Action potential initiation and propagation in layer 5 pyramidal neurons of the rat prefrontal cortex: absence of dopamine modulation.

Abstract

Somatic and dendritic whole-cell recording was used to examine action potential (AP) initiation and propagation in layer 5 pyramidal neurons of the rat prelimbic prefrontal cortex. APs generated by somatic current injection, or via antidromic stimulation, were reliably recorded at apical dendritic locations as far as 480 microm from the soma. Although the backpropagation of single APs into the apical dendrite was robust, frequency-dependent attenuation was observed during AP trains delivered at 10-100 Hz. APs were usually initiated close to the soma (presumably in the axon); however, strong depolarizing input to the apical dendrite could generate dendritic spikes that preceded somatic APs. AP backpropagation was dependent solely on activation of dendritic voltage-gated sodium channels and did not require activation of dendritic calcium channels. Despite not playing a role in AP backpropagation, calcium-imaging experiments demonstrated that dendritic calcium channels are activated by backpropagating APs, leading to transient increases in intracellular calcium. In addition, calcium imaging revealed that AP backpropagation into the distal apical tuft was frequency dependent. Finally, we tested whether dopamine, a prominent neuromodulator associated with prefrontal activity, could alter AP initiation or backpropagation. Bath-applied dopamine (10 or 100 microm) did not effect AP backpropagation, frequency-dependent depression, local dendritic spike initiation, or AP-induced calcium signaling. These data indicate that AP backpropagation in prefrontal layer 5 pyramidal neurons is robust but frequency dependent in the distal tuft, requires dendritic sodium rather than calcium channel activation, and, unlike other aspects of neuronal excitability, insensitive to modulation by dopamine.

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Behavioral/Systems/Cognitive
ActionPotentialInitiationandPropagationinLayer5
PyramidalNeuronsoftheRatPrefrontalCortex:Absenceof
DopamineModulation
AllanT.GulledgeandGregJ.Stuart
DivisionofNeuroscience,JohnCurtinSchoolofMedicalResearch,AustralianNationalUniversity,Canberra,ACT0200,Australia,andPhysiologyInstitute,
UniversityofFreiburg,79104,Freiburg,Germany
Somatic and dendritic whole-cell recording was used to examine action potential (AP) initiation and propagation in layer 5 pyramidal
neuronsoftheratprelimbicprefrontalcortex.APsgeneratedbysomaticcurrentinjection,orviaantidromicstimulation,werereliably
recordedatapicaldendriticlocationsasfaras480?mfromthesoma.AlthoughthebackpropagationofsingleAPsintotheapicaldendrite
wasrobust,frequency-dependentattenuationwasobservedduringAPtrainsdeliveredat10–100Hz.APswereusuallyinitiatedcloseto
the soma (presumably in the axon); however, strong depolarizing input to the apical dendrite could generate dendritic spikes that
precededsomaticAPs.APbackpropagationwasdependentsolelyonactivationofdendriticvoltage-gatedsodiumchannelsanddidnot
require activation of dendritic calcium channels. Despite not playing a role in AP backpropagation, calcium-imaging experiments
demonstrated that dendritic calcium channels are activated by backpropagating APs, leading to transient increases in intracellular
calcium.Inaddition,calciumimagingrevealedthatAPbackpropagationintothedistalapicaltuftwasfrequencydependent.Finally,we
testedwhetherdopamine,aprominentneuromodulatorassociatedwithprefrontalactivity,couldalterAPinitiationorbackpropagation.
Bath-applied dopamine (10 or 100 ?M) did not effect AP backpropagation, frequency-dependent depression, local dendritic spike
initiation, or AP-induced calcium signaling. These data indicate that AP backpropagation in prefrontal layer 5 pyramidal neurons is
robust but frequency dependent in the distal tuft, requires dendritic sodium rather than calcium channel activation, and, unlike other
aspectsofneuronalexcitability,insensitivetomodulationbydopamine.
Keywords:prefrontalcortex;apicaldendrite;actionpotential;workingmemory;dopamine;rat;calciumimaging
Introduction
During the past decade, it has become apparent that the apical
dendrites of cortical pyramidal neurons in the somatosensory
cortex and hippocampus express voltage-gated channels that al-
low active backpropagation of axonally generated action poten-
tials (APs) into the apical dendritic tree (Stuart and Sakmann,
1994;Sprustonetal.,1995;Stuartetal.,1997).Thesebackpropa-
gating APs have important physiological consequences for syn-
aptic integration (Ha ¨usser et al., 2001) and plasticity (Linden,
1999). In addition, the interaction of backpropagating APs with
active dendritic conductances or concurrent synaptic input has
beenshowntopromotehigh-frequencyburstfiringinpyramidal
neurons (Schiller et al., 1997; Schwindt and Crill, 1997; Larkum
et al., 1999a; Magee and Carruth, 1999; Williams and Stuart,
1999).
One cell type in which phasic periods of high-frequency AP
firing are thought to have important functional consequences is
the pyramidal neuron in the prefrontal cortex. This cortical area
is critical for a form of short-term memory known as “working
memory.” Unlike other types of memory such as episodic, se-
mantic, or fear-conditioned memory, working memory is not
thoughttorequirepermanentchangesinthesynapticstrengthof
connections between neurons (i.e., long-term synaptic plastic-
ity). Rather, working memory is believed to involve encoding of
behaviorallyrelevantinformationinthesustainedactivityofspe-
cific subsets of prefrontal pyramidal neurons (Goldman-Rakic,
1995).Thisreverberatoryactivityhasbeenproposedtobedepen-
dent on modulation of specific voltage-dependent membrane
conductances in the somata and dendrites of prefrontal neurons
(Yang and Seamans, 1996; Durstewitz et al., 2000). However, the
active dendritic properties of these neurons have not been fully
characterized.Althoughpreviousstudieshavenotedthepresence
of dendritic voltage-activated sodium and calcium channels in
prefrontal pyramidal neurons (Seamans et al., 1997; Gonzalez-
BurgosandBarrionuevo,2001),theirpotentialphysiologicalrole
in action potential initiation or backpropagation has not been
explored.
