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The Journal of Neuroscience September 1 1997 17176639 6646 Prolonged Sodium Channel Inactivation Contributes to Dendritic Action Potential Attenuation in Hippocampal Pyramidal Neurons HaeYoon Jung Timothy Mickus and Nelson Spruston Department of Neurobiology and Physiology Institute for Neuroscience Northwestern University Evanston Illinois 602083520 During lowfrequency firing action potentials actively invade the dendrites of CA1 pyramidal neurons At higher firing rates however activitydependent processes result in the attenuation of backpropagating action potentials and propagation failures occur at some dendritic branch points We tested two major hypotheses related to this activitydependent attenuation of backpropagating action potentials 1 that it is mediated by a prolonged form of sodium channel inactivation and 2 that it is mediated by a persistent dendritic shunt activated by back propagating action potentials We found no evidence for a persistent shunt but we did find that cumulative prolonged inactivation of sodium channels develops during repetitive ac tion potential firing This inactivation is significant after a single action potential and continues to develop during several action potentials thereafter until a steadystate sodium current is established Recovery from this form of inactivation is much slower than its induction but recovery can be accelerated by hyperpolarization The similarity of these properties to the time and voltage dependence of attenuation and recovery of den dritic action potentials suggests that dendritic sodium channel inactivation contributes to the activity dependence of action potential backpropagation in CA1 neurons Hence the bio physical properties of dendritic sodium channels will be impor tant determinants of action potentialmediated effects on syn aptic integration and plasticity in hippocampal neurons Key words dendrite action potential sodium channels syn aptic integration pyramidal neuron activity dependent Recent experiments using simultaneous somatic and dendritic patchpipette recordings have shown that action potentials are normally initiated in the axon and backpropagate into the den drites ofmany types of CNS neurons for review see Stuart et al 1997 These backpropagating action potentials are likely to provide an important spatial signal that in uences ongoing syn aptic integration and allows for postsynaptic ring in the axon to be associated with presynaptic activity For example the induc tion of activitydependent changes in synaptic strength such as longterm potentiation LTP and longterm depression depend critically on the timing of pre and postsynaptic inputs Levy and Steward 1983 Markram et al 1997 and one form of LTP has been shown to be blocked by preventing action potentials from backpropagating into the dendrites of hippocampal pyramidal neurons Magee and Johnston 1997 These ndings demon strate the importance of understanding the factors that determine the extent and pattern of action potential backpropagation in pyramidal neuron dendrites Action potential backpropagation in CA1 dendrites is com plex At low frequencies action potentials invade most of the dendritic tree in an active fashion whereas at higher frequencies action potentials attenuate more and may fail to actively propa gate into much of the dendritic tree C allaway and Ross 1995 Received May 1 1997 revised June 20 1997 accepted June 23 1997 This manuscript was supported by National Institutes of Health Grant NS35180701 and the Human Frontiers in Science Program Nelson Spruston is a Sloan Fellow We thank Nace Golding and David Ferster for discussion and comments on this manuscript and Arnd Roth for modeling our channel gating scheme TM and H contributed equally to this project Correspondence should be addressed to Dr Nelson Spruston Department of Neurobiology and Physiology Northwestern University 2153 N Campus Drive Evanston IL 6020873520 Copyright 1997 Society for Neuroscience 027076474971766397083S05000 Spruston et al 1995 The degree of action potential attenuation observed during a train of action potentials depends on the location of the recording in the dendritic tree the number of action potentials and the rate of ring ln distal dendrites dra matic attenuation of action potential amplitude typically occurs after one or a few action potentials at 20 Hz and apparent failures of active propagation are often observed at branch points whereas attenuation is smaller and more gradual in proximal dendrites The degree of dendritic action potential attenuation is also known to be dependent on the membrane potential between action potentials Spruston et al 1995 suggesting that the states of dendritic voltagegated channels are important determinants of how action potentials spread through the dendrites The identity