear how cortical excitation and inhibition interact at the threshold level.The results of extracellular studies confirm that the uncertainty of neuronal firing sharply increases at the threshold level[62][63],which is well in accordance with psychoacoustic findings of the low detectability of sound at the hearing threshold[64].Is the cortical excitation and inhibition interaction at threshold levels distinct from that at optimal stimulus level,ie.,poor balanced or completely imbalanced?Clarification of this issue also benefits our understanding of thalamocortical feedforward circuits.Eighteen female C57 mice of 4-5 weeks in age and weighing 17-20 g were employed in our experiments.Anesthesia for the experiments consisted of a ketamine/xylazine mixture.To block action potential firing and improve space clamp,the electrode pipettes were filled with a solution containing sodium channel blocker QX-314 and Cesium.The solution(in mM)consisted of 125 Cs-gluconate,5 TEA-Cl,4 MgATP,0.3 GTP,10 phosphocreatine,10 HEPES,0.5 EGTA,3.5 QX-314(sodium channel blocker),and 2 CsCl.The holding membrane potential was set at-70 mV for recording excitatory postsynaptic currents(EPSC)and at 0 mV for recording inhibitory postsynaptic currents(IPSC).Here,we recorded the EPSCs and IPSCs of layers ?-? neurons in the mouse auditory cortex in response to threshold tones by using in vivo whole-cell patch-clamp.Complete sets of data were successfully sampled in 18 AI neurons.Since the recording area was strictly limited to a range of 400 ?m to 700 ?m below the brain surface,these neurons were considered to be within the thalamocortical recipient layer of the AI.The input resistance was first measured following a successful whole cell patch.Fifteen out of 18 recorded neurons showed relatively lower input resistances when the membrane potentials were held at-70 mV.On average,it was 205.49 ± 123.42 M?.The other three neurons showed larger input resistances.On average,the input resistance of these three neurons was 744.73 ± 620.60M? at-70 mV.They were statistically different(p<0.05).The direction of tone-evoked postsynaptic currents changed from negative to positive following the increase in the holding potential from-90 mV to 0 mV.An example shown in Figure 1 demonstrates that the neuron was well clamped.A tone(at neuronal BF)of 70 dB SPL induced excitatory postsynaptic currents(EPSCs)when the holding potentials were at-90 mV and-70 mV.The tone induced a small EPSC followed by large inhibitory postsynaptic currents(IPSCs)when the holding potential was at-30 mV and induced a pure IPSC when the holding potential was at 0 mV.In line with previous reports[53][8],tone induced EPSC when the membrane potential was held at-70 mV and induced IPSC when held at 0 mV.The tone-evoked EPSCs and IPSCs were coupled in single AI neurons at most frequencies and amplitudes within the neuronal receptive field.The EPSC/IPSC coupling however,was limited at the threshold level.Two examples are shown in Figure 1-2.Neuron A showed a MT at 35 dB SPL.At this level,clear EPSCs and IPSCs were induced by 11 kHz and 12 kHz tone stimuli.Since the EPSC and IPSC to 11 kHz were larger than those to 12 kHz,the BFs and MTs of tone-evoked EPSC and IPSC were respectively 11 kHz and 35 dB SPL,ie.,identical in the BFs and MTs between EPSC and IPSC.In other words,this neuron had a balanced EPSC and IPSC at the threshold level.In contrast,the BF and MT of EPSC in Neuron B were different from those of IPSC(12 kHz and 30 dB SPL vs.13 kHz and 40 dB PSL).The tone induced EPSC but did not induce IPSC at 12 kHz and 30 dB SPL,illustrating an imbalance of EPSC and IPSC at the threshold level.Out of 18 sampled AI neurons,only 1(5%)neuron showed balanced EPSC and IPSC while 17(95%)neurons showed imbalanced EPSC and IPSC(Fig.1-3).The number of imbalanced neurons was much greater than that of balanced neurons.The imbalance between EPSC and IPSC of single AI neurons appeared mostly related to the difference in frequency tunings.Out of these neurons,only 3 AI neurons had EPSC BF(eBF)equal to IPSC BF(iBF)and 15 AI neurons had different eBF and iBF.The number of AI neurons with identical EPSC MT(eMT)and IPSC MT(iMT)was also lower than that with different eMT and iMT(6 vs.12,Fig.1-4).The analysis of the relation between eBFs and iBFs indicated that the AI neurons had iBF higher than eBF in most cases.Out of 15 AI neurons in which eBFs were different from iBFs,the iBF was higher than eBF in 10 neurons and the iBF was lower than eBF in 5 neurons.Notably,10 AI neurons had eBF and iBF difference by 3 kHz or higher and 3 neurons showed the difference by 5 kHz or higher(Fig.4).On average,the difference between eBF and iBF was 3.11±2.67 kHz,p<0.05,ranging from 0 kHz to 7 kHz,(n = 18).Taking into account the hearing range of