It has been hypothesized that dopaminergic regulation of ac-
tive dendritic conductances may be important for modifying the
excitability and overall firing rates of prefrontal pyramidal neu-
rons during behavioral tasks requiring working memory (Yang
ReceivedJuly4,2003;revisedOct.13,2003;acceptedOct.14,2003.
ThisworkwassupportedbyanInternationalResearchFellowAwardfromtheNationalScienceFoundation,the
WellcomeTrust,andtheAlexandervonHumboldtFoundation.
Correspondence should be addressed to Greg J. Stuart, Division of Neuroscience, John Curtain School of
Medical Research, Building 54, Australian National University, Canberra, ACT 0200, Australia. E-mail:
greg.stuart@anu.edu.au.
Copyright©2003SocietyforNeuroscience 0270-6474/03/2311363-10$15.00/0
TheJournalofNeuroscience,December10,2003 • 23(36):11363–11372 • 11363

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and Seamans, 1996; Durstewitz et al., 2000; Dreher et al., 2002).
Although there is little direct evidence for this hypothesis, mod-
ulation of dendritic channels by dopamine would be expected to
have important consequences for synaptic integration in these
neurons. Dopamine has been shown to modulate voltage-
activated sodium and calcium channels in prefrontal neurons
(Geijo-Barrientos and Pastore, 1995; Yang and Seamans, 1996;
Gorelova and Yang, 2000; Maurice et al., 2001), and thus could,
inprinciple,regulatetheinitiationandactivebackpropagationof
APs into the dendrites of these neurons.
We used dual whole-cell recording and calcium-imaging
techniquestodirectlytestthehypothesisthattheapicaldendrites
of prefrontal layer 5 pyramidal neurons express active conduc-
tances capable of supporting AP backpropagation and dendritic
spike generation. In addition, we investigate whether dopamine,
aprominentneuromodulatorassociatedwithprefrontalactivity,
can modulate active dendritic properties in these neurons.
MaterialsandMethods
After halothane anesthesia and decapitation, the brains of 3- to 5-week-
old Wistar rats were removed into an ice-cold solution containing (in
mM): 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 CaCl2, 6 MgCl2,
and 25 glucose (bubbled with 95% O2/5% CO2). Coronal brain slices
(300 ?m thick) containing the prelimbic prefrontal cortex were made
usingavibratingtissueslicer(Dosaka,Kyoto,Japan)andwereincubated
for 30–45 min in a similar solution containing 2 mM CaCl2and 1 mM
MgCl2heated to 35°C. Slices were subsequently stored at room temper-
ature for up to 6 hr before being placed, as needed, into the recording
chamber where they were perfused with the 2 mM CaCl2/1 mM MgCl2
solution heated to 35°C.
Whole-cell somatic and dendritic current-clamp recordings were
made under visual control from layer 5 pyramidal neurons in prelimbic
prefrontalcortexusingmethodsdescribedpreviously(Stuartetal.,1993)
and BVC-700 current-clamp amplifiers (Dagan, Minneapolis, MN). Re-
cording pipettes (4–10 M?) were filled with an internal solution con-
taining (in mM): 135 K-gluconate, 7 NaCl, 2 MgCl2, 10 HEPES, 2
Na2ATP, 0.3 NaGTP, pH 7.2 (KOH). After adjustment of capacitance
neutralization, compensated whole-cell series resistance was generally
between 20 and 40 M? and stable throughout recordings. Cells that
showedlargechangesinseriesresistancewerediscardedfordataanalysis
purposes. Membrane potentials were corrected for the liquid junction
potential (12 mV) and rounded to the nearest millivolt. Voltage and
currentsignalswerefilteredat10kHzandacquiredat30or50kHzusing
anITC-16interface(InstruTech,PortWashington,NY)controlledbyan
Apple PowerPC running Axograph software (Axon Instruments, Foster
City, CA). Data are presented as mean ? SEM. Statistical analysis used
the Student’s t test.
APs were generated via current injection through the somatic or den-
driticrecordingpipetteorviaantidromicstimulation.Antidromicstim-
ulation was achieved using a patch pipette filled with extracellular saline
placed near the axon of the neuron of interest. Stimuli (50 ?sec) were
generated using a custom-built stimulation isolation unit. Unless other-
wise stated, APs were generated at 0.05–0.1 Hz. AP amplitude was mea-
sured from threshold when induced by current injection and from the
resting potential when generated with antidromic stimulation.
For calcium imaging, neurons were loaded with the calcium-sensitive
dye Oregon Green BAPTA-1 (200 ?M; Molecular Probes, Eugene, OR)
and visualized using a confocal microscope (LSM 510; Zeiss, Thorn-
wood,NY).Linescans(0.5–1kHztemporalresolution)weremadeatthe
indicatedsomaticanddendriticlocationsonceperminute,andAPswere
generatedviasomaticcurrentinjection.Rawdatawerebackgroundsub-
tracted, and the change in fluorescence relative to the resting fluores-
cence (?F/F) was calculated using a 100–200 msec baseline before so-
matic current injection. In control experiments, we observed a
progressive decrease in ?F/F over time (mean change in ?F/F after 20
trialsforsingleAPs,?44?7%;fortrainsoffiveAPs,?47?6%;n?3),
whichpresumablyoccurredbecauseofatime-dependentincreaseindye
concentration. Therefore, to assess the potential effect of drug applica-
tions on calcium signaling, we compared the average ?F/F recorded
duringdrugapplicationwiththeaverageofcontroltrialsobtainedbefore
and after drug application.
Drugs (Sigma, St. Louis, MO) were focally or bath applied at the fol-
lowing concentrations: TTX, 1 ?M; CdCl2, 200 ?M; kynurentic acid, 3
mM;bicucullinemethiodide,20?M;dopamine,10or100?M;andnomi-
fensine, 1 ?M. Bath-applied dopamine was made fresh daily and always
includedascorbate(10?M)toreduceoxidation.Forfocaldendriticdrug
applications, drugs were dissolved in extracellular saline and loaded into
a patch pipette positioned ?20 ?m proximal to the dendritic recording
site. CdCl2was dissolved and applied in saline lacking NaH2PO4.Only
one cell receiving a single drug treatment was used from each slice.