of these channels however and their effects on action potential backpropagation are obscure Here we describe experiments that examine the role of voltagegated channels in the back propagation of action potentials into the dendrites of CA1 pyra midal neurons in vitro MATERIALS AND METHODS Slice prqmmtion Hippocampal slices were prepared from the brains of 14 to 45dold Wistar rats which were decapitated after halothane anesthesia Slices were cut 300 pm thick using a vibrating tissue slicer Campden Instruments During the dissection and slicing procedure brains were kept in an icecold physiological solution Slices were trans ferred to a holding chamber containing the same physiological solution at 35737 C for 30745 min and subsequently at room temperature For recording slices were transferred individually to a chamber and perfused with physiological solution at 33737 Cr Patchpipette recording Slices were visualized using infrared differen tial interference microscopy Stuart et al 1993 using a xedstage microscope Zeiss Axiosoop and a Newvicon camera Dage MTI Patchpipette recordings were obtained under visual control on the soma or dendrites of pyramidal neurons in the CA1 region of hippocampus Highresistance seals 2720 G0 were formed using repolished elec 6640 J Neurosci September 1 1997 17176639 6646 A Somatic AP command WW Figure 1 B Dendritic AP command it Jim Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation C 70 mV 2 ms pulses 200 ms Cumulative prolonged inactivation of Na currents in nucleated patches A A command potential consisting of a previously recorded train of somatic action potentials evokes voltagegated Na currents in a nucleated patch Each action potential evokes Na currents that inactivate but do not fully recover during the 50 msec interspike interval leading to a decline in Na current amplitude attributable to cumulative inactivation B A train of action potentials recorded from the apical dendrite 120 Mm from the soma of a diiferent cell also evokes Na currents exhibiting cumulative prolonged inactivation C Cumulative prolonged Na current inactivation is also observed during repetitive depolarizations of 70 mV amplitude and 2 msec duration Recordings in A C are from the same nucleated patch and are averages of three to six trials trodes thickwalled borosilicate glass EN1 Garner Glass Co pulled to tip resistances of 3 8 M BrownFlaming P30 puller Sutter Instru ment Co Experiments were performed in wholecell cellattached or nucleatedpatch con gurations In the wholecell con guration recordings were obtained in the currentclamp mode series resistance 20 50 M9 was compensated with a bridge circuit and capacitance compensation was performed Nucleated patches were obtained by forming wholecell recordings from somata near the surface of the slice and then withdrawing the pipette with negative pressure 05 15 psi applied to the pipette Sather et al 1992 This resulted in the formation of large outsideout patches of membrane surrounding the nucleus Wholecell and nucleatedpatch recordings were performed at 33 37 C dendriteattached patch recordings were performed at 27 36 C For voltageclamp experiments electrodes were coated with Sylgard to reduce electrode capacitance and the remaining patch pipette capacitance was compensated Solutions and drugs External physiological solution consisted of in mM 125 NaCl 25 KCl 25 NaHCO3 125 NaH2P04 1 MgClZ 2 CaClZ 25 dextrose Pipette solutions for the different recording con g urations were as follows in mM wholecell recording 115 potassium gluconate 20 KCl 10 phosphocreatine disodium salt 10 HEPES 10 EGTA 4 MgATP 03 NaGTP pH 73 with KOH cellattached record ing 120 NaCl 3 KCl 10 HEPES 2 CaClZ 1 MgClZ 30 tetraethylam monium chloride TEA 5 4aminopyridine 4AP pH 74 with NaOH nucleatedpatch recording 130 CsCl 10 phosphocreatine disodium salt 2 MgClZ 10 HEPES 02 EGTA 4 NazATP pH 74 with KOH In some cases creatine phosphokinase 50 Uml was also included in the pipette solution In most nucleatedpatch experiments 30 mM TEA and 5 mM 4AP were added to the bath but outward currents were negligible even in the absence of these external K channel blockers Some run down of patch current was observed in nucleatedpatch experiments The rst response in each train was monitored over time and trials were rejected if this rst response was less than twothirds of the amplitude measured at the beginning of the experiment Data acquisition and analysis Currentclamp recordings were obtained with Dagan BVC700 ampli ers voltage was ltered at 5 kHz and digitized at 10 kHz Voltageclamp recordings nucleated and cell attached patches were obtained with Axoclamp 1D and Axoclamp 200A ampli ers current was ltered at 2 kHz and sampled at 50 kHz Data acquisition was performed using Pulse Control software R Bookman University of Miami running under Igor Pro 30 Wavemetrics