the C57 mouse[65],this difference was significantly large.Similar to the difference of eBF and iBF,12 AI neurons had different eMT and iMT.Eleven of them showed higher iMT than eMT and only 1 neuron had lower iMT than eMT.It is also notable that the iMT was 10 dB or higher than eMT in 8 neurons(Fig.4).On average,the difference between eMT and iMT was 5.83±4.92 dB,P<0.05,ranging from 0 to 15 dB(n = 18).The EPSC and IPSC waveforms at the BFs and MTs were characterized using four parameters including latency,peak value,peak time and rising-slope based on postsynaptic conductance converted from the postsynaptic current.For simplicity,the EPSC and IPSC also represent the excitatory and inhibitory postsynaptic conductance.The IPSC latency was significantly longer than EPSC latency(45.47 ± 21.38 ms vs.31.46± 11.58 ms,p<0.05).The rising-slopes of IPSC and EPSC were respectively 0.95 ± 0.59 nS/ms and 0.41 ± 0.35 nS/ms,which were not statistically different(p>0.05).The two components of rising-slope were peak value and time.The peak value of IPSC was also significantly larger than that of EPSC(20.49 ± 11.24 nS vs.10.25 ± 6.53 nS,p<0.05)but the difference in peak times was statistically insignificant(25.22 ± 13.26 ms vs.31.20 ±14.73 ms,p>0.05),It is also noteworthy that the IPSC had longer latency,higher peak and longer duration than the EPSC but its rising-slope was similar to the EPSC.Our data clearly demonstrated that the coupling of cortical excitation and inhibition varied from neuron to neuron at the level of threshold stimulation.Only 5%neurons showed balanced excitation and inhibition while 95%neurons showed imbalanced excitation and inhibition.Furthermore,83.33%neurons showed different BFs of EPSC from those of IPSC(and 61.11%neurons showed higher thresholds of inhibitory responses than those of excitatory ones(iMT>eMT).These findings suggest that the excitation and inhibition are imbalanced when the stimulus only reaches the threshold levels of single cortical neurons.At a stimulus level sufficiently higher than the threshold,our data and many studies with in vivo whole-cell patch-clamp recording demonstrate that a tone-evoked EPSC is always balanced with a tone-evoked IPSC[7][53].Our data together with other findings suggest that the function of this microcircuitry at various sound levels should be dynamic instead of static or mechanical.A challenging but inevitable issue here is how to explain the threshold-level imbalance on the basis of thalamocortical feedforward microcircuitry.Specifically,two questions must be answered;one is why is the IPSC minimum threshold higher than,equal to or lower than EPSC and the other is what causes the BFs differences.An important distinction emerges from the observations of cortical excitatory and inhibitory neurons in response to a tone or thalamic stimulus in either in vivo or in vitro preparations.Cortical inhibitory neurons show more robust responses,ie.,larger postsynaptic potential and higher firing rate,to thalamocortical inputs than excitatory neurons[63][66][67].The synaptic transmission from GABAergic neuron to excitatory neuron is very efficient,and even one action potential is enough to induce changes in postsynaptic potential[68][69].In line with these evidences,our data showed that the IPSC had longer latency and higher peak than but similar slope to the EPSC.The implication here is that greater responsiveness of inhibitory neurons to thalamocortical inputs means that cortical excitatory neurons may have an IPSC minimum threshold equal to or even lower than the EPSC minimum threshold.This is apparently not the case.Our data and examples presented in other studies[53][8][9][70][71][72]showed that most cortical excitatory neurons have the minimum threshold of IPSCs higher than that of EPSCs.To solve this puzzle,one must examine the intricacies of the thalamocortical feedforward circuitry.The fundamental circuit of thalamocortical feedforward excitation and inhibition consists of the direct projection of the thalamic neuron to a cortical excitatory neuron and the direct collateral projections to a cortical inhibitory neuron that in turn sends the inhibitory projection back to the excitatory neuron targeted by the same thalamic neurons[73][74].Three important facts should be considered.The first is that the axons of auditory thalamic neurons primarily terminate at the small or distal dendrites of non-GABAergic neurons.Only a small number of axons reach GABAergic neurons and typically synapse onto the large or proximal dendrites and cell body[75].The second is that cortical excitatory neurons receive inputs from thalamic neurons that have similar tuning properties while the inhibitory neurons receive inputs from thalamic neurons that exhibit a wider range of tuning properties[76][2],This