Results
Actionpotential backpropagation
APinitiationandpropagationwasinvestigatedinlayer5pyrami-
dal neurons from prefrontal prelimbic cortex using dual whole-
cellrecordingsfromthesomaandapicaldendrite(upto480?m
from the soma). In our slices, the apical dendrites of pyramidal
neurons ranged in length from ?500–800 ?m (including the
apical tuft), depending on the superior–inferior location of the
soma. Therefore, our most distal dendritic recordings corre-
spondtomeasurementsfromthedistalpartoftheapicaldendrite
near the base of the apical tuft. Somatic current injections (500
msec) were used to evoke action potentials that were recorded
simultaneously at somatic and dendritic locations (Fig. 1A,B).
When generated in this way, APs were always observed to occur
first at the soma and subsequently at the apical dendritic record-
ing site (Fig. 1B). Average peak-to-peak conduction velocity was
0.47 ? 0.03 m/sec (Fig. 1C,D), which is similar to that found
previouslyinlayer5pyramidalneuronsfromsomatosensorycor-
tex under similar recording conditions (0.5 m/sec; 4-week-old
rats; 35°C) (Stuart et al., 1997). AP propagation in apical den-
drites was robust, with APs showing only modest attenuation
with distance from the soma (Fig. 1E). At distances from 420–
480 ?m from the soma, dendritically recorded APs had ampli-
tudesthatwere57?7%oftheirsomaticvalues(n?4;compare
with ?50% attenuation at 500 ?m in somatosensory layer 5 py-
ramidalneurons)(Stuartetal.,1997).Attenuationofsteady-state
somatic voltage (Fig. 1F) was significantly greater than AP back-
propagation (steady-state attenuation at distances ?420 ?m,
78?3%;n?4;p?0.05),indicatingthatAPbackpropagationis
active.
Frequencydependenceof backpropagation
Backpropagating APs in both CA1 (Callaway and Ross, 1995;
Spruston et al., 1995) and somatosensory layer 5 (Stuart et al.,
1997; Williams and Stuart, 1999; Larkum et al., 1999b, 2001)
pyramidal neurons are sensitive to the frequency of AP genera-
tion, showing increased attenuation during high-frequency AP
trains attributable to cumulative sodium channel inactivation
(Colbert et al., 1997; Jung et al., 1997). To test for frequency-
dependent depression of backpropagating APs, we used anti-
dromic stimulation to produce trains of 10 APs at 10, 20, 50, and
100 Hz. Backpropagating APs were recorded at dendritic loca-
tions up to 335 ?m from the soma (Fig. 2) (n ? 11). During AP
trains,theamplitudeofsuccessiveAPsdecreased,withtheextent
of this decrease dependent on AP frequency (Fig. 2A,B). The
amount of frequency-dependent depression, quantified as the
ratio of the amplitude of the tenth AP relative to that of the first,
was also dependent on the distance of the dendritic recording
from the soma (Fig. 2C), with distal locations showing the great-
estfrequency-dependentattenuation.Incontrasttolayer5pyra-
11364 • J.Neurosci.,December10,2003 • 23(36):11363–11372GulledgeandStuart•APPropagationinPrefrontalApicalDendrites

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midal neurons from somatosensory cortex (Stuart et al., 1997;
Williams and Stuart, 1999; Larkum et al., 1999b, 2001), high-
frequency AP firing was not associated with any obvious den-
dritic calcium electrogenesis even at the most distal dendritic
locations (Fig. 2A).
Roleofsodiumandcalciumchannelsin backpropagation
Previous work in pyramidal neurons from the CA1 region of the
hippocampus (Spruston et al., 1995) and somatosensory cortex
(Stuart and Sakmann, 1994) indicates that AP backpropagation
into apical dendrites depends on activation of dendritic voltage-
activated sodium channels. To determine whether dendritic so-
dium channels participate in AP backpropagation in prefrontal
cortexlayer5pyramidalneurons,wefocallyappliedTTX(1?M)
totheapicaldendritejustproximaltothesiteofdendriticrecord-
ing (Fig. 3A). In these experiments, local TTX application re-
duced AP amplitude by 66 ? 3% while having a negligible effect
on somatic AP amplitude (mean change, 1.2 ? 0.4%; n ? 5).
These data indicate that activation of TTX-sensitive dendritic
sodium channels is required for AP backpropagation in prefron-
tal pyramidal neurons.
To test for activation of dendritic calcium channels by back-
propagatingAPs,wecomparedantidromicAPsgeneratedbefore
andduringbathapplicationofCdCl2(200?M;n?12)(Fig.3B).
Tocontrolforthepotentialeffectofcadmiumapplicationonthe
tonic activation of excitatory and inhibitory dendritic synapses,
insixoftheseexperimentsweincluded3mMkynurenicacidand
20 ?M bicuculline in the recording saline. Because there were no
significantdifferencesbetweenresultsobtainedwithandwithout
synaptic antagonists, the data were grouped together. Cadmium
application caused a small but significant increase in the ampli-
tude of dendritically recorded APs of 6.1 ? 2.4% (n ? 12; p ?
0.05). In addition, while having no significant effect on back-
propagatingAPrisetime(10–90%;meanchange,?4.5?3.3%)
orwidthathalf-amplitude(meanchange,9.7?8.2%),cadmium
application significantly increased the width of the late decay
phase of the AP (Fig. 3B). When dendritic AP base-width was
measured at 15% of peak amplitude, cadmium application pro-
duced a 38 ? 7% increase in AP width (n ? 12; p ? 0.05). These
effects of cadmium are opposite to what would be expected if AP
backpropagation required activation of dendritic voltage-
Figure1.
recordingsfromthesoma(bottom)andapicaldendrite(top;360?mfromthesoma)duringAP
trains generated by depolarizing current steps (500 msec; 650 pA) delivered via the somatic
recordingelectrode.B,ThefirstAPsfromthetrainsshowninAwereenlargedtodemonstrate
thatAPswererecordedfirstatthesomaandsubsequentlyintheapicaldendrite.C,Timedelay
betweensomaticanddendriticAPpeakversusdistancefromthesoma.D,MeanAPconduction
velocityversusdistancefromthesoma.E,PlotofAPattenuationwithdistancefromthesoma.