on Macintosh Power PC computers Apple Computer equipped with ITC16 hardware interfaces Instrutech Corp Capacitance and leak subtraction was performed by adding the current response to the test command with four responses to an inverted command potential one fourth of the test command amplitude ie P4 subtraction Data analysis was performed using Igor Pro All values are reported as mean SEM RESULTS Action potentials recorded in the wholecell con guration from the soma and dendrite 120 am from the soma of different CA1 pyramidal neurons are shown in the top traces of Figure 1AB The activitydependent attenuation of backpropagating action potentials reported previously C allaway and Ross 1995 Sprus ton et al 1995 is evident in this dendritic recording Fig 13 top trace obtained from a neuron maintained at 34 C in a slice from an 18dold rat We tested two major hypotheses regarding the mechanisms underlying this activitydependent action potential attenuation 1 that attenuation is mediated by a form of Na channel inactivation developing as action potentials re and 2 that attenuation is mediated by a dendritic shunt that develops as action potentials re To test the rst hypothesis Na currents were examined in either cellattached or nucleated patches Sather et al 1992 Inward currents were elicited by transient depolarizations using either a previously recorded train of action potentials or a series of 2 5 msec depolarizations as the command potential These currents were mediated by TTXsensitive Na channels see text Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation below and hence are termed 1N3 To measure the currents during realistic depolarizations trains of action potentials recorded from the soma or the dendrite in the wholecell con guration Fig 1AB top traces were stored and later used as command poten tials to depolarize nucleated patches This protocol allowed us to determine the nature of INa that would be owing during normal action potential ring The resulting patch current shows that the INa in response to repetitive action potential ring becomes progressively smaller during the train Fig 1A When a dendriti cally recorded train of action potentials is used as the command in the same patch the current attenuation is greater as predicted because of the progressive decrease in action potential amplitude during the train Fig 13 Although these experiments demonstrate that INa is expected to decrease during successive action potentials in a train the reasons for this remain unclear from this experiment One pos sibility is that some Na channel inactivation occurs that is slow to recover but the slower rise of later action potentials may also contribute to the reduced INa during action potential commands To determine whether inactivation does in fact contribute INa was also examined in response to trains of 2 5 msec step depo larizations at 20 Hz Figure 1C shows that during such a com mand potential signi cant inactivation of INa indeed occurs These ndings support the hypothesis that Na channel inacti vation contributes to the attenuation of backpropagating action potentials The rst action potential leaves a fraction of dendritic Na channels in the inactivated state so the second action potential attenuates more as it propagates along the dendrite At any dendritic location a reduced Na current ows as a result of the prolonged Na channel inactivation and the smaller ampli tude of the upstream action potential That the currents observed in experiments like those shown in Figure 1 were indeed Na currents was con rmed in three nucleatedpatch experiments using TTX The currents evoked by 2 and 5 msec current pulses are shown on an expanded time scale in Figure 2 In all cases in which it was tested 05 MM TTX completely blocked the currents evoked by a 20 Hz train of depolarizations n 3 Fig 2A In response to 5 msec pulses inactivation of INa had a time constant of 078 i 010 msec n 10 and almost complete inactivation occurred by the end of the pulse 89 i 3 n 10 Most experiments were performed using 2 msec depolarizations and inactivation was almost complete during these brief depolarizations as well At 20 Hz however recovery from inactivation was not complete after the 48 msec interpulse interval on average the amplitude of the second response was only 80 of the rst response Table 1 This remaining prolonged form of inactivation accumulated with ad ditional depolarizations but complete inactivation of INa never occurred Fig 1AC During repetitive 20 Hz depolarizations 15 20 pulses of 50 70 mV INa achieved a steady state that was on average 58 of the rst response Table 1 Fast inactivation appeared to be unaffected by the prolonged inactivation because there was no signi cant difference between the inactivation time constants of the rst and last responses in a train pairedsample t test n 10 p gt 