susan e. atkinson and stephen r. williams- postnatal development of dendritic synaptic integration...

8/3/2019 Susan E. Atkinson and Stephen R. Williams- Postnatal Development of Dendritic Synaptic Integration in Rat Neocort

1/18

102:735-751, 2009. First published May 20, 2009; doi:10.1152/jn.00083.2009J NeurophysiolSusan E. Atkinson and Stephen R. Williams

You might find this additional information useful...

for this article can be found at:Supplemental material

http://jn.physiology.org/cgi/content/full/00083.2009/DC1

55 articles, 30 of which you can access free at:This article citeshttp://jn.physiology.org/cgi/content/full/102/2/735#BIBL

including high-resolution figures, can be found at:Updated information and services

http://jn.physiology.org/cgi/content/full/102/2/735

can be found at:Journal of NeurophysiologyaboutAdditional material and information

http://www.the-aps.org/publications/jn

This information is current as of October 27, 2010 .

http://www.the-aps.org/.American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at(monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2009 by the

publishes original articles on the function of the nervous system. It is published 12 times a yearJournal of Neurophysiology
http://jn.physiology.org/cgi/content/full/00083.2009/DC1http://jn.physiology.org/cgi/content/full/102/2/735#BIBLhttp://jn.physiology.org/cgi/content/full/102/2/735http://www.the-aps.org/publications/jnhttp://www.the-aps.org/http://www.the-aps.org/http://www.the-aps.org/http://www.the-aps.org/http://www.the-aps.org/publications/jnhttp://jn.physiology.org/cgi/content/full/102/2/735http://jn.physiology.org/cgi/content/full/102/2/735#BIBLhttp://jn.physiology.org/cgi/content/full/00083.2009/DC1


2/18

Postnatal Development of Dendritic Synaptic Integration in Rat Neocortical

Pyramidal Neurons

Susan E. Atkinson and Stephen R. Williams

Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

Submitted 27 January 2009; accepted in final form 12 May 2009

Atkinson SE, Williams SR. Postnatal development of dendritic synapticintegration in rat neocortical pyramidal neurons. J Neurophysiol 102:735751, 2009. First published May 20, 2009; doi:10.1152/jn.00083.2009.The dendritic tree of layer 5 (L5) pyramidal neurons spans theneocortical layers, allowing the integration of intra- and extracorticalsynaptic inputs. Here we investigate the postnatal development of theintegrative properties of rat L5 pyramidal neurons using simultaneouswhole cell recording from the soma and distal apical dendrite. Inyoung (P9-10) neurons, apical dendritic excitatory synaptic inputpowerfully drove action potential output by efficiently summating at

the axonal site of action potential generation. In contrast, in mature(P25-29) neurons, apical dendritic excitatory input provided littledirect depolarization at the site of action potential generation but wasintegrated locally in the apical dendritic tree leading to the generationof dendritic spikes. Consequently, over the first postnatal month thefraction of action potentials driven by apical dendritic spikes in-creased dramatically. This developmental remodeling of the integra-tive operations of L5 pyramidal neurons was controlled by a 10-foldincrease in the density of apical dendritic Hyperpolarization-activatedcyclic nucleotide (HCN)-gated channels found in cell-attachedpatches or by immunostaining for the HCN channel isoform HCN1.Thus an age-dependent increase in apical dendritic HCN channeldensity ensures that L5 pyramidal neurons develop from compacttemporal integrators to compartmentalized integrators of basal andapical dendritic synaptic input.

I N T R O D U C T I O N

Layer 5 pyramidal neurons are the output neurons of theneocortex and so represent the final site of neocortical process-ing. The dendritic tree of layer 5 pyramidal neurons spans allcortical layers with a prominent dendritic field in layer 1,referred to as the apical dendritic tuft and a basal dendritic fieldin layers 5 and 6. In addition, the ascending apical dendritictrunk of layer 5 pyramidal neurons gives rise to a number ofoblique dendrites that sample synaptic inputs in layers 24(Wise et al. 1979). This anatomical arrangement suggests thatlayer 5 pyramidal neurons integrate divergent intra- and extra-cortical streams of synaptic input to form an action potential

output. As action potentials are initiated in the axon of layer 5pyramidal neurons (Palmer and Stuart 2006), classical modelsof neuronal function suggest that synaptic input must befunneled to this site to influence neuronal output (Rall 1967).The dendritic tree of layer 5 pyramidal neurons, however,enlarges during postnatal development (Miller 1981; Wiseet al. 1979), suggesting that synaptic inputs may undergoincreased voltage attenuation as they spread from dendritic siteof generation to the axonal site of action potential initiation

(Berger et al. 2001; Nevian et al. 2007; Stuart and Spruston1998; Williams and Stuart 2002). Indeed, in mature layer 5pyramidal neurons, the amplitude of dendritic synaptic inputsgenerated at apical and basal dendritic sites are heavily atten-uated as they spread through the dendritic tree (Berger et al.2001; Nevian et al. 2007; Stuart and Spruston 1998; Williamsand Stuart 2002). This arises partly because of passive cablefiltering and partly because of the interaction of synapticpotentials with voltage-activated channels (Berger et al. 2001;

Nevian et al. 2007; Stuart and Spruston 1998; Williams andStuart 2002).

In mature cortical pyramidal neurons, the spread of synapticpotentials through the apical dendritic tree is powerfully con-trolled by hyperpolarization-activated cyclic nucleotide (HCN)-gated channels (Berger et al. 2001; Magee 1998, 1999; Stuartand Spruston 1998; Williams and Stuart 2000, 2003). In addi-tion, HCN channels control the resting membrane potential andapparent input resistance of layer 5 pyramidal neurons (Bergeret al. 2001; Spain et al. 1991; Williams and Stuart 2000). Inadult CA1 and neocortical layer 5 pyramidal neurons, electro-physiological and immunocytochemical approaches haveshown a highly compartmentalized subcellular distribution of

HCN channels with little or no surface expression at somatic,axonal, or basal dendritic sites but high expression at apicaldendritic sites, where HCN channel density progressively in-creases with distance from the soma (Berger et al. 2001;Brewster et al. 2007; Kole et al. 2006; Lorincz et al. 2002;Magee 1998; Williams and Stuart 2000).

Functionally, HCN channels have been shown to control thetime course of synaptic potentials in pyramidal neurons, both atdendritic sites of generation and following their spread to thesoma, by acting as a shunt conductance and through voltage-dependent activation and deactivation properties (Angelo et al.2007; Magee 1999; Nicoll et al. 1993; van Brederode andSpain 1995; Williams and Stuart 2000, 2003). Interestingly, theinteraction between synaptic potentials and HCN channels acts

to compensate for the distance-dependent effects of cablefiltering on the somatic time course of excitatory synapticpotentials in hippocampal CA1 and neocortical layer 5 pyra-midal neurons (Magee 1999; Williams and Stuart 2000). InCA1 pyramidal neurons, this interaction has been suggested tosimplify integrative operations by normalizing the temporal sum-mation of dendritic excitatory postsynaptic potentials (EPSPs) atthe soma (Magee 1999). This finding together with the siteindependence of the somatic amplitude of apical dendriticEPSPs, mediated by scaling of dendritic excitatory synapticconductance (Magee and Cook 2000; Nicholson et al. 2006),suggests that in CA1 pyramidal neurons excitatory synapticinputs generated across the apical dendritic tree will have a

Address for reprint requests and other correspondence: S. R. Williams,Neurobiology Div., MRC Laboratory of Molecular Biology, Hills Road,Cambridge CB2 0QH, UK (E-mail: [email protected]).

J Neurophysiol 102: 735751, 2009.First published May 20, 2009; doi:10.1152/jn.00083.2009.

7350022-3077/09 $8.00 Copyright 2009 The American Physiological Societywww.jn.org


3/18

similar impact on axonal action potential output (Magee 2000).In contrast, in layer 5 pyramidal neurons, the somatic ampli-tude of apical and basal dendritic EPSPs are not normalized butare determined by the site of generation in the dendritic tree(Nevian et al. 2007; Williams and Stuart 2002). EPSPs gener-ated at proximal apical dendritic sites therefore have a largersomatic amplitude than those generated distally in the dendritic

tree (Williams and Stuart 2002). The dendritic site dependenceof EPSP amplitude in layer 5 pyramidal neurons suggests thatthe functional role of HCN channels may not only be tonormalize the somatic time course of dendritically generatedsynaptic potentials. One alternative function may be electricalcompartmentalization.

In mature layer 5 pyramidal neurons, HCN channels ensurethat trains of distal apical dendritic EPSPs are heavily attenu-ated and do not summate as they spread from apical dendriticsites of generation to the soma (Berger et al. 2001; Kole et al.2007; Williams and Stuart 2000). High-frequency barrages ofdistal apical dendritic EPSPs therefore provide a weak directdrive for action potential initiation (Williams 2005). To have asalient signaling role in layer 5 neocortical pyramidal neurons,distal apical dendritic excitatory input must be locally inte-grated in the apical dendritic tree (Larkum and Zhu 2002;Williams 2004; Williams and Stuart 2002; Zhu 2000). Indeed,in mature layer 5 pyramidal neurons, single or barrages ofapical dendritic EPSPs evoke dendritic spikes that robustlyforward propagate through the dendritic tree to initiate actionpotential firing (Larkum and Zhu 2002; Williams 2004; Wil-liams and Stuart 2002; Zhu 2000). Mature layer 5 pyramidalneurons are therefore compartmentalized into axo-somatic anddistal apical dendritic integration compartments, where eachintegration compartment has unique properties (Larkum andZhu 2002; Williams and Stuart 2002). Such compartmentalizedsynaptic integration has been suggested to allow the indepen-

dent integration of synaptic input arriving at basal and apicaldendritic sites (Williams 2004). Here we investigate if thispattern of synaptic integration is a feature of layer 5 pyramidalneurons and so present throughout the development of theneocortical network or if compartmentalized integration devel-ops in response to the postnatal maturation of the neocorticalcircuit. Interestingly, a previous study has shown that earlypostnatal pyramidal neurons are electrotonically compact andbecome increasingly electrically distributed with postnatal age(Zhu 2000). We have therefore investigated the postnataldevelopment of the somatic impact of single and barrages ofsimulated excitatory synaptic input delivered to the soma ordistal apical dendritic sites of layer 5 pyramidal neurons andtheir role in the control of action potential firing. We find an

age-dependent switch in the integrative operations of pyrami-dal neurons, from one analogous to a single-compartmenttemporal integrator early in postnatal development to a highlycompartmentalized integrator after the first postnatal month.This transformation of the integrative operations of layer 5pyramidal neurons was underpinned by a dramatic age-depen-dent increase in the density of apical dendritic HCN channels.