F,Plotofsteady-stateattenuationwithdistancefromthesoma.C–F,n?31.
PropertiesofAPbackpropagationinprefrontalpyramidalneurons.A,Whole-cell
Figure 2.
fromthesoma)ofAPtrainsgeneratedbyantidromicstimulationatfrequenciesof10,20,50,
and 100 Hz. B, Plot of AP amplitude versus timing for the trains shown in A. C, Ratio of the
amplitudeofthetenthversusfirstAPduring50Hztrainsasafunctionofdistancefromthesoma
(n?11).
AP backpropagation is frequency dependent. A, Dendritic recordings (310 ?m
GulledgeandStuart•APPropagationinPrefrontalApicalDendritesJ.Neurosci.,December10,2003 • 23(36):11363–11372 • 11365

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activated calcium channels. The small increase in dendritic AP
amplitude in cadmium may be attributable to a nonspecific ac-
tion of cadmium on fast-inactivating potassium channels (Bek-
kers, 2000), whereas the increase in dendritic AP base-width is
presumablyattributabletoblockofcalcium-activatedpotassium
channels.Becausecadmiumapplicationhadsimilareffectsonthe
APatthesoma,thechangestobackpropagatingAPsincadmium
arelikelytobesecondarytochangesinthesomaticAPwaveform.
Additional experiments will be required to determine the poten-
tial role of dendritic voltage- and calcium-activated potassium
channels in regulation of AP backpropagation in prefrontal py-
ramidal neurons. Despite this complication, our findings with
cadmium demonstrate that backpropagating APs do not require
activation of dendritic calcium channels. This conclusion is con-
sistent with previous reports showing minimal calcium electro-
genesis in the apical dendrites of prefrontal pyramidal neurons
(Seamans et al., 1997; Gonzalez-Burgos and Barrionuevo, 2001).
Together, the above data demonstrate that active backpropaga-
tion of APs into the apical dendrite requires activation of den-
driticsodiumchannelsandisnotassociatedwithsignificantden-
dritic calcium channel activation.
Dendriticspike initiation
When the apical dendrites of somatosensory or CA1 hippocam-
pal pyramidal neurons are sufficiently depolarized, local regen-
erative events can occur that precede somatic AP generation
(Schiller et al., 1997; Stuart et al., 1997; Golding and Spruston,
1998). In somatosensory or CA1 pyramidal neurons, these local
dendritic spikes are mediated by the activation of dendritic so-
dium and calcium channels. To determine whether strong den-
driticdepolarizationcanalsoinitiatedendriticspikesinprefron-
tal pyramidal neurons, we depolarized dendrites via current
injection through the dendritic recording electrode or via distal
synaptic stimulation in layers 2/3 (Fig. 4).
APs generated by brief (3–5 msec) current steps of increasing
amplitude via the apical dendritic recording pipette (120–480
?m from the soma) were observed first by the somatic recording
pipetteandthensubsequentlybythedendriticrecordingpipette,
indicating an axo-somatic site of AP initiation (Fig. 4A, top).
Increasingthemagnitudeofdendriticcurrentinjectionledtothe
initiationofdendriticspikesthatprecededsomaticAPs(Fig.4A,
bottom) (n ? 15). Similarly, synaptic stimulation delivered to
layers 2/3 at low stimulation intensity led to generation of APs
that were observed first by the somatic recording pipette and
subsequently backpropagated into the dendrites (Fig. 4B, top).
Increasing the intensity of synaptic stimulation resulted in the
initiation of dendritic spikes that preceded somatic APs (Fig. 4B,
bottom) (n ? 7). That the synaptic responses in these experi-
ments were of dendritic origin was supported by the observation
that EPSPs recorded at dendritic locations (175–480 ?m from
thesoma)werealwayslargerthanthosedetectedatthesoma(n?
7 of 7 cells). Local dendritic spikes were never observed in isola-
tion and were always followed with a short delay by APs detected
at the somatic recording site, suggesting that dendritic spikes in
prefrontal pyramidal neurons can propagate to the soma and
have a direct impact on axonal AP initiation.
Figure3.
but not calcium channels. A, Somatic (bottom) and dendritic (top; 230 ?m from the soma)
recordingsofsomaticallygeneratedAPsbefore,during,andafterlocaldendriticapplicationof
TTX (1 ?M). Inset, Superimposed traces. B, Dendritic recording (205 ?m from the soma) of
antidromicallygeneratedAPsbefore(left),during(middle),andafter(right)bathapplicationof
cadmium (200 ?M). Note the increased amplitude and base-width at 15% amplitude. Inset,
Superimposedtraces.
DendriticpropagationofAPsisactiveandrequiresvoltage-gatedsodiumchannels
Figure4.
APs. A, Somatic (left), dendritic (middle; 175 ?m from the soma), and superimposed (right;
note different time scale bar) recordings of APs generated by just-suprathreshold dendritic
currentinjection(top)oralargerdendriticcurrentinjectioncapableofgeneratingadendriti-
callyinitiatedspike(bottom).B,Somatic(left)anddendritic(middle;185?mfromthesoma)
responsestoweak(top)orstrong(bottom)distaldendriticsynapticstimulation(deliveredin
layer2/3).Thesuperimposedtraces(right;differenttimescalebar)demonstratethatstrong
synapticinputcangeneratedendriticspikesthatprecedesomaticactionpotentials.