019 This can be seen by the superimposition of the rst response and a scaled version of the last response Fig 2BD Although the inactivation of INa occurred rapidly 50 of the total inactivation was reached after the rst 2 msec pulse recov ery from this form of inactivation was extremely slow with only 72 recovery occurring after 2 sec Fig 3A Table 1 but could be accelerated by hyperpolarization during the recovery period J Neurosci September 1 1997 17176639 6646 6641 A B TTX last first 2 ms last scaled first Figure 2 Na currents in nucleated patches inactivate rapidly and are blocked by TTX A Voltagegated Na currents evoked by 50 mV 5 msec depolarizations Command potentials are shown above the current re sponses The rst large and fteenth small responses in a train of step depolarizations 20 Hz are superimposed revealing the current reduc tion attributable to cumulative prolonged inactivation The response in the presence of 05 MM TTX is also superimposed Imperfect capacitive transient subtraction is apparent at the beginning and end of the re sponses Each current trace is an average of 24 trials B The same data as in A but with the fteenth response scaled dotted line to match the peak amplitude of the rst response revealing the similar time course of fast inactivation C Similar data as in A but from a different nucleated patch and in response to 50 mV 2 msec depolarizations Each current trace is an average of six trials D Data in C with the fteenth response scaled dotted line to match the peak amplitude of the rst response Fig 3B The time course and voltage dependence of the recov ery from prolonged inactivation are similar to that of the recovery of backpropagating action potential amplitude Spruston et al 1995 The rationale for studying the inactivation properties in nucle ated patches was to take advantage of the large currents that can be obtained in this recording con guration Because sodium channels have been shown to be similar in the soma and dendrites of CA1 pyramidal neurons Magee and Johnston 1995 it seems reasonable to make inferences about how the properties of so matic INa will affect action potential propagation in dendrites To determine whether dendritic Na has the same inactivation prop erties as somatic 1N3 however we performed experiments on Na in cellattached dendritic patches As expected from the relative patch size currents in dendriteattached patches were substan tially smaller than in nucleated patches but cumulative pro longed inactivation of INa was still observed Fig 4 On average the steadystate INa in dendritic patches was 44 of the initial current amplitude n 8 28 240 pm from the soma Table 1 6542 J Neurosci September 1 1997 17176639 6646 Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation Table 1 Properties of prolonged sodium channel inactivation in somatic and dendritic patches 20 Hz Train Recovery Second pulse Last pulse 200 msec 500 msec 1000 msec 2000 msec of rst pulse Soma 801 582 664 722 772 864 n30 n30 n13 n17 n14 n8 Dendrite 852 445 600 636 821 767 n8 n8 n2 n n2 n2 Inactivation Soma 201 422 4 282 23 2 144 Dendrite 152 565 400 376 181 247 Recovery Soma 33 3 50 72 Dendrite 24 1 35 2 63 61 Statistics were calculated from peak sodium current amplitudes during 20 Hz trains of 2 msec step depolarizations and from test depolarizations at various times after the train eg Figs 1A 3AB 4 The values of the second and last pulse responses during the train were computed as percentages of the rst response second X 100 rst or last X 100 rst or as the percentage inactivation rst 7 second X 100 rst or rst 7 last X 100 rst Recovery was computed as the percentage of the rst response at the indicated time after the train recovery X 100 rst as the percentage of current that remained inactivated rst 7 recovery X 100 rst or as the percent of inactivated current that had recovered recovery 7 last X 100 rst 7 last Values are mean SEM andn is the total number of patches which are the same for the second and third pairs as for the rst pair of rows Pairedrsamplet tests indicated that responses to the second and last responses were signi cantly smaller than the rst response for both somatic and dendritic patches p lt 0007 which was signi cantly greater than the attenuation observed in somatic patches twosample t testp lt 0006 These results also obviate any concern that the prolonged inactivation of INa might be an artifact of the cytoplasmic dialysis that occurs during nucleatedpatch recording The second hypothesis that activitydependent action poten tial backpropagation could be caused by a shunt developing in the dendrites was tested in two ways First the response to a small hyperpolarizing current pulse was compared before and after a train of action potentials Second EPSP amplitude was compared before and after a train of action potentials If a shunt capable of