M E T H O D S

Brain-slice preparation

Coronal neocortical brain-slices from male Wistar rats (Postnataldays 9 42) were prepared following Institutional and UK. Home

Office guidelines. Brain slices were cut in ice-cold solution of com-position (in mM) 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1CaCl2, 6 MgCl2, 3 Na pyruvate, and 25 glucose bubbled with 95%O2-5% CO2 at a thickness of 300 m for electrophysiological or 60m for immunostaining experiments. For electrophysiological exper-iments, brain-slices were stored in this media for 30 min at 3435Cand then at room temperature.

Electrophysiological recording

Brain-slices were transferred to a recording chamber perfused witha solution of composition (mM) 125 NaCl, 25 NaHCO 3, 3 KCl, 1.25NaH2PO4, 2 CaCl2, 1 MgCl2, 3 Na pyruvate, and 25 glucose at 35 to37C bubbled with 95% O2-5% CO2. The postnatal development ofthe electrophysiological properties of visually identified layer 5Bpyramidal neurons was investigated using simultaneous whole cellcurrent-clamp recordings from the soma and apical dendrite. For eachneuron, the apical dendritic recording pipette was positioned as closeas possible to the nexus of the apical dendritic trunk [58 5 (SE) mfrom the layer 1layer 2 border; n 57]. Whole cell recordingpipettes were filled with (mM) 135 K-gluconate, 7 NaCl, 10 HEPES,2 Na2-ATP, 0.3 Na-GTP, 2 MgCl2, and 0.01 Alexa Fluor 568(Molecular Probes, Eugene, OR; pH 7.37.4; KOH). Somatic record-ing pipettes had a resistance of between 3 and 6 M and pipettes forapical dendritic recordings were between 10 and 12 M. Voltageresponses were recorded using identical current-clamp amplifiers(BVC-700A, Dagan, MN). Voltage recordings were corrected for anexperimentally determined 11-mV liquid junction potential. In allcurrent-clamp experiments, antagonists of excitatory and inhibitoryamino acid receptors were included in the extracellular media[6-cyano-7-nitroquinoxaline-2,3-dione (10 M); 6-imino-3-(4-me-thoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (10 M);D-()-2-amino-5-phosphonopentanoic acid (50 M)]. In some exper-iments, 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidiniumchloride (ZD 7288; 20 M) was added to the perfusion media.Tetrodotoxin (TTX; 1 M) was dissolved in extracellular solution andlocally applied to the region of the apical dendrite by pressure appli-cation (100200 mmHg, 2 s) from a pipette with similar characteris-

tics to those used for dendritic recordings, positioned 2030 m fromthe apical dendritic recording site. At the termination of each wholecell recording, the location of recording pipettes and neuronal mor-phology was examined by fluorescence microscopy and digitallyrecorded (Retiga EXI, QImaging, Burnaby, BC, Canada).

Single excitatory postsynaptic currents (EPSCs) were generated asan ideal current source (peak amplitude: 50100 pA) as the sum oftwo exponential processes with rise and decay time constants of between0.2 and 3 ms and 2 and 30 ms, respectively. Barrages of simulated EPSCswere generated as trains of randomly occurring unitary inputs (peakamplitude, 50500 pA) with rise and decay time constants of 0.2 and 2ms, respectively, generated at mean frequencies of between 50 and 500Hz (Williams 2005). Current and voltage signals were low-pass filtered(DC to 10 kHz) and acquired at 3050 kHz. Data were acquired andanalyzed using AxographX software (AxographX, Sydney, Australia).

Cell-attached patch-clamp recordings were made from either prox-imal (112.3 3.6 m from the soma; n 84 patches) or distal (45 3 m from the layer 1 layer 2 border; n 191 patches) apicaldendritic sites using pipettes of similar resistance (1012 M) filledwith (in mM) 120 KCl, 20 tetraethyammonium, 10 HEPES, 5 4-ami-nopyridine, 3 BaCl, 2 CaCl, 1 MgCl, 1 NiCl, 0.5 CdCl, and 0.001tetrodotoxin (pH 7.4; KOH). Ensemble HCN channel activity wasevoked by the delivery of positive voltage steps from an intra-pipettepotential set to 50 mV using an Axopatch 200B amplifier (Molec-ular Devices, Union City, CA). To map channel density, ensembleHCN currents were generated in response to a 100-mV test step,interleaved with a 20-mV step used for off-line leak subtraction, anapproach previously used to map the subcellular distribution of HCNchannels (Berger et al. 2001; Kole et al. 2006; Williams and Stuart

736 S. E. ATKINSON AND S. R. WILLIAMS

J Neurophysiol VOL 102 AUGUST 2009 www.jn.org


4/18

2000). Current-voltage relationship were constructed by the deliveryof an incremental series of positive voltage steps (40110 mV),interleaved with 10-mV steps for off-line leak subtraction. Aftergathering ensemble HCN channel data, a whole cell recording wasobtained from the neuron, usually at the same apical dendritic site oroccasional at the soma, and the neuron was filled with Alexa Fluor568 to ensure channel data were obtained from the apical dendrite of

a layer 5B pyramidal neuron. In no instance was a recording madefrom the neurite of another neuronal class. In some experiments,simultaneous whole cell and cell-attached recordings were made fromclosely spaced (10 m) apical dendritic sites. In these experiments,the local apical dendritic membrane potential was controlled by theinjection of DC current.

Immunostaining

Brain-slices prepared in an identical manner to those used forelectrophysiological experiments were fixed in 4% paraformaldehydeimmediately after preparation to investigate the subcellular distribu-tion of HCN channel isoforms in preparations that allowed directcomparison with the results of electrophysiological experiments. Fol-

lowing fixation, brain-slices were blocked and permeabalized (1%donkey serum/0.2% Triton) and incubated in 10% dilution of blockingsolution with either Anti-HCN1 N-terminus (host: rabbit; 1:200,Alomone Labs), Anti-HCN1 C-terminus (host: rabbit; 1:200, Abcam,Cambridge, UK), or Anti-HCN2 (host: guinea pig; final concentration4 g/ml) (Notomi and Shigemoto 2004) for 36 h at 4C. Brain-sliceswere then incubated with Alexa Fluor 488 (1:1,000) for 2 h at roomtemperature and mounted using Vectorshield mounting medium (Vec-tor, Burlingame, CA). C- and N-terminus HCN1 antibodies gavesimilar results, and presented data were gathered using the N-terminusHCN1 antibody.

Images of HCN immunostaining of layer 5 pyramidal neurons wereobtained using a laser confocal scanning system (Radiance Plus,Bio-Rad) equipped with a 40 oil-immersion objective lens. Scan-ning parameters including laser power [2.4% of maximal output (5

mW)], iris area (0.32 mm2), gain (30%), and offset (0) were keptconstant, and confocal stacks of 1 m optical sections were taken foreach neuron (Kalman 8, resolution 1,024 pixels2). Eleven opticalsections containing the selected neuron were summed to form a Zprojection. To image the entire apical dendritic tree at 40 magnifi-cation, overlapping areas of the neuron were scanned and landmarkscommon to each adjacent image used to form a complete montage.Scanned images were visualized using a 12-band color LUT to ensurethat the full range of fluorescence intensity was detected. Z projectionswere transformed into grey scale images and regions of interest(ROIs) centered on the soma or apical dendrite (46 m along the axisof the dendrite, 3,512 145 pixels) were selected from the neuronand neighboring background areas (Fig. 6A). Image analysis wasperformed using ImageJ (National Institutes of Health) and numericaldata analyzed using AxographX. A measure of HCN immunostaining

was calculated by subtracting the median pixel intensity in back-ground regions from the median pixel intensity in dendritic regions ofinterest (Fig. 6, A and B). ROIs throughout the apical dendritic treewere selected to detect any regional changes in HCN immunostaining.To reduce variability caused by changes in scanning conditions, suchas changes in slice thickness, neuronal and corresponding backgroundROIs were in close proximity to each other, approximately the samerectangular shape and in the same orientation (Fig. 6, A and B). Tobuild montages, overlapping areas of each high-resolution scan wereimaged twice to allow correct alignment. To check whether fluores-cence in these areas had been reduced, selected ROIs were exposed toa series of four scan sequences (Kalman of 8) and the average pixelintensity after each sequence calculated. No significant reduction inaverage pixel intensity was found.

Statistical analysis

Pooled data are presented as means SE. Statistical analysisincluded ANOVA. Students t-test and Wilcoxon matched pairs test.All curve fitting was performed using AxographX. The evolution ofthe activation of ensemble HCN channel activity was fit with asingle-exponential function. The voltage-dependent activation of en-semble HCN channel activity was fit with a single Boltzmann function

of the form: y 1/[I e(V

1/2 V

/k

)], where V1/2 is the voltage ofhalf-maximal activation and k is a constant. The average amplitude ofbarrages of simulated EPSCs and evoked voltage responses wascalculated by measuring the mean amplitude throughout the timecourse of the injected simulated synaptic current.

