Download Dopamine Increases Excitability of Pyramidal Neurons in Primate

Dopamine Increases Excitability of Pyramidal Neurons in Primate
Prefrontal Cortex
DARRELL A. HENZE, GUILLERMO R. GONZÁLEZ-BURGOS, NATHANIEL N. URBAN, DAVID A. LEWIS,
AND GERMAN BARRIONUEVO
Departments of Neuroscience and Psychiatry and Center for Neural Basis of Cognition, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260
Received 7 June 2000; accepted in final form 10 August 2000
Several lines of evidence indicate that the dorsolateral prefrontal cortex (PFC) plays a critical role in working memory.
First, surgical removal of tissue from the principal sulcal
region of the monkey PFC impairs performance in behavioral
tasks that engage working memory (Butters et al. 1971; Funahashi et al. 1993; Goldman et al. 1971; Levy and GoldmanRakic 1999; Passingham 1975). Second, single units in the
monkey PFC exhibit task-related changes in firing rate during
delayed-response tasks (Fuster and Alexander 1971; Kubota
and Niki 1971). Furthermore PFC units show a delay-related
increase in firing rate that correlates with correct task perfor-
mance (Funahashi et al. 1989; Fuster 1973) and is less sensitive
to disruption by intervening stimuli than delay activity in other
cortical regions (Miller et al. 1996). Together these data indicate that PFC cell activity is a leading candidate for a cellular
basis of working memory (Funahashi and Kubota 1994; Goldman-Rakic 1995).
The neurotransmitter dopamine (DA) appears to play an
important role in the regulation of working memory-related
processes in the PFC. For example, disruption of the PFC DA
system via local depletion or pharmacological antagonism at
the principal sulcus region impairs working memory in monkeys (Brozoski et al. 1979; Sawaguchi and Goldman-Rakic
1991, 1994). Also extracellular levels of DA increase significantly in the monkey PFC during performance of delay tasks
(Watanabe et al. 1997). Finally, the delay-task-related activity
of monkey PFC units is modulated by DA receptor stimulation
(Sawaguchi et al. 1990a,b; Williams and Goldman-Rakic
1995). Thus DA in the PFC seems to play an important role in
both delay-task-related neuronal activity and in working memory performance.
Present understanding of the cellular mechanisms underlying the electrophysiological actions of DA in monkey PFC is
sparse, in part because the currently available evidence comes
only from extracellular recordings in vivo (Sawaguchi et al.
1990a,b; Williams and Goldman-Rakic 1995). All previous in
vitro studies that examined electrophysiological effects of DA
on PFC neurons were performed in the rat medial PFC.
Whereas dorsolateral prefrontal cortical areas in macaque monkeys and humans share multiple characteristics (Petrides and
Pandya 1999), there are significant anatomical and functional
differences between rat medial PFC and monkey dorsolateral
PFC (Preuss 1995). Moreover, the prefrontal DA systems of
primates and rats differ markedly in a number of respects
(Berger et al. 1991; Preuss 1995; Williams and Goldman-Rakic
1998). For example, in rat medial PFC, the density of dopaminergic terminals and receptors is significantly higher in the
deep than in superficial layers, whereas in the monkey and
human dorsolateral PFC, DA receptors and fibers also are
present in high density in the superficial layers (Berger et al.
1991; Lewis and Sesack 1997).
In macaque monkey dorsolateral PFC, the majority (70 –
Address for reprint requests: G. Barrionuevo, Dept. of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260 (E-mail:
[email protected]).
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INTRODUCTION
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0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society
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Henze, Darrell A., Guillermo R. González-Burgos, Nathaniel N.
Urban, David A. Lewis, and German Barrionuevo. Dopamine
increases excitability of pyramidal neurons in primate prefrontal cortex. J Neurophysiol 84: 2799 –2809, 2000. Dopaminergic modulation
of neuronal networks in the dorsolateral prefrontal cortex (PFC) is
believed to play an important role in information processing during
working memory tasks in both humans and nonhuman primates. To
understand the basic cellular mechanisms that underlie these actions
of dopamine (DA), we have investigated the influence of DA on the
cellular properties of layer 3 pyramidal cells in area 46 of the macaque
monkey PFC. Intracellular voltage recordings were obtained with
sharp and whole cell patch-clamp electrodes in a PFC brain-slice
preparation. All of the recorded neurons in layer 3 (n ⫽ 86) exhibited
regular spiking firing properties consistent with those of pyramidal
neurons. We found that DA had no significant effects on resting
membrane potential or input resistance of these cells. However DA, at
concentrations as low as 0.5 ␮M, increased the excitability of PFC
cells in response to depolarizing current steps injected at the soma.
Enhanced excitability was associated with a hyperpolarizing shift in
action potential threshold and a decreased first interspike interval.
These effects required activation of D1-like but not D2-like receptors
since they were inhibited by the D1 receptor antagonist SCH23390 (3
␮M) but not significantly altered by the D2 antagonist sulpiride (2.5
␮M). These results show, for the first time, that DA modulates the
activity of layer 3 pyramidal neurons in area 46 of monkey dorsolateral PFC in vitro. Furthermore the results suggest that, by means of
these effects alone, DA modulation would generally enhance the
response of PFC pyramidal neurons to excitatory currents that reach
the action potential initiation site.