in uencing action potential backpropagation develops as action potentials activate dendritic conductances it should manifest itself as a decrease in the response to a hyperpolarizing current pulse andor a decrease in EPSP amplitude after a train of action potentials Furthermore the shunt should be detectable hundreds of milliseconds after the train when dendritic action potentials remain attenuated Spruston et al 1995 Figure 5 shows that no such decrease in the voltage response to a small hyperpolarizing current pulse is observed The test response began 50 msec after the end of the depolarizing current pulse evoking the train and the steadystate amplitude was measured 2257250 msec after the train with the slow afterhyperpolariza tion subtracted see Fig 5 legend The development of a shunt would be expected to produce a signi cant reduction in the size of the test response We reasoned that this change might be largest in the dendrites where the shunt ought to develop if it were to affect action potential backpropagation so the experi ment was performed in dendritic recordings In contrast to the prediction of a shunt the test responses were almost identical in amplitude to the control responses testcontrol 101 006 after 15725 action potentials n 7 dendritic recordings 56 7210 pm from the soma One complicating factor in the interpretation of this experiment is the sag in the voltage response mediated by the hyperpolarizationactivated conductance In An increase in this conductance eg after Ca2 elevation during the train Hagiwara and lrisawa 1989 could produce a shunt that is masked by its own tendency to reduce the steadystate response to a hyperpolarizing current pulse To test this possibility we also monitored hyperpolarizing responses before and after a train in the presence of 5 mM CsCl to block In Under these conditions we still found no evidence for a shunt testcontrol 098 004 after 15735 action potentials n 3 dendritic recordings 567196 pm from the soma data not shown The prediction that a dendritic shunt would produce a reduc tion of EPSP amplitude was also tested experimentally EPSPs were evoked by stimulation in distal stratum radiatum or in stratum lacunosummoleculare and monitored in somatic record ings This experiment should maximize the likelihood of detect ing a shunt by virtue of the fact that EPSPs generated in the distal apical dendrites must travel a long distance to the soma and could be affected by a shunt anywhere along the way Neverthe less we found no evidence for a shunt EPSPs varied in amplitude from trial to trial in some cases the test EPSP was larger than control and in some cases it was smaller but the average change in EPSP amplitude was insigni cant Fig 6A testcontrol 106 005 n 6 To ensure that the amplitude ofthe test EPSP was not affected by pairedpulse facilitation or depression iride pendent of the train of action potentials control experiments were performed with only a subthreshold depolarization between the two synaptic responses Fig 6B In these experiments the control and test EPSP amplitudes were also similar testcon trol 106 007 n 6 The testcontrol EPSP ratio observed with a train of action potentials was not signi cantly different from the ratio with a subthreshold depolarization pairedsample ttest n 6 gt 09 suggesting that the train of action potentials does not produce a global dendritic shunt Two other possible mechanisms underlying activitydependent attenuation of backpropagating action potentials were also test ed 1 that attenuation is related to voltagegated Ca2 current inactivation and 2 that attenuation is caused by a shunt induced by recurrent synaptic activation These mechanisms we regarded as unlikely albeit formal possibilities Both hypotheses can be excluded by the experiment shown in Figure 7 Application of CdCl2 at a concentration suf cient to block highthreshold Ca2 channels 200 MM did not prevent action potential attenuation in dendritic recordings n 6 In one recording 50 MM NiCl2 was coapplied to block lowthreshold Ca2 channels as well and still Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation A llillllllllllllllllLl llllllllllllllllllll llllllllllllllllllll 25 pA 200 ms Blllllllll llllm JMHMMM l 25 pA 200 ms Figure 3 Na current recovers slowly but is accelerated by hyperpolar ization A Na currents in a nucleated patch three superimposed re sponses below the three command potentials recover very slowly from cumulative prolonged inactivation Test responses 200 1000 and 2000 msec after the initial 20 Hz train of 50 mV 2 msec depolarizations eXhibit 20 40 and 65 recovery respectively of the inactivated portion of the current Each current trace is an average of three to four trials B In a different nucleated patch recovery is accelerated by a 30 mV hyperpo larization during the 500 msec recovery period Recovery is 49 in