Departments of Neuroscience and Psychiatry and Center for Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 µM, 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 µM) but not significantly altered by the D2 antagonist sulpiride (2.5 µM). 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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Goldman-Rakic 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 performance (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 (70-80%) 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-protein-coupled 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 antagonist SCH23390 but not significantly altered by the D2
antagonist sulpiride, indicating that they require activation of
D1-like 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-µm-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 µM 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 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).
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
2 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
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 µM NaMBS added to minimize oxidation. Control bath solution contained 75 µM 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.
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 µm, 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).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data were collected from 86 neurons recorded in layer 3 of area 46 (~350-600 µm 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, ). In addition, an outward rectification was often observed with depolarizing steps that brought the membrane potential near action potential threshold (Fig. 3B,
). 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 (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 µM) 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 concentration 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 10 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 µM) or of the D2 antagonist sulpiride (2.5 µM). Figure 8 illustrates that when 50 µM 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 µM) was applied during blockade of D2 receptors by 2.5 µM 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 D1-like receptors are necessary for the effect of DA, whereas sulpiride-sensitive D2-like receptors are not (Fig. 8).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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 affinity 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 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 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 D1- and 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
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 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.
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 (Gonzalez-Burgos,
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 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
The present results are the first in vitro demonstration
that DA has modulatory effects on the activity of monkey PFC layer 3 pyramidal neurons. Under our experimental conditions, the observed effects of DA are most likely due to direct effects on single cells.
However, under physiological conditions, the DA effects on intrinsic
cell excitability would be combined with any effects it might have on
synaptic transmission. Other data from our laboratory suggest that DA
depresses transmission at glutamatergic synapses onto layer 3 pyramidal
cells in monkey PFC (unpublished results). Whether DA depresses
transmission at excitatory synaptic connections in general or only at a
specific subset of synapses in monkey PFC neurons is still not clear.
In either case, the impact of certain excitatory inputs, those that are
more strongly active or that are not modulated by DA, will be enhanced
relative to that of less active or selectively depressed inputs. This
is consistent with a DA-induced enhancement of the signal-to-noise
ratio during working-memory tasks, which was shown experimentally with
in vivo extracellular recordings from monkey PFC units
(Sawaguchi et al. 1990a,b
). Computational models of
network activity in PFC during working memory tasks have shown that
noise interference can make working-memory storage unreliable
(Camperi and Wang 1998
). Therefore by increasing the
signal-to-noise ratio, DA could make delay-related cell firing less
sensitive to noise and intervening stimuli and thus specifically
improve the storage aspect of working memory (Camperi and Wang
1998
; see also Durstewitz et al. 2000
).
In addition to its role in working memory, PFC neuron activity is
important to other cognitive functions (Miller 1999). In both working memory and other cognitive processes, PFC is likely to act
in dynamic association with other cortices (Fuster 1997
; Miller 1999
; Quintana and Fuster 1999
;
Tomita et al. 1999
). Our present results show that DA
modulates the activity of pyramidal cells in the superficial layers,
which contain most of the cortico-cortical projection neurons of the
monkey PFC (Schwartz and Goldman-Rakic 1984
). Therefore
these findings suggest that DA release in layer 3 is likely to modulate
significantly the interaction between dorsolateral PFC and other
regions of the neocortex.
![]() |
ACKNOWLEDGMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
Address for reprint requests: G. Barrionuevo, Dept. of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260 (E-mail: german{at}pitt.edu).
Received 7 June 2000; accepted in final form 10 August 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|