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HENZE, GONZÁLEZ-BURGOS, URBAN, LEWIS, AND BARRIONUEVO
nist SCH23390 but not significantly altered by the D2 antagonist sulpiride, indicating that they require activation of D1like receptors. Portions of these results have been presented in
abstract form (Henze et al. 1997).
METHODS
PFC slice preparation
PFC slices used in these studies were obtained from 13 young adult
male cynomolgus monkeys (Macaca fascicularis). Animals were
treated according to the guidelines outlined in the National Institutes
of Health Guide to the Care and Use of Animals. Following injections
of ketamine hydrochloride (25 mg/kg), dexamethasone phosphate (0.5
mg/kg) and atropine sulfate (0.05 mg/kg), an endotracheal tube was
inserted, and the animal was placed in a stereotaxic frame. Anesthesia
was maintained with 1% halothane in 28% O2/air. A craniectomy was
performed over the dorsal PFC, and a small block of tissue was
carefully excised containing both medial and lateral banks of the
principal sulcus (area 46) as shown in Fig. 1A. The tissue block
removed was ⬃4 ⫻ 6 ⫻ 4 mm and was placed in an ice-cold modified
artificial cerebrospinal fluid [ACSF; composition was (in mM): 230
sucrose, 1.9 KCl, 1.2 Na2HPO4, 33 NaHCO3, 6 MgCl2, 0.5 CaCl2, 10
glucose, and 2 kynurenic acid; pH 7.3–7.4 when bubbled with 95%
O2-5% CO2 gas mixture]. The animal was treated postoperatively
with analgesics and antibiotics as previously described (Pucak et al.
1996). All animals recovered quickly with no overt behavioral deficits. In most cases, the animals underwent the same procedure 2– 4 wk
later to obtain tissue from the opposite hemisphere. During the second
procedure, after the craniectomy, the animal was given an overdose of
pentobarbital (30 mg/kg) and was perfused through the heart with
ice-cold modified ACSF. A tissue block containing the portions of
areas 9 and 46 nonhomotopic to the first biopsy was quickly excised
(see Fig. 1A). No consistent differences were observed between tissue
obtained on either day. Subsequent treatment of the tissue was the
same for both days. Tissue slices from these same animals also were
used in other studies (Gonzalez-Burgos et al. 2000; unpublished
results).
The PFC tissue blocks were glued to the stage of a vibratome
containing modified ACSF at 0 – 4°C, and 400-␮m-thick slices were
cut in the coronal plane. Slices contained portions of both areas 9 and
46 (see Fig. 1). Slices were maintained in a holding chamber at room
temperature for at least 2 h submerged in a standard ACSF solution
consisting of (in mM) 126 NaCl, 2 KCl, 1.2 Na2HPO4, 10 glucose, 2.5
NaHCO3, 6.0 MgCl2, and 1.0 CaCl2. Individual slices were transferred as needed (between 2 and 24 h following slice preparation) to
a submerged chamber where they were constantly superfused with
oxygenated ACSF at 33°C with 1.5 mM MgCl2 and 2.5 mM CaCl2.
In addition, some incubations and all recordings were done in the
continuous presence of 75 ␮M sodium metabisulfite (NaMBS), a
concentration that is effective to prevent oxidation of DA but has no
detectable effects on pyramidal cell physiology (Sutor and ten
Bruggencate 1990).
Intracellular recordings with sharp microelectrode
Sharp microelectrode recordings were obtained from layer 3 cells in
area 46. Microelectrodes were pulled from 1.0- or 1.5-mm-diam
capillaries. The electrode tips (the entire “taper” plus 2 mm of the
electrode body) were filled with 1 M KMeSO4, and the remainder of
the electrode body was back-filled with 1 M KCl to reduce electrode
potential “drift.” As a result, electrode drift was usually ⬍2 mV.
Electrode resistances ranged from 50 to 200 M⍀. In some experiments, the solution at the tip contained 2% neurobiotin. Recordings
were made using an Axoclamp 2A (Axon Instruments, Foster City,
CA) amplifier in bridge mode. Recordings were accepted for analysis
if the neuron had a resting membrane potential (RMP) more negative
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80%) of pyramidal projection neurons that give origin to cortico-cortical output (association and callosal) are located in
layer 3 (Andersen et al. 1985; Schwartz and Goldman-Rakic
1984). Layers 2/3 also contain most of the pyramidal cells that
provide long-distance intrinsic horizontal connections (Gonzalez-Burgos et al. 2000; Kritzer and Goldman-Rakic 1995;
Levitt et al. 1993; Pucak et al. 1996), which mediate intrinsic
excitation that may be essential to delay-related activity of
monkey PFC neurons (Goldman-Rakic 1995; Lewis and
Anderson 1995). These data suggest that dopaminergic regulation of the activity of layer 3 pyramidal neurons could have
a significant functional impact on local excitation in PFC and
its propagation to other neocortical regions. However, with a
few exceptions (Sawaguchi and Matsumura 1985), most previous in vivo studies in monkey dorsolateral PFC did not report
the laminar localization of the recorded cells. In addition,
because deep layers in rat PFC receive the strongest dopaminergic innervation, the majority of studies of DA actions on
pyramidal neurons in vitro have focused on layers 5 and 6. As
a result, substantial evidence has accumulated that indicates
that in PFC, DA can modulate cell activity in the deep layers,
which send output to subcortical targets. In contrast, direct
evidence for dopaminergic modulation in superficial layers,
which convey output signals to other regions of the cerebral
cortex, is scarce.