Strongdendriticdepolarizationcaninitiatedendriticspikesthatprecedesomatic
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Infourcasestested,focalorbathapplicationofcadmium(200
?M) had no effect on dendritic spikes generated by dendritic
current injection, suggesting that, similar to backpropagating
APs, dendrite spikes are mediated by voltage-activated sodium
channels rather than calcium channel activation (data not
shown).Consistentwiththisobservation,thetimecourseofden-
driticspikeswassimilartothatofbackpropagatingAPs(compare
Figs. 1B and 4A). Together, these data demonstrate that strong
dendritic depolarizations in prefrontal pyramidal neurons can
initiate local dendritic spikes mediated primarily by dendritic
sodium channels, and that these dendritic events can directly
influence axonal AP generation.
DopaminedoesnotmodulateAP backpropagation
The above data suggest a critical role for voltage-gated sodium
channels in both AP backpropagation and dendritic spike gener-
ation in the apical dendrites of prefrontal pyramidal neurons.
Dopamine, an important neuromodulator in prefrontal cortex,
has been reported to both increase (Yang and Seamans, 1996;
Gorelova and Yang, 2000) and decrease (Geijo-Barrientos and
Pastore,1995;Mauriceetal.,2001)sodiumchannelactivationin
prefrontal pyramidal neurons. As a consequence, dopamine
would be expected to modulate AP backpropagation and den-
dritic spike generation. To test this, we investigated the effect of
bath application of dopamine (10 or 100 ?M; 5–10 min) on AP
backpropagation. As reported previously (Penit-Soria et al.,
1987; Geijo-Barrientos and Pastore, 1995; Yang and Seamans,
1996; Shi et al., 1997; Gulledge and Jaffe, 1998, 2001; Wang and
O’Donnell, 2001), dopamine application at 10 ?M induced a va-
riety of effects on prefrontal layer 5 pyramidal neurons in vitro,
including depolarization, a decrease in input resistance, and
modulation of excitability during somatic current injection (Ta-
ble 1). Surprisingly, however, dopamine application did not sig-
nificantly affect AP backpropagation into the apical dendrites of
these neurons (Fig. 5). In the presence of dopamine, the ampli-
tudes of dendritically recorded backpropagating APs (50–480
?mfromthesoma)were98?1%ofbaselinevalues(controlAP
amplitude, 69 ? 3 mV; AP amplitude in dopamine, 68 ? 3 mV;
n ? 25). We also investigated a possible distance dependence to
the action of dopamine by comparing changes in AP amplitude
and half-width during dopamine application as a function of
distance from the soma (Fig. 5B,C). Dopamine had no signifi-
cant effect on either dendritic AP amplitude or half-width at
distancesupto480?mfromthesoma.Inadditionalexperiments
using antidromic stimulation, dopamine also failed to have any
significant effect on the width of backpropagating APs at 15% of
peak amplitude (mean change, ?8.4 ? 3.1%; n ? 11).
To test whether dopamine had an effect on frequency-
dependent depression of backpropagating APs, we bath-applied
dopamine during high-frequency trains of 10 APs generated at
10, 20, 50, and 100 Hz (Fig. 6). Because frequency-dependent
depression of backpropagating APs is attributable to cumulative
sodium channel inactivation (Colbert et al., 1997; Jung et al.,
1997), one would expect that small changes in sodium channel
availability would have a significant effect on the amplitude of
backpropagating APs during high-frequency trains. Dopamine
applied under these conditions might expose an otherwise mod-
estdopamine-inducedmodulationofdendriticsodiumchannels
that is insufficient to affect single backpropagation APs. As seen
inFigure6,however,dopaminehadnosucheffectonfrequency-
dependentdepressionofbackpropagatingAPs(n?11).Thiswas
true for all frequencies (Fig. 6B) and at all dendritic recording
locations (Fig. 6C).
In the above experiments, 10 ?M dopamine had no effect on
AP backpropagation. However, it is possible that 10 ?M is not a
concentration that is high enough to modulate backpropagating
action potentials. To maximally activate dopamine receptors, in
six experiments, we bath-applied dopamine at a high concentra-
tion (100 ?M) in the presence of the dopamine uptake blocker
nomifensine(1?M).Thishigherconcentrationofdopaminehad
no significant effect on AP backpropagation. Mean change in AP
amplitude was ?0.5 ? 1.1% at the soma and ?1.6 ? 0.7% at
dendritic locations (120–410 ?m from the soma). Similarly, we
observed no significant effect of dopamine (100 ? 1 ?M nomi-
fensine) on dendritic AP half-width or rise time (n ? 6; data not
shown).
These experiments rule out a role for dopamine in the modu-
Table1.Summaryofstatisticallysignificanteffectsofdopamine(10?M)onpyramidalneuronphysiology
n(control/DA)Control10?MDAChangeinDA(p?0.05forall)
VM(soma)
VM(dendrite)
RN(soma)
RN(dendrite)
Excitability(withdepolarization)
Excitability(hyperpolarized)
37/25
40/36
8
11
21
12
?80.4?0.5mV
?77.9?0.6mV
88?15M?
55?6M?
5.2?0.4spikes
5.8?0.6spikes
?78.1?0.7mV
?74.8?0.6mV
80?6M?
46?6M?
6.5?0.8spikes
2.7?0.9spikes
2.5?0.4mV
3.0?0.4mV
?7.7?2.5%
?16.9?2.0%
20?9%
?58?13%
Excitabilitywasmeasuredeitherduringdopamine-induceddepolarization(withdepolarization)orwiththemembranepotentialadjustedtocontrollevelswithasomaticDCbiascurrent(hyperpolarized).Excitabilitywasmeasuredas
numberofspikesinresponsetoagivensomaticcurrentinjection(?300pA,1sec).RN,Inputresistance;VM,restingmembranepotential.
Figure5.
(360?mfromthesoma)ofAPsgeneratedbysomaticcurrentinjectionbefore(top)andduring
(middle)bathapplicationof10?Mdopamine.Superimposedtraces(bottom)demonstratethe
absence of an effect of dopamine on AP waveform. B, C, Change in AP amplitude (B) and
half-width(C)duringdopamineapplicationversusdistancefromthesoma.Dashedlinerepre-
sentszerochange.Solidlinesarelinearregressionfitstothedata.