R E S U L T S

Developmental control of the somatic efficacy of dendriticsynaptic input

The majority of synaptic inputs to layer 5 pyramidal neuronsare formed at dendritic sites. To directly influence neuronaloutput, synaptic potentials must spread from their dendritic siteof generation to the site of action potential initiation in the axon(Rall 1967). As the spread of synaptic potentials is controlledby the passive and active properties of the dendritic tree, wefirst investigated how the spread of synaptic potentials throughlayer 5 pyramidal neurons changes during postnatal develop-ment. Simulated EPSPs (sEPSPs) were generated at somatic ordistal apical dendritic sites (58 5 m from the layer 1layer2 border; n 57) by the injection of a series of EPSC-shapedcurrent signals and recorded simultaneously at their site of genera-tion and following spread through the dendritic tree (rise 0.23 ms; decay 230 ms, peak current 100 pA; Fig. 1, Aand B). Targeted dendritic recording allowed us to generateand record sEPSPs at equally remote apical dendritic sitesacross postnatal development (Fig. 1G, E, average distance

from the layer 1layer 2 border was not different between agegroups; ANOVA; P 0.71). In early postnatal layer 5 pyra-midal neurons, sEPSPs were of large amplitude with slowkinetics both at their dendritic site of generation and followingspread to the soma (P9; Fig. 1A). In older animals, however,dendritic sEPSPs generated in response to identical EPSCswere of smaller amplitude with faster kinetics and providedmodest excitation of the soma (P29; Fig. 1B). Indeed, pooleddata showed a steep age-dependent decrease in the somaticamplitude of dendritically generated sEPSPs (Fig. 1, Cand D),a relationship that was apparent for sEPSPs generated across awide range of EPSC kinetics (Fig. 1, AD). Furthermore, thespread of sEPSPs from the soma back into the apical dendritictree was developmentally regulated with a similar age depen-

dence (Fig. 1D). The developmental decrease in the somaticamplitude of dendritic sEPSPs was accompanied by an age-dependent speeding of sEPSPs kinetics, both at dendritic site ofgeneration and following the spread of sEPSPs to the soma(Fig. 1, Eand F). Taken together, these data indicate that singleexcitatory synaptic inputs generated at remote apical dendriticsites powerfully excite the soma of early postnatal layer 5pyramidal neurons but have a progressively weaker somaticimpact as animals mature.

Modeling and experimental studies have indicated that in apassive neuron, the somatic time course of EPSPs is deter-mined by their site of generation within the dendritic tree,increasing as synapses are activated more remotely (Rall 1967;

737DEVELOPMENT OF DENDRITIC INTEGRATION



5/18




6/18

Williams and Stuart 2002). One consequence of this behavioris that trains of EPSPs generated at relatively distal dendriticsites are predicted to show greater temporal summation at thesoma (Magee 1999; Rall 1967; Williams and Stuart 2000). Inmature cortical pyramidal neurons, however, the somatic timecourse of EPSPs generated at the soma and in the apicaldendritic tree are similar, a process described as time-course

normalization (Magee 1999; Williams and Stuart 2000). Thisbehavior cannot arise in a passive system but is shaped by theinteraction of EPSPs with dendritic voltage-activated conduc-tances, in particular HCN channels (Magee 1999; Williams andStuart 2000). We therefore investigated if this relationship heldthroughout postnatal development, by comparing the somatictime course of sEPSPs generated at the soma with thosegenerated distally in the apical dendritic tree (Fig. 2). In earlypostnatal pyramidal neurons, the somatic time course of den-dritically generated sEPSPs was not normalized, but ratherdendritic synaptic inputs produced somatic sEPSPs that wereslower in time course than those generated at the soma (Fig.2A). Importantly, the disparity between the somatic time courseof somatically and dendritically generated sEPSPs was dependenton EPSC kinetics and was greatest for fast EPSCs (decay 2 ms; somatic half-width: soma EPSC: 18.3 1.2 ms; den-dritic EPSC: 26.2 1.6 ms; paired t-test, P 0.01; n 15)but near unity for slow EPSCs (decay 30 ms; somatichalf-width: soma EPSC: 69.2 4.2 ms; dendritic EPSC:67.5 4.2 ms; paired t-test, P 0.24; n 15; Fig. 2B). Thisrelationship was refined during postnatal development so thatin mature pyramidal neurons, the somatic time course of fastbut not slow dendritic EPSPs were similar (P 2629; fastEPSC: somatic half-width: soma EPSC: 10.9 0.4 ms; den-dritic EPSC: 10.8 0.4 ms; paired t-test, P 0.81; slowEPSC: somatic half-width: soma EPSC: 39.2 0.6 ms; den-dritic EPSC: 27.8 1.0 ms; paired t-test, P 0.001; n 12;

Fig. 2B). Indeed, across the first postnatal month, the kineticsof EPSCs that produced sEPSPs that showed time-coursenormalization accelerated from 30 to 2 ms (Fig. 2B). Thesomatic time course of fast sEPSPs that accurately replicate thetime course of spontaneously occurring EPSPs in layer 5 neu-rons (Hausser and Roth 1997; Williams and Stuart 2002) istherefore only normalized in layer 5 pyramidal neurons older than1 month of age. As a consequence, in these mature neurons, thesomatic time course of sEPSPs generated with slow EPSC kinet-ics were heavily constrained and significantly briefer than thosegenerated at the soma (groups significantly different for dendriticEPSCs withdecay of 5, 10, and 30ms; paired t-test, P 0.01; n12; Fig. 2B). These data suggest that the properties and subcellulardistribution of the voltage-activated conductances that control the

time course of dendritic EPSPs are regulated during postnatal

development. Furthermore at a functional level, these data suggestthat trains of dendritic EPSPs in early postnatal layer 5 pyramidalneurons are more likely to show temporal summation at the somathan those in mature neurons.

To directly test this idea, we generated random barrages ofsimulated EPSCs across a wide range of frequencies (fre-quency 50 500 Hz, duration 2 s; unitary amplitude 50

or 100 pA; rise 0.2 ms; decay 2 ms) and injected this barrageof excitatory input either at somatic or apical dendritic record-ing sites. This procedure allowed us to directly record howeffectively barrages of dendritic excitatory synaptic input sum-mate at the soma. In early postnatal pyramidal neurons, bar-rages of dendritic sEPSPs effectively depolarized the soma,with higher frequency barrages producing progressivelygreater somatic depolarization (Fig. 3A). When we comparedthe average somatic depolarization produced by identical bar-rages of synaptic input delivered to the soma or remotely in thedendritic tree, we found that in early postnatal neurons den-dritic input produced voltage responses that were comparablein magnitude to those evoked at the soma (P 910: dendriticresponse 20.3 4.6% less than somatic; n 6; Fig. 3A).Across the first postnatal month, however, the ability of den-dritic excitatory input to depolarize the soma decreased dra-matically (Fig. 3B) so that at P26-29, dendritic excitatory inputonly weakly excited the soma (Fig. 3B). Indeed, in olderanimals, dendritic excitatory input did not summate at the somaand generated only a shallow relationship between somaticdepolarization and injected dendritic synaptic current (2.9 0.7 mV/nA; n 8; Fig. 3B). This is in stark contrast to thesteep relationship generated when synaptic input was deliveredto the soma in older animals (28.9 3.2 mV/nA; n 8; Fig.3B). During the first postnatal month, therefore barrages ofapical dendritic excitatory synaptic input become progressivelyweaker sources of somatic excitation. To quantify this, we com-

pared the slope of the relationship between somatic depolarizationand injected somatic or apical dendritic synaptic current forneurons recorded in six age groups (Fig. 4A). The subthresholdefficacy of dendritic excitatory input decreased with postnatal ageaccording to an exponential relationship with an age constant of 5days (Fig. 4A). This refinement of the electrophysiological prop-erties of layer 5 pyramidal neurons was accompanied by anincrease in the degree of attenuation of voltage signals as theyspread from the soma to the apical dendrite. A dramatic age-dependent increase in somato-dendritic voltage attenuation wasapparent for both single EPSPs (Fig. 1D) and for steady-statevoltage responses (Fig. 4,B and C). Indeed when steps of negativecurrent were injected at the soma to evoke hyperpolarizing re-sponses of 19.9 0.1 mV (n 40), the degree of somato-

dendritic voltage attenuation was found to increase during post-

FIG. 1. Developmental decrease in the somatic impact of apical dendritic excitatory postsynaptic potentials (EPSPs). A and B: simultaneous somatic (Vsoma)and dendritic (Vdend) recording of simulated EPSPs in a postnatal day 9 (P9, A) and P29 (B) layer 5 pyramidal neurons. Simulated EPSPs were generated by theinjection of a series of simulated excitatory postsynaptic currents (EPSCs) at the distal apical dendritic recording site (Idend; bottom traces). The amplitudeand kinetics of simulated EPSPs are transformed with development. Neuronal morphology and the placement of recording electrodes are shown in theinset photomicrographs (scale bars 100 m). C: age-dependent reduction in the somatic amplitude of distal dendritically generated EPSPs. Overlaintraces show simulated EPSPs recorded at the indicated postnatal ages. Fast and slow EPSCs had rise of 0.2 and 3 ms and decay of 2 and 30 ms,respectively. D: pooled data show the age-dependent decrease in the amplitude of EPSPs, generated with fast and slow EPSC kinetics, when generatedat the soma and recorded at an apical dendritic site (E) or vice versa (F). Data have been fit with single exponential functions (). Eand F: developmentalspeeding of the kinetics of simulated EPSPs generated by fast or slow EPSCs. Simulated EPSPs were recorded at their site of generation ( E) and followingspread to the soma (F). Overlain gray symbols show results pooled into 6 age groups (means SE). Grouped data have been fit with single-exponentialfunctions (). G: position of recording electrodes. F, the distance of the apical dendritic recording electrode from the soma of layer 5 pyramidal neurons;E, the distance of the dendritic recording electrode from the layer 1layer 2 border. Grouped data have been fit with a single-exponential function (layer5) or linear regression (layer 1).