It is often assumed that the data from the electrophysiological studies of DA actions on rat medial PFC are applicable to
the primate PFC. Indeed although significant differences in
dopaminergic innervation and receptor distribution separate the
PFC of rats and macaque monkeys, DA actions at the single
cell level could be similar in both species. Given the limited
availability of primate PFC tissue for in vitro studies, it is
extremely important to understand which aspects of the cellular
actions of DA in the PFC can be generalized from rodents to
primates. However, up to this point such comparison has not
been possible. In the CNS, DA acts via activation of G-proteincoupled DA receptors, which are highly conserved across
mammalian species, but no ligand-gated receptor channels are
known for DA (Civelli et al. 1993; Missale et al. 1998).
Therefore DA appears not to mediate fast synaptic transmission but to exert neuromodulatory effects. An important mechanism by which neuromodulators act in the neocortex is by
altering the intrinsic excitability of neurons (Hasselmo 1995).
In rats, recent experiments have shown that DA modulates the
excitability of PFC pyramidal neurons in vitro (Ceci et al.
1999; Geijo-Barrientos 2000; Geijo-Barrientos and Pastore
1995; Gorelova and Yang 2000; Gulledge and Jaffe 1998;
Yang and Seamans 1996).
To gain new insights into the neurophysiology of monkey
PFC neurons, recently we have developed a monkey PFC brain
slice preparation (Gonzalez-Burgos et al. 2000). In the present
study, we used this preparation to obtain intracellular voltage
recordings from pyramidal cells in layer 3 of area 46 of the
macaque monkey dorsolateral PFC, using sharp and whole cell
patch-clamp electrodes. We examined whether DA had modulatory effects on the intrinsic excitability and found that DA
enhances the firing response of these neurons to somatic injection of depolarizing current. The increase in excitability is
reflected by a hyperpolarizing shift in action potential threshold
and decreased inter-spike interval during depolarizing current
steps. These effects were blocked by the D1 receptor antago-
DOPAMINE INCREASES EXCITABILITY OF MONKEY PFC NEURONS
than ⫺65 mV and action potentials that crossed 0 mV. All recordings
were digitized at 10 kHz and stored on computer hard-disk for later
analysis using custom designed software in LabView (National Instruments) and Origin (Microcal).
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Whole cell recordings
To obtain whole cell recordings, pyramidal neurons in superficial/
middle layer 3 were identified visually with infrared illumination and
differential interference contrast optics (Stuart et al. 1993) as described previously (Gonzalez-Burgos et al. 2000). Patch pipettes
(⬃4 –7 M⍀) were filled with (in mM): 120 K-methylsulphate, 10 KCl,
10 HEPES, 0.5 EGTA, 4.5 ATP, 0.3 GTP, and 14 phosphocreatine.
Patch-pipette voltage recordings were obtained with an Axoclamp-2A
amplifier (Axon Instruments) operating in bridge mode. Membrane
potential was not corrected for changes in junction potential after
break-in. Whole cell recordings were accepted only if seal resistance
was ⱖ2 G⍀ and if the resting membrane potential was more negative
than ⫺65 mV. Signals were low-pass filtered at 3 kHz, digitized at 10
kHz, and stored on disk for off-line analysis. Data acquisition and
analysis were performed using LabView (National Instruments, Austin, TX).
Data collection and analysis
FIG. 1. Prefrontal cortex (PFC) tissue used in these studies. A: schematic
drawing of the dorsal surface of monkey PFC. The shaded areas indicate the
location and approximate size of the tissue blocks that were removed for these
studies. Numerals and thin lines indicate the approximate locations of different
regions of the PFC. AS, arcuate sulcus; PS, principal sulcus. B: schematic
drawing of a tissue slice used for these experiments. The locations of recordings (all from layer 3 in area 46) are indicated by the dark gray shaded
electrodes. C: photomicrograph of a neurobiotin-labeled layer 3 pyramidal cell
representative of the neurons recorded in the present experiments. Note the
prominent ascending apical dendrite and the high-density of dendritic spines,
characteristic features of cortical pyramidal neurons. In addition, a significant
number of local axonal arborizations with putative synaptic varicosities or
boutons are observed (arrows). Calibration bar ⫽ 150 ␮m.
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Once a satisfactory recording was obtained, input resistance (Rinput)
was measured by passing 300-ms current steps from ⫺0.3 to ⫺0.5 nA
in 0.05- or 0.02-nA steps. Three sweeps were collected at each
amplitude and averaged. The “steady-state” input resistance reported
is the slope of the best-fit line to the linear portion of the relation
between the injected current and the membrane potential at the end of
the step. In some cells, the larger amplitude steps showed some
inward rectification and so were excluded from the fitting procedure.