DopaminedoesnotmodulatesinglebackpropagatingAPs.A,Dendriticrecording
GulledgeandStuart•APPropagationinPrefrontalApicalDendritesJ.Neurosci.,December10,2003 • 23(36):11363–11372 • 11367

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lation of backpropagating APs. To test whether dopamine could
affect the local generation of dendritic spikes, we bath-applied
dopamine (10 ?M) under conditions in which dendritic spikes
weregeneratedbypowerfuldendriticcurrentinjectionorsynap-
tic stimulation. In seven of seven cells, the presence of dopamine
had no effect on the initiation of dendritic spikes by strong den-
dritic depolarization via either direct current injection (Fig. 7A)
(n ? 5) or strong synaptic activation of the apical dendrite (Fig.
7B) (n ? 2).
Calciumimagingofbackpropagating APs
Although the experiments described above demonstrate that
dendriticvoltage-activatedcalciumchannelsdonotplayasignif-
icantroleinAPbackpropagationanddendriticspikeinitiationin
prefrontal cortex pyramidal neurons, previous data in both hip-
pocampal and somatosensory cortical pyramidal neurons indi-
cate that activation of dendritic calcium channels by backpropa-
gating APs can lead to a significant rise in intracellular calcium
(Jaffeetal.,1992;Schilleretal.,1995).Toinvestigatewhetherthis
isalsothecaseinprefrontalpyramidalneurons,weusedconfocal
microscopy and a calcium-sensitive fluorescent dye (Oregon
Green BAPTA-1; 200 ?M) to image changes in intracellular cal-
cium at different locations along the somatodendritic axis (Fig.
8A): at the intersection of the soma and apical dendrite (n ? 5),
in the apical trunk (?200 ?m from the soma; n ? 5), and on
primary or secondary branches of the apical tuft (400–600 ?m
from the soma; n ? 5). Somatic current injection was used to
generate either single APs or trains of three to five APs (50 Hz)
andlinescansusedtoaccesschangesinfluorescence(Fig.8B).As
is the case in both hippocampal and somatosensory cortical py-
ramidal neurons, backpropagating APs generated transient rises
in intracellular calcium at all dendritic locations examined (Fig.
8C). This indicates that backpropagating APs in prefrontal pyra-
midalneuronsactivatedendriticvoltage-activatedcalciumchan-
nels, although their activation is not required to sustain back-
propagation. The magnitude of AP-induced calcium signals was
dependent on location, presumably resulting from differences in
surface/volume ratio, local dye concentration, and the extent of
AP backpropagation. Importantly, in the proximal tuft, we ob-
served that the response to five APs was often similar to, or not
muchlargerthan,theresponsegeneratedbyasingleAP(Fig.8C,
bottom). This is presumably a consequence of frequency-
dependent attenuation of AP backpropagation at these locations
(Fig. 2).
Previous data suggest that dopamine can downregulate cal-
cium channels in the apical dendrites of prefrontal pyramidal
neurons(YangandSeamans,1996).Wethereforetestedthepos-
sibility that dopamine might modulate the rise in intracellular
calcium generated by backpropagating APs (Fig. 9). To correct
for time-dependent decreases in ?F/F (Fig. 9A), which could
influence the interpretation of experiments with dopamine, we
compared the calcium signals induced by backpropagating APs
in the presence of dopamine with the average of AP-induced
calcium transients observed before and after dopamine applica-
tion (Fig. 9B,C) (see Materials and Methods). As summarized in
Figure 9D, no significant effect of dopamine was observed on
AP-induced calcium influx for either single or multiple APs at
any of the tested dendritic locations (n ? 5 for each location). In
separate control experiments, focal TTX application (1 ?M) to
theapicaldendrite(200?mfromthesoma)reversiblydecreased
backpropagatingAP-inducedcalciuminfluxduringtrainsoffive
APsby84?9%(n?3;datanotshown),indicatingthatchanges
in backpropagating AP-induced calcium influx can be detected
with our system. The absence of an effect of dopamine on back-
Figure6.
ingAPs.A,TrainsofantidromicAPs(10,20,50,and100Hz)recordedintheapicaldendrite(250
?mfromthesoma)before(black)andduring(gray;offset)bathapplicationof10?Mdopa-
mine. B, Plot of AP amplitude versus timing for the four trains shown in A. C, Ratio of the
amplitudeofthetenthversusfirstAPduring50Hztrainsasafunctionofdistancefromthesoma
incontrol(black)anddopamine(gray)conditions.
Dopaminedoesnotmodulatefrequency-dependentdepressionofbackpropagat-
Figure7.
(middle; 210 ?m from the soma), and superimposed (right; note different time scale bar)
responses to a strong dendritic current injection in the presence of 10 ?M dopamine. B, Re-
sponsesfromthesoma(left)anddendrite(middle;300?mfromthesoma)tostrongsynaptic
stimulation delivered to layer 2/3 in the presence of 10 ?M dopamine. Superimposed traces
(right)showadendriticspikeprecedingthesomaticAP.
Dopaminedoesnotblockinitiationofdendriticspikes.A,Somatic(left),dendritic
11368 • J.Neurosci.,December10,2003 • 23(36):11363–11372GulledgeandStuart•APPropagationinPrefrontalApicalDendrites

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propagating AP-induced calcium influx is consistent with our
observation of no effect of dopamine on electrically recorded
backpropagating APs. In addition, these imaging data demon-
strate that dopamine does not modulate dendritic voltage-
activated calcium channels activated by backpropagating APs.
Finally, we explored AP backpropagation into the distal tuft
near the outer edge of layer 1 (Fig. 10A) (150–250 ?m from the
base of the apical tuft). At these distal locations, single APs or AP
trainsatlowfrequencies(50Hz)failedtoproduceanyobservable
rise in intracellular calcium. Detectable rises in intracellular cal-
ciumwereonlyobservedduringhigherfrequencyAPbursts(Fig.