suggests that the thalamocortical projections to cortical excitatory neurons are restricted in single frequency channels while those to inhibitory neurons have the inputs from various frequency channels.The third is that the single thalamocortical synapses onto both excitatory and inhibitory neurons appears weak in function and that the synchronous activities of multiple thalamocortical inputs are required to drive the cortical neurons[77].Taken together,these features of the thalamocortical circuit allow us to outline an enriched model of thalamocortical feedforward excitation and inhibition[60][5][78].Four new properties appear plausible.Several thalamocortical excitatory pathways share a feedforward inhibitory pathway.In other words,the cortical inhibitory neurons receive thalamocortical inputs from different frequency and amplitude channels while cortical excitatory neurons receive inputs primarily from single frequency and amplitude channels.Secondly,synchronous activities of thalamocortical inputs are required to drive the postsynaptic activities of both cortical excitatory and inhibitory neurons.Thirdly,thalamocortical synapses onto inhibitory neurons exhibit relatively high efficiency.Finally,cortical inhibitory neurons project back to all cortical excitatory neurons.These excitatory neurons and inhibitory neurons share the inputs from the same thalamic neurons that belong to different frequency/amplitude channels.This enriched model could account for the differences in a number of properties between the EPSC and IPSC of single cortical neurons.The longer latency of the IPSC indicates more synaptic relays for inhibition.The similar rising-slopes of the IPSC and EPSC suggest the efficiencies of the excitatory and inhibitory synapses on the target excitatory neuron are relatively uniform.The longer IPSC duration is possibly more interesting,suggesting that the thalamocortical inputs to GABAergic neurons are relatively less synchronized than those to excitatory neurons.This weaker synchronization could result from diverse thalamocortical inputs originating from different frequency/amplitude channels as proposed in this model.Our model does not exclude the possibility of the varied strengths of the involved synapses;this may also contribute to the imbalance of cortical excitation and inhibition at the threshold level.Balanced excitation and inhibition of single cortical neurons play a critical role in shaping temporal processing,which leads to more uniform timing of neuronal action potentials,ie.,more phasic firing[7].The imbalance of cortical excitation and inhibition may underlie the larger variation in firing probability and the timing of cortical neurons in response to threshold sound[79][12].Our ongoing investigations of imbalanced threshold-level excitation and inhibition significantly enhance the knowledge of sensory information processing and neural plasticity development in the auditory cortex.We show that the excitation and inhibition of cortical neurons were largely imbalanced at the threshold levels.All the contents I have wrote above had been published in Frontiers in Neural Circuits.The citation is“Zhao Y,Zhang Z,Liu X,Xiong C,Xiao Z and Yan J(2015)Imbalance of excitation and inhibition at threshold level in the auditory cortex.Front.Neural Circuits 9:11.doi:10.3389/fncir.2015.00011".2.Imbalance of receptive field of EPSC and IPSC in C-57 mouse with Ml receptor knockoutAcetylcholine is a very important neuromodulators.Acetylcholine projections mainly originates from the basal nucleus.It is known to play a regulatory role in cortical functions ranging from attention,learning,and memory,to neural processing in the sensory cortex[13][14].In addition,Alzheimer's disease and cortical dysfunction of cholinergic nerves are closely linked[15].Damaged cortical cholinergic nerve fibers have been shown to impair external stimuli detection,identification,and location,as well as a reduction in learning,memory,and sensory discrimination tasks[16][17].Structurally,acetylcholine receptors are divided into two types,the muscarinic receptor(mAChRs)and nicotinic receptors(nAChRs).Muscarinic cholinergic receptor have 5 subtypes,M1,M2,M3,M4 and M5[18][19][20].In the brains of adult animals,all muscarinic receptor are expressed.The most cortical muscarinic receptor subtypes expressed in Adult animals is M1 receptors[18][2]],in the surface and deep layers of the cortex[22][23].During the initial stages of development both muscarinic receptor and nicotinic receptors are expressed in mammalian cortex.While nicotinic receptor expression remains constant throughout,during later stages of cortical development(within a few weeks after birth)mRNA transcription of the muscarinic receptor gene was found to change drastically.Normally,high