control left and 96 with hyperpolarization right Each current trace is an average of three trials no effect was observed on action potential attenuation Fig 7 Although the ring pattern sometimes changed slightly in the presence of these blockers perhaps because of effects on Ca activated K channels activitydependent backpropagation al ways persisted indicating that neither highthreshold voltage gated Ca2 channels nor Ca2activated K channels are required for activityinduced changes in the ability of action potentials to invade CA1 dendrites Blocking Ca2 channels also completely blocked synaptic transmission Fig 7 thereby ruling out the possibility that action potential attenuation is mediated by recurrent synaptic activity eg attributable to a shunt induced by recurrent inhibition Similar observations were made using blockers of synaptic transmission such as DAP5 CNQX and bicuculline n 4 DISCUSSION Our results suggest that the activitydependent attenuation of action potentials that occurs as spikes backpropagate into the dendrites of CA1 pyramidal neurons is mediated by a cumulative prolonged form of Na channel inactivation Action potentials J Neurosci September 1 1997 17176639 6646 6643 5 pA 200 ms B Figure 4 Na currents in dendriteattached patches also eXhibit cumu lative prolonged inactivation A Na currents in a dendriteattached patch 203 um from the soma evoked by a 20 Hz train of 50 mV 2 msec depolarizations Steadystate current is 20 of the rst response and 38 of the inactivated current recovered after 500 msec The current trace is an average of 13 trials Recording was performed at 35 C B The rst and last responses in the train shown inA are superimposed and displayed on an eXpanded time scale are initiated in the axon of CA1 cells probably in the rst node of Ranvier Colbert and Johnston 1996a Initiation is likely to occur at this site because of a high density of Na 1 channels there compared with the somatic and dendritic membrane Mainen et al 1995 Rapp et al 1996 As action potentials invade the soma and dendrites they encounter a lower density of Na channels which is suf cient to support active backpropagation but with signi cant attenuation of action potential amplitude as a function of distance from the soma Spruston et al 1995 The data presented here indicate that as the rst action potential in a train invades the dendrites a fraction of the dendritic Na channels becomes inactivated At typical ring rates for CA1 pyramidal neurons eg 20 Hz the interspike interval is too short for complete recovery to occur from the prolonged form of inactiva tion so the next spike will encounter an even lower density of available Na channels and hence this spike will attenuate more than the rst spike as it propagates along the dendrite Axonal action potentials are unlikely to be affected by prolonged Na channel inactivation however because it is never complete and 6644 J Neurosci September 1 1997 17176639 6646 30 mV 200 ms F control test 10mV 1W1 l i 200 ms C D control test subtracted Figure 5 Small hyperpolarizing current injections in the dendrites do not reveal a global shunt after a train of action potentials A Single trace response top showing the effect of a train of 33 action potentials in 1 sec on the response to a small hyperpolarizing current pulse bottom hyper polarizing current injections are 10 pA and depolarizing current injection is 400 pA Apical dendritic recording is 168 um from the soma B Average of eight responses like the one in A C Enlarged view of the average control hyperpolarization before the train of action potentials D Enlarged view of the average test hyperpolarization after the train of action potentials The afterhyperpolarization measured in the absence of a test pulse was subtracted from the response with the test pulse the density of Na channels in the axon is likely to be so high that it is not signi cantly affected by the 40 inactivation at steady state Slow inactivation of Na channels has been described pre viously in several neuronal preparations Narahashi 1964 Adel man and Palti 1969 Chandler and Meves 1970 Schauf et al 1976 Rudy 1978 1981 Belluzi and Sacchi 1986 Ogata et al 1990 Ruben et al 1992 Colbert and Johnston 1996b Fleider vish et al 1996 In some cases the rates of both inactivation and recovery are comparably slow whereas in other cases as we describe here entry into the slow inactivated state is faster than recovery from this state Slow inactivation has been proposed to account for slow adaptation of action potential ring in Myxicola axons Rudy 1981 and in neocortical pyramidal neurons Flei dervish et al 1996 This adaptation requires inactivation of sodium currents after several seconds of depolarization whereas the molecular mechanism underlying activitydependent action potential backpropagation must occur more rapidly because backpropagating