7/18

natal development (somatic voltage responses were of similaramplitude in each age group; ANOVA; P 0.05; Fig. 4, B andC). Interestingly this effect was accompanied by an age-dependentdecrease in the somatic apparent input resistance of layer 5pyramidal neurons (Fig. 4C). Taken together, these data show thatthe electrotonic architecture of layer 5 pyramidal neurons issignificantly remodeled over the first postnatal month.

The electrotonic architecture of mature cortical pyramidalneurons is critically controlled by the subcellular distribu-tion and density of voltage-activated channels (Migliore and

Shepherd 2005). Previous observations have indicated inmature layer 5 pyramidal neurons that HCN channels play a

major role in controlling the attenuation of synaptic poten-tials as they spread through the apical dendritic tree (Bergeret al. 2001; Stuart and Spruston 1998; Williams and Stuart2000, 2003). To highlight the constraint of the somaticimpact of dendritic excitatory inputs by HCN channels inmature pyramidal neurons, we explored the influence ofblocking HCN channels on the somatic efficacy of dendriticexcitatory synaptic input (Fig. 5A). The somatic depolariza-tion produced by barrages of distal apical dendritic sEPSPswas transformed by the pharmacological block of HCN

channels with ZD 7288 (P26-29; dendritic recordings 84

17 m from the layer 1layer 2 border; n 6; Fig. 5A).

FIG. 2. Postnatal development of EPSP time course normalization. A: overlain somatic recording of simulated EPSPs (sEPSPs) evoked by the injection offast EPSCs (rise 0.2 ms, decay 2 ms) at somatic (black traces) or distal apical dendritic (gray traces) sites in a P10 and a P29 layer 5 pyramidal neuron.In each case, the dendritic EPSC generates smaller-amplitude EPSPs at the soma. The marked gray traces represent the digital scaling of the dendriticallygenerated sEPSP. Note in the P10 neuron that the time course (time at half-amplitude) of the scaled dendritic sEPSPs is longer than the somatically generated

sEPSP. B: developmental refinement of time-course normalization. Relationship between the somatic half-width (time at half-amplitude) of sEPSPs generatedat dendritic and somatic sites with EPSCs of different kinetics (deacy time constant) in the indicated age groups. Note that simulated EPSPs with similar somatictime course are generated by EPSCs with faster kinetics in older neurons. Values represent means SE; the horizontal reference line indicates a ratio of 1.

FIG. 3. Developmental reduction of EPSP temporal summation. A and B: somatic voltage responses evoked by the injection of identical patterns of simulatedEPSCs (bottom traces) at somatic and apical dendritic sites. Note in the P10 layer 5 pyramidal neuron (A) that the somatic voltage response evoked by EPSCsinjected at somatic and dendritic sites are similar in amplitude. In contrast, in the P27 neuron (B) dendritic EPSCs only weakly depolarized the soma. Barragesof simulated EPSCs of different frequency and the corresponding voltage responses are delineated by gray scale for clarity. Inset: photomicrographs show themorphology of the illustrated neurons (scale bars 100 m). Summary graphs show the relationship between the magnitude of injected simulated synapticcurrent and evoked somatic voltage responses, when identical barrages of simulated EPSCs were generated at somatic (E) or apical dendritic sites (F). Data hasbeen pooled (means SE) for neurons from the indicated age groups. Values of injected current and evoked voltage responses represent the average amplitudethroughout the barrage. Note the similar slope of these relationships in P9-10 neurons ( n 6), but the weak somatic impact of dendritic input in P26-29 neurons(n 8). Relationships were fit for subthreshold voltage responses by linear regression (lines).




8/18

Indeed when HCN channels were blocked, the slope of therelationship between somatic depolarization and dendriticcurrent injection was similar to that obtained under controlwhen sEPSPs were injected at the level of the soma (den-dritic: control: 3.2 0.7 mV/nA; ZD 7288: 24.9 3.4mV/nA; n 6; soma: control: 28.9 3.2 mV/nA; n 8;compare Fig. 5B with Fig. 3B). These values were calculatedfrom similar somatic and dendritic membrane potentials,following the delivery of positive DC current through re-cordings electrodes to offset the membrane potential hyper-polarization produced by the blockade of HCN channels (Wil-

liams and Stuart 2000, 2003). HCN channels therefore powerfullycontrol the subthreshold somatic efficacy of dendritic excitation inmature layer 5 pyramidal neurons. This finding prompted us toinvestigate how the density and sub-cellular distribution of thisimportant class of voltage-activated ion channel was refined dur-ing postnatal development.

Development of HCN channel expression and targeting

We examined the postnatal refinement of HCN channelexpression in layer 5 pyramidal neurons using a combination of

FIG. 4. Developmental refinement of the electrotonic properties of layer 5 pyramidal neurons. A: subthreshold dendritic efficacy decreases with postnatal age.Subthreshold dendritic efficacy was calculated as the fraction of somatic depolarization produced by identical barrages of synaptic inputs generated at somaticand distal apical dendritic sites across a broad range of injected synaptic current (Fig. 3). Data have been pooled for the indicated age groups (means SE)., a fit to grouped data by a single-exponential function. B: simultaneous somato-dendritic recording of voltage responses evoked by the injection of a currentstep at the soma in P10 and P27 layer 5 pyramidal neurons. Note the minimal somato-dendritic voltage attenuation at the negative peak ( E) and at steady state(F) in the P10 neuron. In contrast in the P27 neuron, dramatic somato-dendritic voltage attenuation is evident. In each neuron, a similar amplitude somatic voltage

response was generated in response to a step of negative current, the smaller current step was injected in the P10 neuron ( bottom traces). C: summary datadescribing the age-dependent increase in somato-dendritic voltage attenuation when measured at peak and steady state. The grey symbols show the age-dependentincrease in apparent input conductance. Data are pooled for 6 age groups (means SE). Lines represent fits with single-exponential functions.

FIG. 5. Blockade of HCN channels in-creases the somatic impact of dendritic excita-tory input. A: somatic voltage traces (Vsoma)evoked by barrages of simulated EPSCs injected at a distal apical dendritic site (Idend)under control and in the presence of ZD 7288(20 M). Traces are gray-scaled for clarity.The morphology of the neuron is shown in the

inset(scale bar 100 m). B: summary data(P26-29; n 6; means SE) describing thesomatic depolarization generated by dendriticsimulated synaptic current under control (E)and in the presence of ZD 7288 (F). Values ofinjected current and evoked voltage responsesrepresent the average amplitude throughout thebarrage. Data were fit by linear regression ().




9/18

immunostaining of HCN channel isoforms and cell-attachedpatch-clamp recording of functional ensemble HCN channelactivity. In confirmation of a previous study (Lorincz et al.2002), we observed in adult animals (P42) a striking apicaldendritic distribution of HCN channels in confocal montages

of layer 5 neurons resolved by anti-HCN1/Alexa 488 immu-nostaining. We calculated the optical density of HCN1 immu-nostaining in regions of interest placed at apical dendritic sitesprogressively distal from the soma of individual layer 5 pyra-midal neurons (Fig. 6A).




10/18

The subtraction of autofluorescence from adjacent back-ground areas revealed an increase in HCN1 channel density atprogressively distal apical dendritic sites (Fig. 6, A and B).Next we charted the postnatal development of apical dendriticHCN1 channel expression. At early postnatal ages (P10), weobserved diffuse and low levels of HCN1 expression with nosignificant apical dendritic targeting (n 5 neurons; Supple-

mentary Fig. S11). A prominent apical dendritic gradient ofHCN1 channel expression was first apparent around P14-16(Fig. 6, C and D). At this age background-subtracted averagepixel intensity from dendritic regions of interest showed adistance-dependent increase in the level of HCN1 immuno-staining at progressively distal apical dendritic sites (P14-16:proximal ROI: 125.1 6.8 m from the soma; 6.8 1.1 AU;distal ROI: 942.0 19.7 m from the soma; 19.7 3.0 AU;ANOVA P 0.001; n 10). As animals matured, the apicaldendritic polarization of HCN1 immunostaining was refined, aprocess characterized by an age-dependent increase in thedensity of channels at distal but not proximal apical dendriticsites (P42: proximal: 143.91 7.6 m from the soma; 13.2 1.7 AU; distal: 989.0 42.0 m from the soma; 59.7 7.6AU; n 9 neurons; Fig. 6, C and D). When we plotted theintensity of HCN1 immunostaining at apical dendritic sites asa function of postnatal age, we observed a steep age-dependentincrease in density at distal but not proximal sites (Fig. 6E). Atdistal apical dendritic sites, this age-dependent relationshipwas well described by a bounded exponential function with anage constant of 15 days (Fig. 6E).

As the HCN1 antibodies we used target intracellular sites ofthe channel, and so required tissue permeabilization, we ex-plored if HCN1 immunostaining was located at or close to theplasma membrane or at intracellular sites. In single confocalsections (1 m), the optical density along line profiles drawnthrough the soma and apical dendrites of HCN1 immuno-

stained layer 5 pyramidal neurons was plotted (SupplementaryFig. S2). For comparison, we filled layer 5 pyramidal neuronswith the fluorescent dye Alexa 568, following somatic wholecell recording, a dye that does not partition into the neuronalmembrane (Supplementary Fig. S2). At distal apical dendriticsites of P 42 layer 5 pyramidal neurons, HCN1 immunostain-ing was restricted to sites close to the plasma membrane, asshown by the clear peaks in the line profiles (500 m fromthe soma, n 6; Supplementary Fig. S2A, left). This lineprofile was in contrast to the unimodal intracellular peak in opticaldensity observed at distal apical dendritic sites in Alexa 568 filledneurons (Supplementary Fig. S2A, right). In somatic regions,however, line profiles revealed that HCN1 immunostaining wasboth intracellular and membrane-bound, judged by the similar, but

broader, line profiles obtained for Alexa-568-filled and HCN1-

stained neurons (n 5; Supplementary Fig. S2B). This intracel-lular accumulation of HCN1 immunostaining rapidly dissipated atproximal apical dendritic sites (Fig. 6, A and C). These datasuggest that HCN1 immunostaining at apical dendritic sites re-ports the surface density of HCN channels.