10B,C) (3–6 APs at 200 Hz; n ? 5). By analogy with similar
findingsinlayer5pyramidalneuronsfromsomatosensorycortex
(Larkum et al., 1999b), this suggests that AP backpropagation
into the distal tuft of layer 5 pyramidal neurons in the prefrontal
cortex is frequency dependent and requires high-frequency (200
Hz) AP burst firing. In general, peak ?F/F values were smaller at
moredistallocationsandrequiredagreaternumberofAPsinthe
burst to evoke a detectable rise in calcium (Fig. 10D). In four of
theseexperiments,dopamine(10?M)wasbathappliedtotestfor
modulation of calcium signaling in the distal tuft. As observed at
more proximal locations, dopamine had no significant effect on
theoccurrenceormagnitudeofcalciumsignalsatthesedistaltuft
locations (Fig. 10E,F).
Discussion
Activepropagationin dendrites
The ability of axonally generated APs to actively propagate into
dendriteshasbeenassessedinanumberofneuronaltypesfroma
varietyofbrainregionsandhasbeenfoundtobestronglydepen-
dent on the specific type of neuron studied. Some neurons, such
ascerebellarPurkinjecells,exhibitnoactiveAPbackpropagation
(StuartandHa ¨usser,1994),whereasotherneurons,suchasmid-
brain dopaminergic neurons (Ha ¨usser et al., 1995) and mitral
cellsintheolfactorycortex(BischofbergerandJonas,1997;Chen
et al., 1997), show robust nondecremental AP backpropagation.
Pyramidal neurons from the somatosensory cortex and CA1 re-
gionofthehippocampusexhibitmoderateAPpropagation(Stu-
art and Sakmann, 1994; Spruston et al., 1995), which is strongly
dependent on ion channel expression and morphology (Magee
and Carruth, 1999; Golding et al., 2001; Vetter et al., 2001; Tsay
and Yuste, 2002).
Figure8.
prefrontalneurons.A,Areconstructedprefrontallayer5pyramidalneuronshowingsomatic,
dendritic,andtuftlocationsoflinescansusedtodetectcalciumsignalsinresponsetosomatic
APs.B,InacellfilledwithOregonGreenBAPTA-1(200?M),somaticcurrentinjectionproduces
APs(top)thatresultinacalcium-dependentchangeinfluorescenceimagedusingalinescanat
the intersection of the soma and apical dendrite (bottom). C, Plot of ?F/F in response to a
similarspiketrainimagedatthreelocations:theintersectionofthesomaandapicaldendrite
(top),intheapicaldendrite(middle;?200?mfromthesoma),andinaproximalbranchofthe
apicaltuft(bottom;?500?mfromthesoma).Dataarefromthreedifferentneurons.
BackpropagatingsomaticAPscausecalciumtransientsintheapicaldendritesof
Figure9.
normalizedaveragepeak?F/F(?SEM;n?3)inresponsetosomaticAPsimaged200?m
fromthesoma.Notethetime-dependentdecreasein?F/F.B,Plotofpeak?F/Fduringsingle
(triangles) and trains of five APs (circles) during dopamine (DA) application at the indicated
time(bar).C,Comparisonofaveraged?F/Ftracesincontrolconditions(black;beforeandafter
dopamine)andduringdopamineapplication(gray).Tracesshownareaveragesoftrialsatthe
timesindicatedbytheblackandgrayarrowsinB.D,Summarygraphsshowingalackofeffectof
dopamineondendriticcalciumtransientsevokedbysingleAPsortrainsoffiveAPsatthesoma
(top),200?mintotheapicaldendrite(middle),andintheproximalapicaltuft(bottom).
Dopaminedoesnotmodulatecalciumsignalingintheapicaldendrite.A,Plotof
GulledgeandStuart•APPropagationinPrefrontalApicalDendritesJ.Neurosci.,December10,2003 • 23(36):11363–11372 • 11369

Page 8

Here, we show that the apical dendrites of layer 5 pyramidal
neurons of the prefrontal cortex are also capable of supporting
active AP backpropagation, suggesting that AP backpropagation
may be a common feature of all pyramidal neurons. AP back-
propagation into the apical dendrites of prefrontal neurons was
found to have several characteristics similar to AP backpropaga-
tion observed in somatosensory cortex. Backpropagating AP
conductionspeedandmagnitudeofAPattenuationduringsingle
APs, as well as AP-induced calcium influx, were similar to previ-
ous findings in somatosensory layer 5 pyramidal neurons
(Schiller et al., 1995; Stuart et al., 1997). In addition, AP back-
propagation into the most distal dendrites was found to be fre-
quency dependent, requiring high-frequency AP bursts, as is the
case in distal dendrites of pyramidal neurons from somatosen-
sory cortex (Larkum et al. 1999b; Williams and Stuart, 2000). In
contrast, we found no evidence for either dendritic calcium elec-
trogenesis during high-frequency AP firing or initiation of den-
dritic calcium spikes, which are common features of the distal
apical dendrites of layer 5 pyramidal neurons from somatosen-
sory cortex (Amitai et al., 1993; Kim and Connors, 1993; Schiller
et al., 1995; Stuart et al., 1997; Larkum et al., 1999a,b, 2001; Wil-
liams and Stuart, 1999, 2000, 2002). These differences suggest
that dendritic calcium electrogenesis is significantly reduced in
prefrontal cortex layer 5 pyramidal neurons in comparison with
layer 5 pyramidal neurons from somatosensory cortex. This
conclusion is consistent with previous reports of little, if any,
calcium electrogenesis in the apical dendrite of prefrontal py-
ramidal neurons (Seamans et al., 1997; Gonzalez-Burgos and
Barrionuevo, 2001).
Lackofmodulationby dopamine
Because dopamine has previously been reported to either facili-
tate (Yang and Seamans, 1996; Gorelova and Yang, 2000) or de-
press (Geijo-Barrientos and Pastore, 1995; Maurice et al., 2001)
sodium currents in prefrontal pyramidal neurons, we were espe-
ciallyinterestedinexaminingtheeffectofdopamineonAPback-
propagation. In our experiments, bath application of dopamine
ledtochangesinrestingpotential,inputresistance,andexcitabil-
ity(Table1),consistentwiththeactionsofdopamineonprefron-
tal cortex pyramidal neurons reported previously (Penit-Soria et
al.,1987;Geijo-BarrientosandPastore,1995;YangandSeamans,
1996; Shi et al., 1997; Gulledge and Jaffe, 1998, 2001; Wang and
O’Donnell, 2001). Despite these actions of dopamine, we ob-
served no change in the amplitude or kinetics of dendritically
recorded backpropagating APs or any effect on dendritic spike
initiation. In addition, dopamine had no effect on frequency-
dependent attenuation of backpropagating APs during high-
frequency AP trains, in which small changes in dendritic sodium
channel availability should be easily detected. Consistent with
previousfindings(GulledgeandJaffe,2001),wealsoobservedno
effect of dopamine on the amplitude or kinetics of somatic APs.