Action potential threshold and peak amplitude were measured for
each spike in a train evoked by depolarizing current injections. Action
potentials were detected and subsequently measured by first examining the first derivative of the membrane potential for peaks. Once a
peak was detected in the first derivative, the actual peak of the AP was
determined from the point where the first derivative crossed back
through zero. The foot of the AP was determined by looking backward
from the peak in the first derivative to the point where the third
derivative of the membrane potential changed sign from negative to
positive (Fig. 2). If necessary, the RMP was manually clamped at a
“constant” value by passing tonic bias current. This “current clamping” of the RMP was needed because Rinput varied as a function of
RMP (see RESULTS), and thus spontaneous changes in RMP could
result in Rinput changes.
All group data are presented as means ⫾ SE unless otherwise
indicated.
All drugs were prepared as concentrated stocks and added directly
to the perfusing medium. DA stock solutions were prepared fresh
(1000:1) in boiled distilled water with 75 ␮M NaMBS added to
minimize oxidation. Control bath solution contained 75 ␮M NaMBS.
Dopamine, SCH23390, and sulpiride were obtained from RBI
(Natick, MA). All other drugs were from Sigma (St. Louis, MO). In
the experiments in which the time course of DA action was examined,
the delay introduced by the dead space in the perfusion line was
compensated for so that the indicated time of DA application matches
the time in which DA starts to enter and leave the recording chamber.
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HENZE, GONZÁLEZ-BURGOS, URBAN, LEWIS, AND BARRIONUEVO
RESULTS
Histological procedures
After recordings with neurobiotin-filled electrodes were finished,
slices were incubated for 1–3 h in low Ca2⫹-ACSF at room temperature to allow for intracellular diffusion and extracellular washout of
the label. The tissue slices were then immersed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline for 12–16 h. The fixed slices
were then transferred to 0.1 M Na-phosphate buffer, serially resectioned at 50 ␮m, and processed for visualization of the neurobiotin
label using the Vectastain Elite ABC kit and diaminobenzidine, as
previously described (Pucak et al. 1996). Pyramidal cells were identified by the presence of an apical dendrite and a high-density of
dendritic spines (Fig. 1C).
FIG. 3. Membrane responses from layer 3 pyramidal cells to intracellular current steps and dependence of the input resistance of layer 3 pyramidal cells with the cells’ membrane potential.
A: single sweeps showing responses to hyperpolarizing current steps (0.05-nA step increments) from
rest (⫺78 mV). The inward rectification is indicated by 1. B: superimposed single sweeps showing responses to depolarizing steps that bring the
membrane potential near threshold for eliciting
action potential. Note the outward rectification indicated by the 2. The action potentials are
clipped. C: superimposed single sweeps in response to depolarizing steps that exceeded threshold. The cell in A–C had an Rinput of 71.7 M⍀, an
AP peak potential of 4.5 mV, a membrane time
constant of 27 ms, and a threshold of ⫺40.9 mV.
Calibration bars in A, B, and C; vertical: 20 mV,
horizontal: 50 ms. D: resting potential (measured
in the absence of tonic intracellular current injection) vs. Rinput by cell. Each point represents the
resting membrane potential of a single cell in the
database. The best-fit line is indicated and has an R
value of 0.57, P ⬍ 0.0001. E: input resistance data
presented by cell across holding potentials.
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FIG. 2. Determination of action potential threshold. The intracellular action
potential (Vm) is shown in filled squares and solid line. The 1st derivative of the
Vm (V⬘m) is shown in open circles and solid line. The 3rd derivative of the Vm
(V⵮
m) is shown in open triangles and dotted line. The black arrow indicates the
point where the 1st derivative crosses 0 after its peak. The point indicated by
the black arrow was used to assign the peak of the AP (adjacent dotted line).
The gray arrow indicates the point where the 3rd derivative changes from
negative to positive values before the peak of the 1st derivative. The point at
the gray arrow was used to assign the threshold of the action potential (adjacent
dotted line).
Data were collected from 86 neurons recorded in layer 3 of
area 46 (⬃350 – 600 ␮m from the pial surface) as shown
schematically in Fig. 1. None of the recorded cells fired spontaneously at rest. In response to injection of suprathreshold
depolarizing currents, all of the cells exhibited a regular spiking firing mode with marked spike frequency accommodation
(see Figs. 6A and 7), which is typical of pyramidal cells
(Connors and Gutnick 1990). In most cases, the spikes also
showed broadening during the depolarization induced firing
(e.g., Figs. 3 and 6). In every case in which the cells were
labeled intracellularly with neurobiotin, the recovered neuron
had the characteristic morphological features of pyramidal
cells, including an ascending apical dendrite and a high-density
of dendritic spines. A representative example of a neuron
labeled intracellularly with neurobiotin is shown in Fig. 1C.