Electrophysiological techniques were used to independentlymap the postnatal development of the subcellular distributionof functional HCN channels. In P11-12 animals, the amplitudeof ensemble HCN channel activity in cell-attached patchesfrom distal apical dendritic sites was small (current amplitudeat steady state 10.3 1.9 pA; distance from layer 1layer2 border: 79 10 m; n 11; Fig. 7, A and B). Thesteady-state amplitude of ensemble HCN channel activity inpatches made at distal apical dendritic sites, however, in-creased with postnatal age (P36-40: 121 10 pA; distancefrom layer 1layer 2 border: 49 8 m; n 12; Fig. 7, A andB). Indeed the age-dependent increase in the density of ensem-ble HCN channel activity evoked in response to a standard100-mV test step could be well fit by a single boundedexponential function with an age-constant of 15.7 days (P11 to

P40; 45 3 m from the layer 1layer 2 border; n 191; Fig.7B). In contrast, we found that the amplitude of ensemble HCNchannel activity in patches made from proximal apical den-dritic recordings sites remained at low levels throughout thisperiod of postnatal development (P12P40; 112 4 m fromlayer 5; n 84; linear regression: 0.006 pA/day; Fig. 7B).

Although we found a close parallel between the age-depen-dent increase in the density of functional HCN channels andimmunostaining for the HCN1 channel isoform at distal apicaldendritic sites, the activation properties of functional HCNchannels were found to change during postnatal development(Fig. 7, A and C). In response to a 100-mV voltage step, theactivation kinetics of ensemble HCN channel activity acceler-ated with postnatal development (Fig. 7A). Indeed when weplotted the relationship between postnatal age and the activa-tion kinetics of ensemble channel activity, we observed anage-dependent speeding of current responses that could beapproximated by an exponential function with an age-constantof 18.6 days (Fig. 7C).

In expression systems, the activation kinetics of ensemblehomomeric HCN channel activity is determined by channelisoform, with HCN1 channels exhibiting fast and HCN2 chan-nels relatively slow activation kinetics (Ludwig et al. 1998;Santoro and Tibbs 1999; Santoro et al. 1998). In the neocortex,high HCN1 and HCN2 mRNA and protein levels have beendetected (Kole et al. 2007; Notomi and Shigemoto 2004;Santoro et al. 2000). The developmental speeding of the

activation kinetics of ensemble HCN channel activity in layer5 pyramidal neurons may therefore reflect a developmentalchange in the subunit composition of HCN channels. To1 The online version of this article contains supplemental data.

FIG. 6. Developmental increase in the density of apical dendritic HCN1 channels in layer 5 pyramidal neurons. A: the density of HCN1 immunostaining (darkreaction) increases at distal apical dendritic sites in P42 animals. The montage is formed from a series of high-resolution confocal stacks. Regions of interest(ROIs) are highlighted by white squares. Two ROIs from proximal and distal apical dendritic sites are magnified and pixel intensities calculated from dendritic(white border) and background areas (black border). B: intensity histograms calculated from background and dendritic (gray) ROIs. In this P42 neuron, there isclear separation between intensity measured from background and distal apical dendritic sites (2 and 3). In contrast, at a proximal apical dendritic site (1), a largeoverlap of fluorescent intensity between background and dendrite is apparent. C: confocal montage illustrating HCN1 immunostaining of P26 and P14 layer 5pyramidal neurons. D: summary graphs describe the increase in apical dendritic HCN1 optical density with distance from the soma. Values were calculated bysubtracting pixel intensity of background area from dendritic regions of interests. Data in the 3 indicated age groups have been pooled from the indicated numberof neurons (means SE). Lines represent fits to the data with single bounded exponentials. E: summary data describing the age-dependent increase in the densityof HCN1 immunostaining at distal apical dendritic sites (black symbols). In contrast, at proximal apical dendritic sites HCN1 immunostaining does not increasewith age (gray symbols). Lines represent fits to the data with single bounded exponential function (distal) or linear regression (proximal).




11/18

explore this in more detail, we investigated the voltage-depen-dent properties of ensemble HCN channel activity in twodevelopmental epochs: young: P13-16 (n 22) and mature:P25-40 (n 25). As cell-attached recordings do not control theneurons membrane potential, we first measured the distalapical dendritic resting membrane potential in young andmature neurons, a value that will be influenced by the age-dependent increase in HCN channel density. Dendritic wholecell recording revealed that the local distal apical dendriticmembrane potential of young layer 5 pyramidal neurons wassignificantly hyperpolarized compared with mature neurons(P13P16: 71.3 0.8 mV; n 23; P25P30: 66.2 0.8

mV; n 17; t-test; P 0.001). Tail current analysis revealeda negative shift in the voltage at half-maximal activation ofensemble HCN channel activity between young and maturegroups when activation curves were fit with a single Boltzmannfunction (young: V1/296.3 mV; slope 11.2; old: V1/287.8mV; slope 10.6; Fig. 8, A and B). Furthermore, when we fitthe activation kinetics of ensemble channel activity generatedacross the current-voltage relationship, we found a significantspeeding of activation across a wide voltage range (Fig. 8C).The observed difference in apical dendritic resting membranepotential is unlikely to explain the developmental speeding ofensemble HCN channel activation kinetics. The delivery ofpositive voltage steps in a cell-attached recording from a rel-

atively hyperpolarized membrane potential would be expectedto lead to the activation of faster ensemble current responses,as the activation kinetics of ensemble HCN channels acceleratewith membrane hyperpolarization (Fig. 8, A and C). To con-firm this intuition, we made simultaneous whole cell current-clamp and cell-attached voltage-clamp recordings from closelyspaced (10 m) sites in the distal apical dendrite of layer 5pyramidal neurons (P25-29; 39 12 m from the layer 1layer2 border; n 11). The activation time constant of ensembleHCN channel activity generated in response to a 100-mVtest step was accelerated by 23.3 8.3% following a 9.5 0.7 mV tonic hyperpolarization of the local dendritic mem-

brane (membrane potential hyperpolarized from 64.2 0.5 to 74.0 0.8 mV by the injection of0.65 0.05 nADC current, data not shown). Similarly, membrane hyper-polarization led to an increase in the steady-state amplitudeof current responses (23.9 3.1%; data not shown). Thesedata suggest that differences in the resting membrane po-tential of layer 5 pyramidal neurons cannot explain theage-dependent changes in the activation properties of en-semble HCN channel activity.

We therefore explored if this behavior arose because of achange in the subunit composition of HCN channels. Immu-nostaining for HCN2 channels in both young (P13-14) andmature (P25) slices revealed somatic labeling throughout neo-

FIG. 7. Developmental increase in thedensity of functional HCN channels at distalapical dendritic sites. A: cell-attached re-cording of leak-subtracted ensemble HCNchannel activity evoked by a 100-mV volt-age step (bottom trace). Note the increase in

current amplitude and acceleration of activa-tion kinetics with development [postnatalage is indicated, apical dendritic recordingswere made at 55 (P11), 70 (P15); 15 (P26)and 30 m (P36) from the layer 1layer 2border]. Leak subtraction was performedoff-line by the subtraction of scaled re-sponses generated by interleaved 20-mVvoltage steps. B: summary data describingthe age-dependent increase in HCN channeldensity with age at distal but not proximalapical dendritic sites. Each data point isrepresented by the smaller symbols (distaldendritic, gray; proximal dendritic, open).The number of recorded patches is indicated.Pooled data are represented by the largesymbols (distal, black; proximal, gray;means SE). Pooled data have been fit by abounded exponential function for distal andlinear regression for proximal apical den-dritic recordings. C: pooled data describingthe acceleration of ensemble HCN channelkinetics with postnatal development. Eachgray data point represents the kinetics ofensemble activity generated in an apical den-dritic patch, approximated by a single-expo-nential fit to the evolution of the current, inresponse to a 100-mV voltage step. Largeblack symbols show results pooled into agegroups (means SE). Pooled data havebeen fit with an exponential function (line).




12/18

cortical layers (Fig. 8, D and E). However, we did not observedendritic immunostaining in either young or mature groups,despite the clear immunostaining of the apical dendritic tree oflayer 5 pyramidal neurons by HCN1 antibodies in adjacentsections (Fig. 8E). Immunostaining results therefore do notsupport a prominent alteration in the subunit composition ofHCN channels with postnatal development in the neocortex.