Together, these data demonstrate that dopamine does not mod-
ulate the sodium channels that sustain AP initiation or back-
propagation in prefrontal layer 5 pyramidal neurons. Dopamine
also fails to modulate sodium channel-dependent boosting of
artificial EPSPs generated by current injection into the apical
dendritesofprefrontalpyramidalneurons(G.Gonzalez-Burgos,
personalcommunication).Theexplanationforwhyweobserved
no effect of dopamine on AP initiation and backpropagation,
despite previous work showing dopaminergic regulation of so-
dium channels in prefrontal pyramidal neurons (Geijo-
BarrientosandPastore,1995;YangandSeamans,1996;Gorelova
and Yang, 2000; Maurice et al., 2001), remains to be determined.
Becausedopaminehasbeenshowntoreducecalciumchannel
activation in the prefrontal cortex (Yang and Seamans, 1996) as
well as other cells types (Sah and Bean, 1994), we suspected that
dopamine might depress dendritic calcium transients generated
bybackpropagatingAPs.However,calciumimagingexperiments
demonstrated no such effect of dopamine at any of the dendritic
locations examined. Thus, we find no evidence in favor of the
notion that dopamine modulates active conductances in the api-
caldendritesofprefrontalpyramidalneurons.Thisconclusionis
consistent with the finding that dopaminergic input to the pre-
frontal cortex is made preferentially to the deeper cortical layers,
closetothesomaoflayer5neurons,ratherthaninthesuperficial
layers containing the apical dendrites of these neurons (Berger et
al., 1991). However, it should be noted that our data do not rule
Figure10.
prefrontal layer 5 pyramidal neuron showing location of line scans in the distal apical tuft
(470–780?mfromthesoma).B,OnetofiveAPsat200Hzweregeneratedbybriefsomatic
current injections (B1), whereas simultaneous line scans taken across dendrites in the distal
apicaltuftproducethecorrespondingcalciumtransients(B2).C,Plotsofaverage?F/Fforfive
trialssimilartothatshowninB.NumbersrefertosomaticAPsgeneratedpertrial.Notethata
detectablecalciumsignalisonlyobservedduringburstsofthreeormoreAPs.D,Plotofpeak
?F/Fduringhigh-frequencyAPburstsasafunctionofdistancefromthesoma.Thenumbers
indicate the minimum number of APs needed to evoke a detectable calcium signal at each
location. E, Plot of average ?F/F during AP bursts of three to six APs (200 Hz) under control
conditions(black)andduringbathapplicationof10?Mdopamine(gray).F,Summaryofpeak
?F/Fincontrolanddopamine(10?M;n?4)conditionsatdistaltuftlocationsduringhigh-
frequencyAPbursts.TheminimumnumberofAPsneededtoevokeadetectablecalciumsignal
undercontrolconditionswasusedforcomparisonofcontrolanddopamineresponses.
APbackpropagationintothedistaltuftisfrequencydependent.A,Reconstructed
11370 • J.Neurosci.,December10,2003 • 23(36):11363–11372GulledgeandStuart•APPropagationinPrefrontalApicalDendrites

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outthepossibilitythatdopaminemightmodulateAPbackpropa-
gation or initiation in more developmentally mature animals
(i.e., those more than 5 weeks of age).
Functional implications
One hypothesis regarding dopaminergic action during work-
ing memory tasks is that dopamine “uncouples” inputs to the
apical dendrite from the rest of the neuron by downregulating
active dendritic conductances (Yang and Seamans, 1996;
Durstewitz et al., 2000; Dreher et al., 2002). The data pre-
sented here argue against this hypothesis, because we find no
evidence for dopamine modulation of the dendritic sodium
conductances that support AP backpropagation in prefrontal
pyramidal neurons. Furthermore, we find no evidence for
modulation of the dendritic calcium conductances activated
by backpropagating APs and responsible for AP-induced in-
creases in dendritic calcium.
Although our data suggest that dopaminergic modulation of
dendritic conductances does not underlie the persistent activity
associatedwithworkingmemory,APbackpropagationitselfmay
play an important role in maintaining sustained activity in pre-
frontal pyramidal neurons during the delay periods associated
with working memory tasks. Working memory delay periods are
associated with irregularly timed high-frequency burst dis-
charges(Compteetal.,2003).Suchirregularhigh-frequencydis-
charges promote AP backpropagation into the distal apical den-
dritesofsomatosensorylayer5pyramidalneurons(Williamsand
Stuart,2000),andourdatasuggestthisisalsothecaseinprefron-
tallayer5pyramidalneurons(Fig.10).AmplificationofAPback-
propagation into distal dendrites during high-frequency dis-
chargeswillincreasetheinteractionofbackpropagatingAPswith
concurrent excitatory synaptic input, which could, in principle,
further enhance the rate of AP output (Larkum et al., 1999a;
Stuart and Ha ¨usser, 2001). Such interactions between back-
propagating APs and excitatory synaptic inputs onto the distal
dendrites of prefrontal pyramidal neurons is likely to be en-
hanced during working memory tasks, because transient in-
creases in extracellular dopamine (Watanabe et al., 1997) may
enhance the NMDA component of excitatory responses (Sea-
mansetal.,2001;WangandO’Donnell,2001).Inthisway,active
AP backpropagation in prefrontal pyramidal neurons may facil-
itatethedevelopmentandmaintenanceofpersistentactivitydur-
ing tasks requiring working memory.
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