Several interesting membrane properties were observed in
response to injected current steps. First, a slight inward rectification was usually observed with large hyperpolarizing steps
(Fig. 3A, 1). In addition, an outward rectification was often
observed with depolarizing steps that brought the membrane
potential near action potential threshold (Fig. 3B, 2). Finally,
for single or multiple APs following a long inter-spike interval,
the afterhyperpolarization (AHP) exhibited a slow transition
from the fast to slow varieties of the AHP as can be seen by the
lack of a sharp inflection following the AP (Fig. 3, B and C).
The recorded cells (n ⫽ 86) had average (⫾SD) resting
potentials of ⫺77.2 ⫾ 6.9 mV, for sharp electrode recordings
(n ⫽ 64) and ⫺72.7 ⫾ 4.8 mV for whole cell recordings (n ⫽
22). Average action potential peak potential was 15.8 ⫾ 9.2
(SD) mV. The average (⫾SD) input resistance (Rinput) at rest
was 66.9 ⫾ 18.1 M⍀ for sharp electrode recordings and
56.3 ⫾ 18.8 M⍀ for whole cell recordings. The average (⫾SD)
membrane time constant measured with sharp electrodes
(0.10-nA steps hyperpolarizing from rest) was 22.6 ⫾ 5.8 and
23.5 ⫾ 7.7 ms when measured with whole cell electrodes
DOPAMINE INCREASES EXCITABILITY OF MONKEY PFC NEURONS
tration was increased sequentially for each cell to increase the
yield of collected data, given the limited availability of monkey
PFC tissue slices. Therefore the trend toward greater effects
with larger DA concentrations could be explained by the total
duration of DA exposure. Alternatively, because each concentration was applied for ⱖ10 min (although not 30 min), the
prolonged exposure to DA could have lead to desensitization of
DA receptors and underestimation of the effects observed at
higher concentrations. Indeed as observed in Fig. 5B, in some
neurons the decrease in action potential threshold for a given
DA concentration seemed to be attenuated when the cell experienced a previous exposure to DA.
In a separate series of experiments, we examined the time
course of changes in layer 3 pyramidal cell excitability induced
by a 5-min bath application of DA at a single concentration,
followed by washout. As shown in Fig. 6, the changes in
excitability developed late compared with the timing of application, being generally observed only after ⬃ 10 min following
the onset of application. Therefore with 5-min applications, the
effect was observed during the early washout period (Fig. 6). In
addition, in five of six layer 3 neurons the DA effect was not
reversed after as much as 30 – 40 min of washout (Fig. 6).
Figure 7 shows the frequency-current (F-I) curves for the
waveforms shown in Fig. 5A. The data are presented as instantaneous frequency for each interspike interval since the firing
rate varied during the 350-ms steps. Note that the frequency of
all spikes increased in the presence of DA. DA had no consistent effect on the amplitude or time course of either the inward
or outward rectification that was sometimes observed in control
conditions (see Fig. 3).
We then asked whether activation of D1- or D2-like receptors was necessary for the observed increase in cellular excitability by DA. To address this question, DA was applied in the
presence of either the D1 antagonist SCH23390 (3 ␮M) or of
the D2 antagonist sulpiride (2.5 ␮M). Figure 8 illustrates that
when 50 ␮M DA is added in the continued presence of
SCH23390, there is no statistically significant effect on cell
excitability as measured by changes in threshold (Fig. 8A) or
first ISI (Fig. 8B). In addition, when DA (50 ␮M) was applied
during blockade of D2 receptors by 2.5 ␮M sulpiride, neuron
excitability as measured by spike threshold was still enhanced
significantly. In the presence of sulpiride, DA tended to shorten
the first ISI, although this effect was not statistically significant. Therefore our data suggest that SCH23390-sensitive D1like receptors are necessary for the effect of DA, whereas
sulpiride-sensitive D2-like receptors are not (Fig. 8).
DISCUSSION
FIG. 4. Dopamine (DA) has no effect on the input resistance of layer 3
pyramidal cells. Series of hyperpolarizing and depolarizing current steps (350
ms) were injected in control conditions and after application of DA to the bath.
Input resistance was measured at the resting membrane potential as described
in methods. A: input resistance measured by cell across various DA concentrations. B: group data (⫾SE).
In summary, we have demonstrated that the excitability of
layer 3 pyramidal cells in monkey dorsolateral PFC is modulated by DA. Interestingly, although DA does not directly alter
the resting state of these neurons, it can increase their excitability at low concentrations (as low as 500 nM). The increase
in excitability is reflected by a hyperpolarizing shift in first
spike threshold and a decrease in first ISI for action potentials
evoked by somatic depolarizing current injections. These effects of DA were prevented by SCH23390, suggesting that
they require activation of D1-like receptors but not of receptors
of the D2 family.
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(0.04- to 0.10-nA steps hyperpolarizing from rest). Interestingly, we observed that the Rinput varied as a function of the
membrane potential of the cell, such that it decreased when the
cells were hyperpolarized (Fig. 3, C and D). This was true
across all cells for both the resting potential measured without
intracellular current injection and for a range of holding potentials for a single cell (Fig. 3, D and E). The changes in Rinput
with membrane potential were statistically significant: Rinput at
⫺80 mV ⫽ 63 ⫾ 20 M⍀ (n ⫽ 13); Rinput at ⫺70 mV ⫽ 72 ⫾
24 M⍀ (n ⫽ 12); and Rinput at ⫺60 mV ⫽ 79 ⫾ 23 M⍀ (n ⫽
13) *,# (*, significantly different from Rinput at ⫺80 mV; #,
significantly different from Rinput value at ⫺70 mV, paired
t-test, P ⬍ 0.05). These results are consistent with previously
reported data obtained from hippocampal (Spruston and
Johnston 1992) and neocortical pyramidal cells (Deisz et al.