action potential failure can occur after even a single action potential Spruston et al 1995 The inactivation we Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation A Suprathreshold a f l I 15 mV T 200 ms b control EPSP 0 test EPSP subtracted B Subthreshold l I A i 200 ms 0 test EPSP subtracted 15mV Figure 6 Somatically recorded EPSPs also do not reveal a global shunt after a train of action potentials A Average of 12 EPSPs before and after 180 pA current injections evoking trains of 16 action potentials on average a The control EPSP b and test EPSP after the train C have similar amplitudes The afterhyperpolarization measured in the absence of a test EPSP has been subtracted from the test EPSP B Average of 11 EPSPs before and after subthreshold depolarizations evoked by a 120 pA current pulse a The control EPSP b and test EPSP C have amplitudes similar to one another and to those shown in A The dashed lines are drawn at the same levels relative to baseline inA and B describe here has this important property and we therefore refer to this inactivation as prolonged rather than slow It is nev ertheless possible that the rapidly induced yet prolonged inacti vation we describe here is functionally similar to the slow inacti vation described by others For example although Fleidervish and colleagues 1996 found that 200 msec depolarizations pro duced an amount of inactivation similar to what we observed with only 2 msec depolarizations Rudy reports that in Myxicola axons long depolarizations are no more effective than much shorter depolarizations at producing slow inactivation presumably be cause the slow inactivated state is primarily entered from the open state Rudy 1981 In agreement with our data Rudy showed that sodium channels accumulate in the slow inactivated state only if repetitive depolarizations are applied Rudy 1981 Our data suggest some interesting gating properties of Na channels in CA1 neurons A gating scheme that can explain the salient features of the prolonged inactivation we describe is presented in Figure 8 The model has four states closed C open 0 inactivated I and prolonged inactivated P1 In developing the model we focused on the observation that cumu lative inactivation of INa occurs rapidly and reaches a substantial steady state during repetitive brief depolarizations Figs 1 3 4 Jung et al 0 Prolonged Dendritic Sodium Channel Inactivation A Control B 200 uM CdCI2 20 W 50 uM Niel2 200 ms Figure 7 Blocking voltagegated Ca2 channels with CdC12 and NiC12 does not prevent activitydependent spike attenuation in dendrites A Action potentials evoked by a 400 pA current injection in an apical dendritic recording 174 um from the soma exhibit activitydependent attenuation Synaptic responses evoked before and after the train of action potentials were used to monitor the effects of CdC12 and NiC12 in B B Activitydependent action potential attenuation is not affected by the application of 200 MM CdCl2 and 50 MM NiCl2 to block high and lowthreshold calcium channels respectively The effectiveness of these blockers is indicated by the elimination of the synaptic responses before and after the train only stimulus artifacts are visible despite the fact that recovery from the inactivated state is much slower than the rate at which the inactivated state is reached These observations are surprising and require some special con siderations Simpler models in which entry into the P1 state is proportional to the number of channels that open during a pulse ie the O6P1 transition is prominent and the recovery P16 C is proportional to the number of channels already in the P1 state predict either a smaller steadystate INa or a faster recovery from prolonged inactivation A key feature of the gating scheme shown in Figure 8 is that the total amount of prolonged inactivation during repetitive depolarizations is limited by the fact that depolarization promotes the P161 transition and repo larization promotes the 16C transition The P161 transition is therefore central to the model without it more Na channels would accumulate in the P1 state and smaller currents would be observed during repetitive depolarizations The model also ex plains the acceleration of recovery by hyperpolarization by pro moting the P16C transition Hence our data are consistent with a model of prolonged inactivation whereby recovery from this J Neurosci September 1 1997 17176639 6646 6645 I A P V C Figure 8 Model of a Na channel gating scheme consistent with the properties of prolonged inactivation in hippocampal CA1 pyramidal neu rons The states are closed C open 0 inactivated I and prolonged inactivated PI Transitions promoted by depolarization move upward and transitions promoted by hyperpolarization move downward See Discussion for details 0 state is promoted by both hyperpolarization and depolarization Although we