Dendritic synaptic integration

The age-dependent increase in the density and apical den-dritic polarization of HCN channels provides a basis for therefinement of the electrotonic structure of layer 5 pyramidalneurons. Classical models of synaptic integration indicate thatthe direct impact of dendritic synaptic inputs at the site ofaction potential initiation solely determines their influence onneuronal output (Rall 1967). However, recent evidence sug-gests that in mature neocortical pyramidal neurons, distalapical dendritic excitatory inputs influence action potentialoutput not only by providing direct depolarization at the site of

action potential initiation but also by local integration in thedendritic tree that leads to the initiation of regenerative den-dritic electrical activity termed apical dendritic spikes (Kimand Connors 1993; Larkum and Zhu 2002; Williams and Stuart2002). In mature layer 5 pyramidal neurons, apical dendriticspikes have been shown to forward propagate through the

dendritic tree to trigger axonal action potential firing (Larkumand Zhu 2002; Williams 2004; Williams and Stuart 2002; Zhu2000). However, previous observations have indicated thatapical dendritic spikes are absent or only manifest weakly inearly postnatal neurons (Zhu 2000). Moreover, once generatedin immature neurons, dendritic spikes have been shown to beunreliable triggers for the initiation of action potential outputand are often confined to the distal apical dendritic tree, failingto faithfully forward propagate (Schiller et al. 1997; Zhu 2000).We therefore investigated how efficaciously barrages of distalapical dendritic excitatory synaptic input drove axonal actionpotential firing across the first month of postnatal development.To do this, we directly compared how effectively barrages of

FIG. 8. Developmental refinement of theactivation properties of distal apical den-dritic HCN channels. A: families of leak-subtracted cell-attached current responsesevoked by an incremental series of voltagesteps (bottom traces) recorded from an api-

cal dendritic site of a P14 (70 m from thelayer 1layer 2 border) and P36 layer 5pyramidal neuron (30 m from the layer1layer 2 border). Leak subtraction was per-formed off-line by the subtraction of scaledresponses generated by interleaved 10-mVvoltage steps. B: voltage-dependent activa-tion curves of HCN channel activity calcu-lated from tail currents in cell-attachedpatches made from young (P13-16, E;means SE) and mature (P25 40, F) layer5 pyramidal neurons. Voltage refers to cal-culated membrane potential (see RESULTS fordetails). Relationship have been fit with sin-gle Boltzmann functions (). Note the rel-atively hyperpolarized voltage at half-maxi-mal activation in the young group. C: acti-vation kinetics of ensemble HCN channelactivity are different between young andmature layer 5 pyramidal neurons across awide voltage range. Note that in each groupactivation kinetics speed as the size of thepipette negative voltage step increases. Dataare fit by single-exponential functions ().The statistical significance between groupsis indicated by stars (P values 0.01,2-tailed t-test). D: HCN2 immunostaining(dark reaction) of the neocortex from a P14animal; inset: the cell bodies of layer 2/3neurons at a higher magnification. E: HCN1and HCN2 immunostaining (dark reaction)in consecutive neocortical slices from a P25animal. Note the prominent HCN1 immuno-

staining of the apical dendrites of layer 5pyramidal neurons, and the somatic HCN2immunostaining of layer 2/3 neurons. Mon-tages were formed from a series of high-resolution confocal stacks.




13/18

EPSCs generated at somatic or distal apical dendritic sitesdrove action potential output, calculating the number of actionpotentials evoked by the delivery of series of identical 2-s-longbarrages of EPSCs generated at each site (Fig. 9). In earlypostnatal layer 5 pyramidal neurons, the delivery of barrages ofEPSCs to the soma or apical dendrite resulted in the generationof a similar number of action potentials across a wide range of

injected synaptic current (P9-10; dendritic recording 78 13m from the layer 1layer 2 border; n 6; Fig. 9A). Thereforeat this age, current-discharge relationship had similar slopes,when synaptic input was generated at the soma or distal apicaldendrite (Fig. 9B). In progressively older animals, the numberof action potentials generated by the apical dendritic synapticinput became less than those generated by the delivery ofidentical simulated synaptic input to the soma (Fig. 9, C, E, andG). This resulted in a disparity between the slope of current-discharge relationship (Fig. 9, D, F, and H). Interestingly, inmature pyramidal neurons, robust action potential output couldbe evoked by distal dendritic barrages of sEPSPs, a finding thatcould not be predicted by the heavy constraint of subthreshold

voltage responses (Fig. 9, G and H). We therefore explored ifbarrages of dendritic excitatory input triggered action potentialoutput not only by directly spreading to the site of actionpotential initiation but also by the generation of dendriticspikes. To do this, we overlaid voltage recordings for eachaction potential generated by somatic or dendritic barrages ofsimulated synaptic input (Fig. 10, A and B). When excitatoryinput was delivered to the soma, each action potential was firstrecorded from the somatic recording electrode and then sub-sequently at the dendritic site, consistent with the axonalinitiation of action potential firing and subsequent back-prop-agation into the apical dendritic tree (data not shown) (Stuartand Sakmann 1994). Similarly, in early postnatal neurons whensynaptic excitation was generated at distal apical dendritic

sites, the vast majority of action potentials were recorded firstat somatic sites (P9 10; 92.7 4.6%; n 6; Fig. 10, AC). Inneurons older than P11, the majority of somatically recordedaction potentials were however preceded by dendritic spikes(Fig. 10B). The fraction of somatically recorded action poten-tials that were preceded by a dendritic spike increased in anage-dependent manner. Indeed in mature pyramidal neurons,

the vast majority of action potentials were driven by dendriticspikes (Fig. 10C). This relationship could be fit with a boundedexponential function with an age-constant of just 2.4 days (Fig.10C). These data indicate that after postnatal day 11, dendriticsynaptic integration significantly enhances the impact of distalapical dendritic synaptic inputs on action potential output. Toquantify this, we plotted the relative efficacy of dendriticsynaptic inputs in the sub- and suprathreshold range (Fig.10D). Subthreshold efficacy was calculated as the ratio of theslope between somatic depolarization and injected currentwhen barrages of EPSCs were injected at the soma or distalapical dendritic sites (Figs. 3 and 4A). Suprathreshold efficacywas calculated as the ratio of the current-discharge relationship

evoked by barrages of somatic or distal apical dendritic EPSCs(Fig. 9). This comparison revealed a significant increase in theefficacy of dendritic synaptic inputs in the suprathresholddomain after postnatal day 13, indicating that after the first 2wk of age, dendritic synaptic integration profoundly contrib-utes to the output of layer 5 pyramidal neurons (Fig. 10D).

To directly test if apical dendritic spikes in layer 5 pyramidalneurons from immature animals drove action potential output,we locally pharmacologically blocked apical dendritic spikesby the brief pressure application of TTX (1 M, in pufferpipette) delivered from a pipette placed close to (2030 m)the apical dendritic recording site in P13-14 layer 5 pyramidalneurons (Fig. 11, inset). To ensure that TTX had effects onlylocally, we measured the amplitude of somatic and back-

FIG. 9. Developmental refinement of the suprathreshold efficacy of apical dendritic excitation. A, C, E, and G: somatic voltage recordings of the actionpotential output generated in response to identical barrages of EPSCs injected at somatic ( left) and apical dendritic sites (right). Recordings were made from layer5 pyramidal neurons of the indicated postnatal age. B, D, F, and H: current-discharge relationship generated when barrages of EPSCs were delivered at somatic(F) and distal apical dendritic sites (E). Data have been pooled in the indicated postnatal age groups. Each value represents means SE Note that in earlypostnatal neurons the action potential output is similar when synaptic current is injected at somatic and apical dendritic sites.




14/18

propagating action potentials (BPAP) generated by somaticcurrent injection and found that somatic action potentials wereunaffected, but BPAPs were significantly attenuated by thelocal apical dendritic application of TTX (soma: control 87.3 1.3 mV; TTX 86.9 1.8 mV, n 6; not signifi-cantly different; dendrite: control 41.2 4.7 mV; TTX 25.7 1.5 mV, n 6, P 0.01; paired t-test; Fig. 11C). Inresponse to a barrage of apical dendritic simulated synapticinput (535 17 m from the soma; n 6), 87.0 8.0% ofaction potentials were preceded by an apical dendritic spike(Fig. 11, A and B). The local application of TTX blocked apical

dendritic spikes and resulted in a 71.4 11.7% reduction inthe number of action potentials evoked by the distal apicaldendritic excitatory input (Fig. 11, A and B). In neuronsrecorded from older animals, however, the local apical den-dritic application of TTX blocked the generation of dendriticspikes evoked by distal apical dendritic excitatory input, andresulted in somatic voltage responses that were sub-thresholdfor the generation of axonal action potentials (P2729; n 3;not shown). Taken together, these data indicate that apicaldendritic spikes powerfully drive neuronal output in layer 5pyramidal neurons.

The proceeding results suggest that the development in-crease in the density of apical dendritic HCN channels

sculpts the integrative operations of layer 5 pyramidalneurons. Early in postnatal development (P9) excitatorysynaptic input to the apical dendrite is funneled to the somaand axon to influence action potential firing. In contrast, inmature layer 5 pyramidal neurons little direct depolarizationreaches the soma and axon, and apical dendritic excitatoryinputs are locally integrated to evoke dendritic spikes that inturn drive action potential output. We therefore explored ifthe blockade of HCN channels in mature layer 5 pyramidalneurons could transform this pattern of compartmentalizedintegration and result in behavior reminiscent of early post-

natal neurons where synaptic input to the apical dendrite isintegrated at the axon. When the distal apical dendrites ofmature layer 5 pyramidal neurons was depolarized by bar-rages of simulated excitatory synaptic input, prominentapical dendritic spikes were evoked that drove 98.7 1.0%of action potential output (n 237 action potentials; 533 8 m from the soma; n 6; P2729; Fig. 12, AD). Thisbehavior was transformed by the pharmacological blockadeof HCN channels with ZD 7288 (20 M; Fig. 12, AD).When HCN channels were blocked, and the somatic andapical dendritic membrane potential repolarized to controllevels by the delivery of tonic DC through the recordingelectrodes, the vast majority of apical dendritic excitatory

FIG. 10. Developmental increase in apical dendritic synaptic integration. A: simultaneous somatic and dendritic voltage recordings of action potential outputevoked by the injection of barrages of EPSCs at distal apical dendritic sites. For neurons of the indicated postnatal age, somatic (black trace) and dendritic (gray)voltage records have been overlain. B: action potential waveforms recorded from somatic (black) and apical dendritic sites (gray). Action potentials were alignedat the peak of somatically recorded events and averaged for all events generated across current-discharge relationship. Note in the P9 neuron that the averageaction potential waveform is recorded first at the soma and then backpropagates to the dendritic recording site. In contrast, recordings from older animals indicatethat somatically recorded action potentials are preceded by dendritic spikes (P1229). C: summary graph describing the percentage of somatically recorded actionpotentials that were preceded by a dendritic spike. Results have been pooled in the indicated age groups (means SE). The line represents a fit to the data witha single bounded exponential function. Note the age-dependent increase in dendritic spike generation. D: comparison of the efficacy of dendritic inputs in thesub- and supra-threshold domain (see RESULTS for details). Significant differences are indicated by stars (P values 0.05, Wilcoxon matched pairs test).