1991; Sutor and Hablitz 1989).
Bath application of DA (0.5–50 ␮M) produced no significant changes in the RMP of layer 3 pyramidal cells (⫺75.1 ⫾
2.2 mV, RMP during DA application: ⫺76.4 ⫾ 2.5 mV, n ⫽
15; 2-tailed t-test, P ⬎ 0.05). Consistent with an absence of DA
modulation of conductances opened at or near the resting
membrane potential, DA did not produce changes in the Rinput
of the cells, as shown in Fig. 4.
DA application increased the excitability of the cells in
response to depolarizing current steps. Figure 5A illustrates
that in the presence of DA at a concentration as low as 500 nM,
the number of spikes in response to a current injection of fixed
amplitude increased. Significant effects were observed after a
10-min application at the lowest concentration (500 nM). DA
decreased the voltage threshold at which the first action potential was initiated and decreased the inter-spike interval (ISI)
between the first and second evoked spikes (Fig. 5, B and C).
As expected for a receptor-mediated effect, higher concentrations of DA tended to produce larger effects on PFC neuron
excitability. However, in most of the experiments, DA concen-
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HENZE, GONZÁLEZ-BURGOS, URBAN, LEWIS, AND BARRIONUEVO
Potential mechanisms of DA-mediated enhanced excitability
In cortical pyramidal cells, action potentials are usually
initiated in the axon near the soma, suggesting that this membrane region is a critical target for regulation of excitability
(Colbert and Johnston 1996; Stuart et al. 1997). Interestingly,
DA receptor proteins are present in the axon hillock and
adjacent segments of the axon of layer 3 pyramidal cells in
monkey PFC (Bergson et al. 1995). However, dopaminergic
fibers do not frequently contact the soma or proximal axon of
monkey PFC pyramidal neurons (Sesack et al. 1995; Smiley
and Goldman-Rakic 1993). Therefore most axonal or somatic
DA receptors must be located at a distance from the DA
terminals. Indeed most of the DA receptors located in the
dendrites of monkey PFC neurons are located at a distance
from DA containing terminals (Smiley et al. 1994). Therefore
activation of all DA receptors in vivo must be nonsynaptic with
endogenous DA activating receptors far away from the release
sites via a volume transmission mechanism (Zoli et al. 1999).
In general, volume transmission requires binding to high af-
finity receptors, like the G-protein-coupled DA receptors (Civelli et al. 1993; Missale et al. 1998). The D1 excitatory actions
of DA in the rat striatum in vivo have been reported to be
mediated via such a volume transmission mechanism (Gonon
1997).
The conductances present in the initial portions of the axon
are poorly characterized, but Na⫹ channels are known to be
present (Colbert and Johnston 1996). In pyramidal neurons of
rat PFC, the D1-receptor-mediated enhancement of excitability
was proposed to be mediated in part through a shift in the
activation voltage of a persistent Na⫹ current to more hyperpolarized voltages (Gorelova and Yang 2000; Yang and Seamans 1996; but see Geijo-Barrientos and Pastore 1995). Theoretically such a shift in Na⫹ current gating could by itself
explain the change in threshold for action potential initiation
observed in the present study (Dilmore et al. 1999). In neurons
from rat neostriatum, DA exerts a complex effect on excitability via a combined modulation of Ca2⫹ and K⫹ conductances
(Hernandez-Lopez et al. 1997). In rat PFC neurons, DA also
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FIG. 5. Dopamine increases the excitability of layer 3 pyramidal cells. A: responses from a representative pyramidal cell evoked
by a range of depolarizing current steps (0.3–1.0 nA steps in
0.1-nA increments) before and in the presence of DA. B: decrease
in action potential threshold in the presence of increasing concentrations of DA. C: group data for action potential threshold (⫾SE).
D: decrease in the 1st inter-spike interval in the presence of
increasing concentrations of DA. E: group data for 1st inter-spike
interval (⫾SE).
DOPAMINE INCREASES EXCITABILITY OF MONKEY PFC NEURONS
2805
FIG. 6. Time course of the effect of DA on pyramidal neuron excitability.
A: representative voltage recordings obtained from a layer 3 pyramidal neuron
before, during, and after the application of DA (5 ␮M) to the bath. In every
trace, responses were elicited by a 0.4-nA depolarizing step. B: dopamine
induced an increase in the number of action potentials elicited by the step,
which was delayed compared with the timing of DA application. C: a delayed
decrease in spike threshold also was observed. D: the 1st inter-spike interval
was decreased in a delayed manner by DA application. E: the input resistance
did not change significantly during the course of the experiment. None of the
observed changes were reversed after DA was washed out of the chamber.