emphasize that this model is preliminary and cannot explain all of the available data in the literature on Na channel gating it demonstrates some interesting features that more com plete models should take into account An alternative to such a complex gating scheme however is that multiple populations of channels may exist some that un dergo prolonged inactivation and others that do not Such distinct subpopulations of Na channels could arise because of different 1 subunits accessory subunits or posttranslational modi cations possibly differentially distributed or susceptible to neuromodu lation Distinguishing between these possibilities will require additional experiments Understanding whether the prolonged inactivation properties of INa are determined by one population or multiple populations of Na channels will also help to eluci date the mechanisms underlying the diiference in the prolonged inactivation of somatic and dendritic 1N3 One possibility is that the increased prolonged inactivation of dendritic INa is caused by differential posttranslational modi cation of the same gene prod uct found at the soma alternatively multiple gene products could be differentially distributed along the somatodendritic axis Prolonged Na channel inactivation has been shown to pro duce attenuation of backpropagating action potentials in a com putational model Migliore 1996 The degree of inactivation used in the model however is substantially greater than we observed so it remains to be determined whether Na channel inactivation alone is suf cient to explain all observed features of the backpropagation during trains of action potentials including complexities such as asymmetrical branch point failures Spruston et al 1995 The model also showed that a dendritic shunt could be responsible for activitydependent attenuation of back propagating action potentials We nd no evidence for such a shunt but a few caveats should be noted regarding this interpre tation First the method we used relies on an ability to discern changes in input conductance as a change in the response to a hyperpolarizing current pulse or an EPSP Although we would expect a shunt to be measurable by these methods if it is suf cient to alter the nature of spike backpropagation we have not tested 6646 J Neurosci September 1 1997 17176639 6646 this explicitly with a computational model The fact that we cannot measure a shunt associated with the slow afterhyperpo larization suggests that such shunt conductances are small under our conditions but it also indicates that the sensitivity of our method is limited Second a shunt could be activated at potentials reached during an action potential but could be inactive at rest such a mechanism was included in the computational model mentioned previously Migliore 1996 Although this is theoret ically possible it requires a very particular biophysical mechanism eg a strongly rectifying shunt conductance Finally we cannot rule out the possibility that small localized shunts could affect backpropagation but might be dif cult to measure in the whole cell For example local hot spots of voltagegated K4r channels could theoretically mediate asymmetrical propagation of action potentials into different regions of the dendritic tree Our experiments examine the question of which mechanisms are responsible for action potential backpropagation in the rest ing state in vitro In vivo other considerations are likely to come into play For example shunting and hyperpolarization attribut able to inhibition have been shown to limit the backpropagation of action potentials into CA1 dendrites Tsubokawa and Ross 1996 This may provide a mechanism for selectively allowing backpropagating action potentials to reach certain dendritic compartments but not others under de ned conditions of inter neuron activity Such a mechanism might function to selectively promote or inhibit associative plasticity in restricted sets of syn aptic inputs onto CA1 dendrites Determining how spike back propagation might be regulated by inhibition or neuromodulation of Na channel inactivation will be an important direction for future studies aimed at understanding the complex process of synaptic integration in these and other neurons REFERENCES Adelman WJ Palti Y 1969 The effects of external potassium and long duration voltage conditioning on the amplitude of sodium currents in the giant axon of the squid Loligo pealei J Gen Physiol 5425897606 Belluzi O Sacchi O 1986 A quantitative description of the sodium current in the rat sympathetic neurone J Physiol Lond 38022757291 Callaway JC Ross WN 1995 Frequencydependent propagation of so dium action potentials in dendrites of hippocampal CA1 pyramidal neurons J Neurophysiol 742139571403 Chandler WK Meves H 1970 Slow changes in membrane permeability and 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