15/18

input was integrated at the axon, with only a small fractionof action potentials preceded by dendritic spikes (2.4 0.9%; n 916 action potentials; Fig. 12, AD). Moreover,in the presence of ZD 7288 the structure of the current-discharge relationship generated by distal apical dendriticbarrages of simulated EPSCs resembled that produced by

excitation delivered to the soma under control conditions(compare Fig. 12C with Fig. 9H). Taken together these dataindicate that the high-density of apical dendritic HCN chan-

nels enforces compartmentalized integration in mature layer5 pyramidal neurons.

D I S C U S S I O N

In this study, we find an age-dependent switch in the inte-

grative operations of layer 5 pyramidal neurons, from oneanalogous to a single-compartment temporal-integrator early inpostnatal development to a highly compartmentalized integra-

FIG. 11. Dendritic spikes drive action potential output in immature layer 5 pyramidal neurons. A: the local apical dendritic application of TTX

(1 M in a puffer pipette) blocks the action potential output evoked by apical dendritic excitatory input in a P13 layer 5 pyramidal neuron. Simultan-eously recorded somatic (black traces) and dendritic (gray traces) voltage responses evoked by the injection of a barrage of EPSCs ( bottom trace) atdistal apical dendritic sites under control and following the local apical dendritic application of TTX. Note that under control dendritic spikes precededaction potential output, the sequence of regenerative events is shown in B, inset. The photomicrograph shows the morphology of this pyramidal neuronand the placement of pipettes. B: pooled data describing the reduction in the frequency of action potential output produced by the local apical dendri-tic application of TTX (n 6; P13- 14). C: pooled data describing the selective attenuation of the amplitude of back-propagating action potentials bylocal apical dendritic TTX (n 6). Inset: the influence of TTX on the waveforms of the somatic and dendritic action potentials, same neuron as shownin A.

FIG. 12. Hyperpolarization-activated cy-clic nucleotide (HCN)-gated channels enforcedendritic synaptic integration. A: simultaneoussomatic (black traces) and dendritic (550 mfrom the soma, gray traces) voltage recordingsof action potential output evoked by the injec-tion of barrages of EPSCs (bottom traces) atdistal apicaldendritic sites under control and inthe presence of ZD 7288 (20 M) in a P27layer 5 pyramidal neuron. B: averaged actionpotential waveforms recorded from somatic(black) and apical dendritic sites (gray) under

control (top traces) and in ZD 7288. Actionpotentials were aligned at the peak of somati-cally recorded events and averaged for allevents generated across current-discharge rela-tionship. Under control the somatic action po-tential was preceded by a dendritic spike,while in ZD 7288 somatic action potentialswere followed by dendritic back-propagatingaction potentials. Data from the same cell as A.C: blockade of HCN channels transforms thecurrent-discharge relationship evoked by bar-rages of distal apical dendritic simulated syn-aptic current. Data has been averaged from 6neurons (P2729). D: the percentage of actionpotentials driven by dendritic spikes undercontrol and in the presence of ZD 7288.




16/18

tor after the first postnatal month. This transformation of theintegrative operations of pyramidal neurons was underpinnedby a dramatic age-dependent increase in the density of apicaldendritic HCN channels.

Functional classes of layer 5 pyramidal neurons are identi-fiable morphologically around the first postnatal week (Kasperet al. 1994). After this age, thick/tufted layer 5 pyramidal

neurons can be differentiated by the presence of an apicaldendritic tuft in layer 1 from slender/untufted layer 5 pyramidalneurons (Kasper et al. 1994). This morphological divisioncorrelates with the cell body position of layer 5 pyramidalneurons in sublayer 5B and layer 5A, respectively, in thesomatosensory cortices (Frick et al. 2008; Wise and Jones1977). In this study, we visualized the apical dendritic tree oflayer 5 pyramidal neurons under infra-red differential interfer-ence contrast microscopy prior to electrophysiological record-ing, allowing us to target layer 5 pyramidal neurons thatexhibited a prominent apical dendritic tuft in layer 1. Thus thepresented data represents the postnatal development of thick/tufted layer 5B pyramidal neurons.

Key morphological features of the postnatal development oflayer 5 pyramidal neurons are the age-dependent increases inthe size of the dendritic tree and the density of dendritic spines,which reach adult levels by postnatal day 30 (Miller 1981;Wise et al. 1979). Analysis of Golgi-impregnated layer 5pyramidal neurons has shown a low spine density at basal andapical dendritic sites of early postnatal (P3-5) neurons consis-tent with a low number of excitatory synaptic inputs (Miller1981; Wise et al. 1979). Dendritic spine density, however,increases with age in abrupt steps around P15 and P21 in layer5 pyramidal neurons of the visual cortices (Miller 1981).Interestingly, until P21, the density of spines is uniform acrossthe apical dendritic trunk of layer 5 pyramidal neurons (Miller1981). However, after P21, spine density is low at proximal but

high at relatively distal apical dendritic sites (Miller 1981). Ourelectrophysiological results provide a functional framework inwhich to interpret these morphological data.

In early postnatal layer 5 neurons (P9-10), the somaticamplitude of dendritically generated simulated EPSPs waslarge. Moreover, barrages of apical dendritic sEPSPs power-fully summated at the soma, depolarizing the soma onlyslightly less efficiently than identical barrages of EPSPs gen-erated somatically. Distal apical dendritic and somatic excita-tory synaptic input therefore drove action potential firing withalmost equal efficacy where each action potential was initiatedaxonally. These properties indicate that a relatively smallnumber of excitatory inputs are required to generate actionpotential firing in early postnatal layer 5 pyramidal neurons

consistent with the low dendritic spine density at this age. Thusin early postnatal neurons, excitatory synaptic activity pro-duces prominent voltage and conductance changes that spreadthroughout the neuron. Excitatory synaptic activity generatedat basal and apical dendritic sites is therefore expected tointeract by decreasing the driving force at even distant excita-tory synapses. Similarly, the compact electrotonic structurewill allow the wide spatial distribution of synaptic conductanceas conductance is compartmentalized with a length constant onthe order of half the voltage length constant in the apicaldendrite of layer 5 pyramidal neurons (Williams 2004). Volt-age interactions will, however, be more influential in earlypostnatal neurons because the time course of sEPSPs greatly

outlasts that of the underlying synaptic current. Early postnatallayer 5 pyramidal neurons can therefore be considered elect-rotonically compact temporal integrators, allowing, on onehand, remote synaptic inputs to powerfully influence actionpotential output but on the other, promoting voltage interactionbetween distant excitatory synapses. If this situation was main-tained during development as spine density increases, intersyn-

aptic voltage interactions would decrease the efficacy of syn-aptic signaling, Moreover, intersynaptic depolarization wouldenhance the NMDA receptor-dependent calcium entry at acti-vated excitatory synapses (Markram et al. 1997; Thomson et al.1989), hampering the input-specificity of synaptic plasticity(Andersen et al. 1977; Engert and Bonhoeffer 1997), andpotentially leading to neurotoxic levels of calcium entry (Lip-ton and Rosenberg 1994). Therefore as dendritic spine densityincreases, postsynaptic mechanisms must be in place to ensuretranslation of dendritic excitatory input into neuronal outputand damp undesirable intersynaptic interactions. The physicalexpansion of the dendritic tree in part allows for this, bycreating longer dendritic cables between areas of the tree, forexample basal and apical dendrites. Similarly the relatively lownumbers of excitatory synapses at proximal apical dendriticsites in mature ( P 21) layer 5 pyramidal neurons will help tominimize direct interaction between basal and apical dendriticexcitatory synapses. The electrical compartmentalization ofareas of the dendritic tree will also be increased by theage-dependent increase in the density of HCN channels.

HCN channel density was found to increase by 10-fold atdistal apical dendritic sites of layer 5 pyramidal neurons from P11to P40. This dramatic rise in apical dendritic channel density hada profound influence on the spread of synaptic potential throughthe dendritic tree. As postnatal age increased, single or barrages ofdistal apical dendritic sEPSPs become progressively weakersources of somatic excitation. Indeed we found an age-dependent

constraint of the somatic time course of apical dendriticallygenerated sEPSPs that resulted in a progressive reduction in thetemporal summation of barrages of dendritic sEPSPs at the soma.Consequently in mature layer 5 pyramidal neurons, the pharma-cological blockade of HCN channels dramatically increased thesomatic depolarization produced by apical dendritic sEPSPs. Theage-dependent increase in HCN channel density therefore pro-foundly increases electrical compartmentalization. At a functionallevel, such compartmentalization ensures that synaptic activity indifferent areas of the dendritic tree have minimal voltage andconductance interactions (Williams 2004). However, the highdensity of apical dendritic HCN channels also ensures that distalapical dendritic synaptic inputs provide only a weak direct sourceof axo-somatic excitation.