Similar results were observed in 5 other of a total of 6 layer 3 pyramidal
neurons.
appears to regulate Ca2⫹- and K⫹ -dependent potentials that
may regulate neuronal excitability (Yang and Seamans 1996).
Further work is required to determine the ionic mechanism of
the excitatory effects of DA in primate PFC.
Both D1- and D2-like receptors are present in monkey
dorsolateral PFC, but D1-like binding sites are much more
abundant than D2-like (Goldman-Rakic et al. 1990; Lidow et
al. 1991). Consistent with this receptor distribution, D1- but
not D2-like receptor antagonists impaired working memory
function and antagonized the electrophysiological effects of
DA in vivo (Sawaguchi and Goldman-Rakic 1991, 1994;
Sawaguchi et al. 1990a), suggesting a predominant role of
receptors with D1-like pharmacology in mediating DA effects
in PFC.
However, other work indicates that the pharmacology of D1
and D2 receptors in PFC may be more complex in that the
FIG. 7. Instantaneous frequency intensity curves from a representative pyramidal cell before and after DA. A: control. B: 0.5 ␮M DA. C: 5 ␮M DA.
Each line represents the reciprocal of the inter-spike interval for a certain spike
number in the depolarizing current-evoked spike train. The data for these plots
are from the waveforms shown in Fig. 6A.
Downloaded from http://jn.physiology.org/ by 10.220.33.1 on April 29, 2017
effects of so called “specific” agonists and antagonists do not
always agree with one another (Ceci et al. 1999; Godbout et al.
1991; Sesack and Bunney 1989; Shi et al. 1997). Also certain
effects of DA in rat PFC neurons are mediated by D1-D2
receptor co-activation (Otani et al. 1998, 1999; Rorig et al.
1995; Sugahara and Shiraishi 1999; Vincent et al. 1995). In
monkey PFC, individual pyramidal neurons express both D1and D2-like receptors (Bergson et al. 1995; Mrzljak et al.
1996), and iontophoresis of either D1 or D2 antagonists can
inhibit cell firing of PFC neurons recorded in vivo (Williams
and Goldman-Rakic 1995). The limited availability of monkey
PFC tissue precluded the ability to perform an extensive pharmacological study, therefore the main goal of the present study
was to determine whether DA had any effect on the excitability
of monkey PFC neurons. Our present results suggest that, in
layer 3 neurons of the monkey dorsolateral PFC in vitro,
DA-induced changes in intrinsic excitability are mediated by
D1-like receptors.
In agreement with previous studies in rat PFC (Gorelova and
Yang 2000; Zheng et al. 1999), we found that in most of the
neurons in which we examined the time course of the actions
of DA, the effect was persistent after DA washout. However, in
previous studies of striatal neurons, D1-like receptor-mediated
effects were found to be readily reversed after drug wash out
(Surmeier et al. 1995; Zhang et al. 1998). The mechanisms for
2806
HENZE, GONZÁLEZ-BURGOS, URBAN, LEWIS, AND BARRIONUEVO
the long-lasting effects are at present unclear. D1-like receptors
are traditionally associated with activation of the adenylate
cyclase-cAMP-protein kinase A signaling cascade (Missale et
al. 1998). In striatal neurons, protein kinase A phosphorylates
DARPP32, and phospho-DARPP-32 inhibits phosphatase 1
activity, contributing to the electrophysiological effects of DA
(Schiffmann et al. 1998). Simultaneous protein kinase A activation and phosphatase 1 inhibition may result in persistent
phosphorylation of substrates and thus persistent effects. Interestingly, however, DARPP32 could not be detected in pyramidal neurons from adult monkey PFC in a previous study
(Berger et al. 1990). Layer 3 monkey PFC neurons also seem
to lack D2 receptors that can inhibit adenylate cyclase activity
(Missale et al. 1998) that may reverse the D1 effects in other
neurons. An interesting idea that remains to be tested is that D1
effects are reversed by signal transduction cascades activated
by receptors for other neuromodulators that have been proposed to be critical for correct PFC-dependent short-term
memory function, among them norepinephrine and serotonin
(Arnsten 1998; Goldman-Rakic 1999). Activation of G-protein-linked neurotransmitter receptors typically requires release
by bursts of presynaptic action potentials (Gonon 1997), suggesting that there is little activation of G-protein-linked receptors by spontaneous release of endogenous modulators in brain
slices. Therefore since neuromodulators other than dopamine
were not applied, it is probable that nondopaminergic receptors
were not activated in our slice preparation.
Comparison with previous results in monkey PFC
All the previous studies on the effects of DA on monkey
PFC cell activity were performed using in vivo extracellular
Comparison with previous results in rat PFC
In rat PFC, dopaminergic input is most dense in the deep
layers (Berger et al. 1991), therefore the majority of previous
studies in vitro have been focused on layers 5 and 6. Furthermore early studies have suggested that pyramidal cells in
superficial layers of rat PFC are not responsive to DA receptor
activation (Bunney and Aghajanian 1976; Sesack and Bunney
1989). In contrast, more recent studies indicate that a proportion of neurons in the superficial layers of rat medial frontal
cortex do express DA receptors and are responsive to DA
receptor stimulation (Ariano et al. 1997; Gaspar et al. 1995; Le
Moine and Gaspar 1998; Zhou and Hablitz 1999). These and
our present results support the idea that dopaminergic modulation of superficial layer cell activity is generally found in PFC
across species but that perhaps it is more robust in primates
than in rodents.