levels of mAChR expression correspond to the expression of a variety of muscarinic receptor subtypes in many organ systems.It has been reported,the most common muscarinic receptor subtypes expressed in the brain are M1,M2 and M4.M1 is the maximum expression in the cortex.Five days after birth,M1 protein was detected in the brain of mice.The expression of M1 protein is predominately in layer 4 in the first 14 days after birth[22][23],while M2 mainly in layer 2/3 and 5.The evidence suggests that during development,the distribution of M1 and M2 subtypes may play an important role in the formation process of neurons in the cortex functional differentiation.During development,one important phenomenon is the timing of the formation of projections.Interestingly,the growth of thalamic projections to the cortex occurs at the same time as the growth of cholinergic basal forebrain projections the auditory cortex[24][25].Moreover,during cortex maturation and synapse formation of the active phase,cortical cholinergic projections reach their cortical destinations as well[26][27].This evidence suggests that cholinergic neurons may play an important role in cortex maturation and synapse formation.Morphological studies suggest roles in the development for early pre-deprivation basal forebrain cholinergic neurons,dysfunctions of which can cause abnormal cortical structures[27].Furthermore,M1 receptor has an important role in the structure forming of neuron.In comparison with the control group,the length of dendrites in granulosa cells of M1 knockout group were extremely shorter in auditory cortex layer 4 than control group[28].The function of the auditory system is to detect,identify,locate and act according to specific sound stimuli[29].For the auditory cortex,effects of acetylcholine include increased firing rate of action potentials evoked by pure tone.Experiments have confirmed that acetylcholine(ACh)and muscrinic receptor agonists have a similar effect,they can enhance the auditory neurons in response to sound stimuli[30].Acetylcholine can reduce response threshold of the neurons,which can be reversed completely with the addition of atropine(muscrinic receptor antagonist).The muscarinic acetylcholine receptors have an important regulatory role on cholinergic neurons in auditory cortex.The postsynaptic excitatory and inhibitory inputs onto an auditory cortex neuron are integrated and reflected by its output.According feed forward circuit,cortical excitatory neurons directly receive excitatory input from thalamus,and also accept inhibitory from cortex interneurons(which themselves also accept thalamic excitatory projection).Therefore,the balance of excitatory and inhibitory inputs will change the integration of the postsynaptic neuron,and ultimately affect reaction of the sensory system to sensory stimuli.Experiments demonstrate that endogenous or exogenous acetylcholine release,for the primary auditory cortex is mainly affected by the role of muscrinic receptor[38][39][40][41][36].We hypothesized that during development,M1 cholinergic receptors regulate cortical neuron firing through postsynaptic mechanisms.We recorded the EPSC and IPSC in layer 3/4 of primary auditory cortex of muscrinic receptor knockout mice by using in vivo whole-cell patch-clamp recording.When the membrane potential was clamped at-70mV,we recorded excitatory postsynaptic currents(EPSC);when membrane potential clamped at OmV,we recorded inhibitory postsynaptic currents(IPSC).By comparing the frequency-intensity receptive field of EPSC and IPSC,we can determine role of M1 cholinergic receptor in excitatory and inhibitory synaptic inputs.In a comparison of EPSC and IPSC receptive field areas,the overlap region of their receptive fields decreased(the difference of the receptive field area increases in Ml group)but did not change the fact that the EPSC receptive field area was greater than IPSCs.There are three possible reasons for the differences in receptive field area:one is that the IPSC area reduced,two is that the EPSC area increased,and three is that while the areas may be constant,the overlapping portion EPSC and IPSC decreased.According to the feedforward circuit theory,excitatory projections from the auditory thalamus project to excitatory and inhibitory neurons in the cortex.Then,inhibitory neurons project back to excitatory neurons.Additionally,excitatory neurons in the auditory cortex receive excitation and inhibition between the cortices.If the receptive field area of IPSC is reduced,it suggests that connections between the excitatory neurons of the auditory thalamus and auditory cortical inhibitory neurons is reduced.It's been found that M1 cholinergic receptor knockouts decreased the overlap region.If receptive field area of EPSC is increased,it