Interestingly, we find that the activation properties of en-semble HCN channel activity in cell-attached patches fromdistal apical dendritic sites changes with age; with the activa-tion, kinetics speeding and the voltage dependence of activa-tion become relatively depolarized as the channel densityincreases with age. Whole cell recordings from the somata ofCA1 hippocampal pyramidal neurons have similarly shownthat the properties of IH, the macroscopic manifestation ofHCN channel activity, are developmentally regulated, withactivation kinetics speeding and the voltage dependence ofactivation becoming more depolarized with age (Surges et al.2006; Vasilyev and Barish 2002). Informatively, in area CA1,an age-dependent increase in HCN1 mRNA, protein level and




17/18

apical dendritic HCN1 immunostaining has been found (Brew-ster et al. 2007). This increase in HCN1 expression is accom-panied by an age-dependent reduction in HCN4 mRNA andprotein levels (Brewster et al. 2007). In contrast to the apicaldendritic expression of HCN1, however, HCN4 immunostain-ing is restricted to the somata of CA1 pyramidal neurons(Brewster et al. 2007), that together with the predominately

somatic HCN2 isoform acts to slow the kinetics of somaticallyrecorded IH in early postnatal neurons (Brewster et al. 2007;Surges et al. 2006; Vasilyev and Barish 2002). Our cell-attached recording approach, however, directly indicates anage-dependent modification of the properties of ensemblechannel activity at apical dendritic sites and is not complicated byvoltage-clamp errors apparent during somatic whole cell record-ing (Williams and Mitchell 2008) or by the subcellular distribu-tion of HCN channel isoforms. Combined electrophysiologicaland immunostaining showed that the age-dependent increase inthe density of functional channels at distal apical dendritic sites oflayer 5 pyramidal neurons was paralleled by an increase in thedensity of HCN1 immunostaining. In the neocortex, little HCN4expression is detectable with HCN1 and HCN2 being the abun-dant channel isoforms (Notomi and Shigemoto 2004; Santoroet al. 2000). As HCN2 channels produce macroscopic currentswith relatively slow kinetics (Santoro et al. 2000), we explored ifthe HCN2 channel isoform was expressed at distal apical dendriticsites preferentially during early postnatal development. However,we were unable to detect significant apical dendritic HCN2immunostaining in either young or mature animals. A previousstudy has, however, reported relatively weak HCN2 expression atdistal apical dendritic sites in 5- to 10-wk-old layer 5 pyramidalneurons (Notomi and Shigemoto 2004). Interestingly, in HCN1knockout mice, somatic whole cell recordings of layer 5 pyrami-dal neurons have shown a prominent IH current with sloweractivation kinetics and relatively hyperpolarized voltage depen-

dence compared with wild-type mice consistent with a functionalrole of HCN2 isoforms (Chen et al. 2008). We suggest thereforethat the age-dependent refinement of the properties of ensembleHCN channels at distal apical dendritic sites is consistent with anage-dependent alteration in HCN channel subunit composition(Santoro and Tibbs 1999). Alternatively, our data are consistentwith a developmental regulation of HCN channel properties byinteracting proteins (Santoro et al. 2004; Yu et al. 2001) and/or bythe channels phosphorylation state (Huang et al. 2008). Interest-ingly, a putative subunit of the HCN channel, MiRP1 has beenshown to regulate the activation kinetics of HCN channel re-sponses (Yu et al. 2001). We consider it unlikely that differencesin basal intracellular levels of cyclic nucleotides, which modulatethe activation properties of HCN channels, underlie the develop-

mental regulation of HCN channel function because HCN1 chan-nels are weakly influenced by cyclic nucleotide levels (Santoroet al. 1998) and age-dependent differences in the activation prop-erties of ensemble HCN channel activation in cell-attachedpatches were apparent during simultaneous whole cell and cell-attached recordings where intracellular nucleotide levels are dom-inated by the intra-pipette filling solution (data not shown).

The electrical compartmentalization of layer 5 pyramidal neu-rons imposed by the high density of HCN channels ensures thatdistal apical dendritic excitatory and inhibitory synaptic inputevoke minimal voltage responses at the axonal site of actionpotential generation in mature animals (Berger et al. 2001; Stuartand Spruston 1998; Williams and Stuart 2000, 2003). Indeed

modeling studies have shown that single EPSPs generated in theapical tuft, are attenuated by100-fold as they spread to the soma(Stuart and Spruston 1998). Here we find that even a high-frequency barrage of distal apical dendritic sEPSPs provides lessthan 500 V of somatic depolarization in mature layer 5 pyrami-dal neurons. As both cortico-cortical and subcortical synapticinput is conveyed in layer 1 of the neocortex (Bernander et al.

1994; Zhu and Zhu 2004), these properties suggest that such distalapical dendritic excitatory inputs are ineffective direct drivers ofaction potential output. We however, found an age-dependentswitch in the way distal apical dendritic synaptic input influencedneuronal output. As postnatal age increased, the fraction of actionpotentials that were driven by apical dendritic spikes increased.Therefore we find that active dendritic synaptic integration, lead-ing to the initiation and forward propagation of dendritic spikes,acts to compensate for the distance-dependent attenuation ofapical dendritic input. Interestingly, in comparison with a previousstudy that probed the excitability of distal apical dendrites of layer5 neurons by the injection of simple positive current steps (Zhu2000), we find that apical dendritic integration becomes dominantafter postnatal day 11, when studied in response to more invivo-like, time-varying barrages of simulated synaptic input. In-deed during a development period between P11 and P22, we findthat the apical dendrite can be considered to be hyperexcitable;electrical compartmentalization is incomplete at this age, resultingin a significant spread of depolarization from the apical dendrite tothe soma and axon that is complemented by the robust forwardprorogation of apical dendritic spikes. An idea supported by thereduction, but not abolition, of action potential output evoked bydistal apical dendritic excitatory input when dendritic spike gen-eration was blocked by local apical dendritic application of TTXin P1314 neurons. In mature layer 5 pyramidal neurons, how-ever, the high density of apical dendritic HCN channels ensuresthat little synaptic depolarization flows from the apical dendrite to

the soma, and integration is largely restricted to distal apicaldendritic sites, resulting in the initiation of dendritic spikes. Aconclusion supported by the transformation of the site of integra-tion of distal apical dendritic synaptic input from apical dendriticto axonal following the pharmacological blockade of HCN chan-nels in mature neurons. Functionally, the high density of HCNchannels provides a platform for compartmentalized synapticintegration (Williams 2004) as well as allowing interaction be-tween axo-somatic and apical dendritic integration compartmentsthrough regenerative activity, such as BAC firing (Larkum et al.1999; Williams 2005). During the first month of postnatal devel-opment therefore, the integrative properties of layer 5 pyramidalneurons are transformed, from electronically compact temporalintegrators of synaptic input to highly compartmentalized integra-

tors of basal and apical dendritic synaptic input.

A C K N O W L E D G M E N T S

We are grateful to Prof. R. Shigemoto for the gift of an HCN2 antibody.S. E. Atkinson performed immunostaining and S. R. Willliams performedelectrophysiological experiments.

G R A N T S

This work was supported by the Medical Research Council.

R E F E R E N C E S

Andersen P, Sundberg SH, Sveen O, Wigstrom H. Specific long-lastingpotentiation of synaptic transmission in hippocampal slices. Nature 266:736737, 1977.




18/18

Angelo K, London M, Christensen SR, Hausser M. Local and global effectsof I(h) distribution in dendrites of mammalian neurons. J Neurosci 27:86438653, 2007.

Berger T, Larkum ME, Luscher HR. High I(h) channel density in the distalapical dendrite of layer V pyramidal cells increases bidirectional attenuationof EPSPs. J Neurophysiol 85: 855868, 2001.

Bernander O, Koch C, Douglas RJ. Amplification and linearization of distalsynaptic input to cortical pyramidal cells. J Neurophysiol 72: 27432753, 1994.

Brewster AL, Chen Y, Bender RA, Yeh A, Shigemoto R, Baram TZ.Quantitative analysis and subcellular distribution of mRNA and protein expres-sion of the hyperpolarization-activated cyclic nucleotide-gated channelsthroughout development in rat hippocampus. Cereb Cortex 17: 702712, 2007.

Chen X, Shu S, Kennedy DP, Willcox SC, Bayliss DA. Subunit-specificeffects of isoflurane on neuronal Ih in HCN1 knockout mice. J Neurophysiol29: 129140, 2008.

Engert F, Bonhoeffer T. Synapse specificity of long-term potentiation breaksdown at short distances. Nature 388: 279284, 1997.

Frick A, Feldmeyer D, Helmstaedter M, Sakmann B. Monosynaptic con-

nections between pairs of L5A pyramidal neurons in columns of juvenile ratsomatosensory cortex. Cereb Cortex 18: 397406, 2008.

Hausser M, Roth A. Estimating the time course of the excitatory synapticconductance in neocortical pyramidal cells using a novel voltage jumpmethod. J Neurosci 17: 76067625, 1997.

Huang J, Huang A, Zhang Q, Lin YC, Yu HG. Novel mechanism for

suppression of hyperpolarization-activated cyclic nucleotide-gated pace-maker channels by receptor-like tyrosine phosphatase-alpha. J Biol Chem283: 2991229919, 2008.

Kasper EM, Larkman AU, Lubke J, Blakemore C. Pyramidal neurons inlayer 5 of the rat visual cortex. II. Development of electrophysiologicalproperties. J Comp Neurol 339: 475494, 1994.

Kim HG, Connors BW. Apical dendrites of the neocortex: correlationbetween sodium- and calcium-dependent spiking and pyramidal cell mor-phology. J Neurosci 13: 53015311, 1993.

Kole MH, Brauer AU, Stuart GJ. Inherited cortical HCN1 channel lossamplifies dendritic calcium electrogenesis and burst firing in a rat absenceepilepsy model. J Physiol 578: 507525, 2007.

Kole MH, Hallermann S, Stuart GJ. Single IH channels in pyramidal neurondendrites: properties, distribution, and impact on action potential output.

J Neurosci 26: 16771687, 2006.Larkum ME, Zhu JJ. Signaling of layer 1 and whisker-evoked Ca2 and Na

action potentials in distal and terminal dendrites of rat neocortical pyramidal

neurons in vitro and in vivo. J Neurosci 22: 69917005, 2002.Larkum ME, Zhu JJ, Sakmann B. A new cellular mechanism for coupling

inputs arriving at different cortical layers. Nature 398: 338341, 1999.Lipton SA, Rosenberg PA. Excitatory amino acids as a final common

pathway for neurologic disorders. N Engl J Med 330: 613622, 1994.Lorincz A, Notomi T, Tamas G, Shigemoto R, Nusser Z. Polarized and

compartment-dependent distribution of HCN1 in pyramidal cell dendrites. Nat Neurosci 5: 11851193, 2002.

Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpo-larization-activated mammali

susan e. atkinson and stephen r. williams- postnatal development of dendritic synaptic integration...

Documents