Our present results, together with recent data obtained from
rat brain slices (Ceci et al. 1999; Gorelova and Yang 2000;
Yang and Seamans 1996), show that an important effect of DA
on pyramidal neurons in PFC is an enhancement of intrinsic
pyramidal cell excitability, mediated by activation of D1-like
receptors. However, different effects of DA on cellular excitability in rat PFC in vitro were reported across laboratories,
making cross-species comparisons difficult. We (GonzalezBurgos, unpublished results) and others observed that activation of DA receptors can decrease the excitability of pyramidal
neurons in rat medial frontal cortex (Geijo-Barrientos 2000;
Geijo-Barrientos and Pastore 1995; Gulledge and Jaffe 1998;
Zhou and Hablitz 1999). Our present data are in agreement
with the enhanced excitability reported previously by several
groups (Ceci et al. 1999; Gorelova and Yang 2000; Penit-Soria
et al. 1987; Yang and Seamans 1996). An interesting possibility is that the decrease in cell excitability is mediated by
D2-like receptors, as suggested by Gulledge and Jaffe (1998).
In monkey PFC layer 3 pyramidal neurons, this D2-like effect
was not observed with bath application of DA alone or during
blockade of D1-like receptors by SCH23390, suggesting that
D2-like receptors do not depress excitability in these cells.
Indeed in the monkey dorsolateral PFC, D2-like receptors are
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FIG. 8. Effects of DA receptor antagonists SCH23390 and sulpiride on the
effects of DA in layer 3 pyramidal cell excitability. Action potential threshold
and 1st inter-spike interval were measured sequentially, first in control artificial
cerebrospinal fluid, then after 10 min application of the antagonists alone and
then after a 10 min co-application of the antagonist and DA. A1: blockade of
D1 receptors by SCH23390 5 ␮M prevents the change in spike threshold by
DA 50 ␮M. A2: SCH23390 blocks the effect of DA on first inter-spike interval.
B1: sulpiride 2.5 ␮M did not prevent the decrease in spike threshold induced
by DA (50 ␮M). B2: in the presence of sulpiride (2.5 ␮M), DA tended to
produce a decrease in first inter-spike interval, although this change was not
significant. (*, significantly different from control, P ⬍ 0.05. Repeated-measures ANOVA, followed by comparison by Dunnett’s method; n ⫽ 7 cells for
SCH23390 experiments, n ⫽ 9 for sulpiride experiments.)
recordings and iontophoretic application of drugs (Sawaguchi
et al. 1988, 1990b; Williams and Goldman-Rakic 1995). In
some of these studies, DA receptor agonists and antagonists
were shown to enhance or depress, respectively, the firing of
PFC units in awake monkeys (Sawaguchi et al. 1988, 1990a,b).
These findings are consistent with the increased excitability
found in the present in vitro experiments. Williams and Goldman-Rakic (1995) reported that unit firing is decreased by high
doses and increased by low doses of D1 antagonists. One
possible explanation for those results is that depending on the
level of receptor occupancy, DA triggers different mechanisms
at the single-cell level, leading to opposite changes in firing
rate. Alternatively, the changes in firing observed during different levels of D1 antagonism in vivo may result from other
effects of DA in the PFC network, like modulation of excitatory and inhibitory synaptic inputs, as suggested recently based
on immunohistochemical findings (Muly et al. 1998). The lack
of a differential effect of DA concentration in our studies
suggests that the complex effects reported by Williams and
Goldman-Rakic (1995) are mediated by network effects.
DOPAMINE INCREASES EXCITABILITY OF MONKEY PFC NEURONS
found in layer 5 but are barely detectable in the superficial
layers (Goldman-Rakic et al. 1990; Lidow et al. 1991, 1998).
Thus it remains to be tested if in pyramidal neurons from the
deep layers of monkey PFC, DA has D2-like inhibitory effects
such as those reported by Gulledge and Jaffe (1998) for deep
layer PFC neurons in rats.
Functional implications of DA modulation of layer 3
pyramidal cells in primate PFC
We thank D. Melchitzky and M. Brady for histology and reconstruction of
cell morphology.
This work was supported by National Institute of Mental Health (NIMH)
Predoctoral Fellowship MH-10474 to D. A. Henze, a Howard Hughes Medical
Institute Predoctoral Fellowship to N. N. Urban, NIMH Grant MH-51234, and
NIMH Center for the Neuroscience of Mental Disorders Grant MH-45156.
Present addresses: D. A. Henze, Center for Molecular and Behavioral
Neuroscience, Aidekman Research Center, Rutgers University, 197 University
Ave., Newark, NJ 07102; N. N. Urban, Max-Planck-Institut für medizinische
Forschung, Abteilung Zellphysiologie, Jahnstrasse 29, D-69120 Heidelberg,
Germany.
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