may be due to the lack of M1 cholinergic receptors(excitatory synaptic connections between cortical inhibition diminished).The increase occurs in the receptive field periphery,suggesting cholinergic inputs predominately affects non-CF inputs.We also analyze the frequency-selective receptive field of EPSC and IPSC through Q10 and BW10.Q10 = BF/BW10,the stronger Q10 value,the greater the frequency selectivity,the sharper the bottom of the curve.Found no significant difference in the frequency selectivity of the EPSC and IPSC in M1 group.However,the frequency selectivity of EPSC in the WT group is better than M1 group.We also used BW10(frequency response width at 10dB SPL above threshold)to compare the frequency selectivity of EPSC and IPSC.Smaller BW10 value the frequency selectivity is stronger,sharper at the bottom of the curve.In recorded 15 neurons of M1 group,EPSC was 12.6 ± 5.28kHz,IPSC was 9.0 ± 3.74kHz,there is a significant difference between the two,p<0.05(n=15).In M1 group,the bottom of the frequency response curve of EPSC is wider than the bottom of IPSC.It suggests auditory thalamacortical excitability information increased.In addition,no significant differences between eBF and iBF in M1 group.However,Ml group eMT the mean average of 67.33± 9.98 dB SPL and iMT was 72.0±10.99 dB,p<0.05,indicating there was a significant difference(n=15)between the two.Tip neurons M1 receptor knockout group of BF consistency EPSC and IPSC relatively high.However,IPSC MT is higher than the EPSC of MT.In addition,no significant differences between eBF and iBF in Ml group.However,in M1 group,eMT is 67.33±9.98dB SPL and iMT was 72.0 ±10.99 dB,P<0.05,there was a significant difference between them(n=15).It indicates the consistency of BF of EPSC and IPSC relatively high in M1 receptor knockout group.However,MT of IPSC is higher than the MT of EPSC.We used four indicators the latency,peak value,peak time,the rising slope to describe the best frequency and minimal threshold levels of inhibitory postsynaptic conductance(IPSC)and excitatory postsynaptic conductance(EPSC)waveform characteristics.The latency of IPSC(42.42±7.02 ms vs.37.97 ±4.36 ms,p<0.05 compared EPSC in M1group,with significant differences.Rising slopes of IPSC and EPSC were 0.55 ± 0.28 nS/ms and 0.30 ± 0.16 nS/ms(p<0.05),with significant differences.The peak value of IPSC(20.26 ± 11.97 nS)compared with EPSC(8.55±5.15 nS)in 15 neurons of M1 group also have significant differences(p<0.05).However,there is no significant difference between IPSC rise time 37.95 ± 36.89 ms and EPSC rise time 24.27 ± 9.79 ms,p>0.05,(n=15).It indicated that IPSC with a longer latency,faster and higher peak rising slope compared to EPSC.The longer latency of IPSCs is likely due to the physical distance of the thalamocortical feedforward circuit model.The cortical inhibitory neuron accepts excitatory input from the thalamus then projecting inhibitory to the excitatory neurons.Therefore,the inhibitory input onto an excitatory neurons,longer than excitatory input,resulting in IPSC long incubation period.In addition,it may be because of inhibitory neurons need a long time to integrate information before the output.It indicates inhibitory neurons may accept more information than excitatory neurons.Further,IPSC with high peaks and steep rising slopes indicate that the inhibitory inputs are processed with higher efficiency of excitatory input;it can generate a stronger reaction in a short time.In summary,differences of receptive fields of EPSC and IPSC increased in M1 group,suggesting that during development,the lack of regulation of acetylcholine by M1 cholinergic receptors may expand the scope of EPSC's receptive field,or narrow range of IPSC receptive fields.In addition,overlapping areas of EPSC and IPSC reduced,showing that acetylcholine is involved in regulating the formation of auditory cortex neurons receptive fields during development.At 10dB above threshold level the EPSC responses significantly expanded,suggesting that muscrinic acetylcholine receptors are involved in modulating thalamus-to-cortical excitatory function during development.IPSC has a longer latency,higher peak value,and faster rising slope,suggesting that synaptic efficiency of IPSC is higher than the EPSC.This is consistent with the characteristics of these features of IPSC in WT group,which suggests that M1 cholinergic receptors for the regulation of cortical inhibitory input were limited.Therefore,according to our results,the absence or dysfunction of M1 cholinergic receptors in development,mainly affect the thalamus-cortical excitability projection.It may change the ratio of cortical neurons excitatory and inhibitory projections,and ultimately impact the formation of cortical neuron receptive fields. |
PDF Full Text Request