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The Journal of Neuroscience, March 15, 2000, 20(6):2332-2345
Amphetamine Withdrawal Alters Bistable States and Cellular
Coupling in Rat Prefrontal Cortex and Nucleus Accumbens Neurons
Recorded In Vivo
Shao-Pii
Onn and
Anthony A.
Grace
Departments of Neuroscience and Psychiatry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260
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ABSTRACT |
Repeated amphetamine administration is known to produce changes in
corticoaccumbens function that persist beyond termination of drug
administration. We have found previously that long-term alteration in
dopamine systems leads to changes in gap junction communication,
expressed as dye coupling, between striatal neurons. In this study, the
cellular bases of amphetamine-induced changes were examined using
in vivo intracellular recordings and dye injection in
ventral prefrontal-accumbens system neurons of control and amphetamine-treated rats. Rats that had been withdrawn from repeated amphetamine displayed a significant increase in the incidence of dye
coupling in the prefrontal cortex and nucleus accumbens, which
persisted for up to 28 d after withdrawal. The increased coupling
was limited to projection neurons in both prefrontal cortical and
accumbens brain regions, as identified by their axonal trajectory or
the absence of interneuron-selective immunocytochemical markers. These
changes occurred with no substantial loss of tyrosine hydroxylase-immunoreactive terminals in these cortical and accumbens regions, ruling out dopamine degeneration as a precipitating factor. Previous studies showed that nitric oxide plays a role in the regulation of coupling; however, amphetamine-withdrawn rats had fewer
numbers of neurons and processes that stained for nitric oxide synthase
immunoreactivity. In amphetamine-treated rats, a higher proportion of
cortical cells fired in bursts, and a larger proportion of accumbens
and prefrontal cortical neurons exhibited bistable membrane
oscillations. By increasing corticoaccumbens transmission, amphetamine
withdrawal may lead to neuronal synchronization via gap junctions.
Furthermore, this adaptation to amphetamine treatment persists long
after the drug is withdrawn.
Key words:
addiction; psychostimulant; amphetamine; prefrontal
cortex; nucleus accumbens; electrophysiology; electrotonic
transmission; synchronization
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INTRODUCTION |
The prefrontal cortex (PFC) and
ventral striatum receive extensive dopaminergic (DA) inputs from the
ventral tegmental area (Fuxe et al., 1974 ; Berger, 1992; Williams and
Goldman-Rakic, 1998), and these regions regulate motivation-related
behaviors (Koob and Bloom, 1988; Schultz et al., 1992; Wise, 1996).
Psychostimulants such as amphetamine increase extracellular DA levels
in the PFC and nucleus accumbens (Kalivas and Duffy, 1990; Reid et al.,
1997; Rawls and McGinty, 1998) via blockade of the uptake and enhancing nonvesicular release of amines (Ritz et al., 1987; Kuhar et al., 1991;
Sulzer et al., 1995). Repeated intermittent administration of
psychostimulants causes a progressive augmentation of motor responses
that can be demonstrated with a psychostimulant challenge even weeks
after terminating the repeated dosing paradigm. This phenomenon is
called behavioral sensitization (Robinson and Becker, 1986; Kalivas and
Stewart, 1991; Prasad et al., 1999). Amphetamine-induced sensitization
is persistent for at least 1 year after withdrawal (Paulson et al.,
1991; White and Wolf, 1991; Valadez and Schenk, 1994). The anatomical
adaptations that underlie behavioral sensitization in rats may model
the adaptive process that contributes to drug craving that occurs
during withdrawal in humans after amphetamine binging. Human drug
craving corresponds to an activation of frontal cortical regions (Grant
et al., 1996; Maas et al., 1998; Childress et al., 1999), and
alterations within the prefrontal-accumbens circuit are proposed to
play a central role in the sensitization, cognitive dysfunction,
negative affect, and drug-seeking behavior associated with drug abuse
(Robbins, 1991; Jentsch and Taylor, 1999).
One response that often occurs after long-term manipulations of DA
transmission in the striatum is an alteration in dye coupling. Dye
coupling is believed to reflect gap junctional conductance between
adjacent neurons (Eghbali et al., 1990; Moreno et al., 1991), providing
a means for synchronization in electrical activity and exchange of
small molecules between linked cells (Bruzzone et al., 1996). As such,
coupling modulates neuronal network function by increasing information
flow between adjacent neurons within a given brain nucleus. Increases
in dye coupling occur in the striatum after long-term manipulations of
the DA system, including withdrawal from repeated antipsychotic drug
administration (O'Donnell and Grace, 1995; Onn and Grace,
1995a) and after recovery from DA-depleting brain lesions
(Cepeda et al., 1989; Onn and Grace, 1999). Dye coupling also can be
augmented by corticostriatal activation, and this occurs via a nitric
oxide-dependent mechanism (O'Donnell and Grace, 1997). Therefore,
long-term alterations in the DA system that affect corticostriatal
transmission appear to produce changes in dye coupling in striatal
regions that persist after removal of the precipitating stimulus.
In this study, we used in vivo intracellular recording and
staining to examine the effects of acute and repeated amphetamine treatment on dye coupling in limbic cortical and striatal regions in the rat. In particular, we wanted to examine the neuronal
adaptations that persisted in the drug-free condition after the
withdrawal of amphetamine from the rat. Given the evidence for
corticoaccumbens involvement in drug-mediated behaviors, combined with
our evidence for corticostriatal modulation of dye coupling, we
examined whether any alterations in dye coupling after amphetamine
treatment were associated with changes in limbic cortical afferents to
the accumbens and whether these changes correlate with nitric oxide
synthase (NOS) activity.
Portions of these data were presented previously in abstract form (Onn
and Grace, 1995b).
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MATERIALS AND METHODS |
Animals and drug treatment protocol. All experimental
procedures were performed in accordance with the United States Public Health Service publication Guide for the Care and Use of
Laboratory Animals and were approved by the Institutional Animal
Care and Use Committee at the University of Pittsburgh. A total of 144 male Sprague Dawley rats, consisting of 72 vehicle-treated control rats
and 72 rats treated with amphetamine, were used in this study. The
control rats were treated with 0.9% saline and were
subjected to treatment withdrawal in parallel with each group of the
amphetamine-treated rats. One set of rats (n = 42)
weighing 150-175 gm at the start of treatment were given daily
injections of amphetamine (1-3 mg/kg, i.p.) in their home cages for
2-4 weeks. This dosing protocol has been reported to elicit
sensitization with respect to stimulant-evoked locomotor behavior in
rats (Robinson and Becker, 1986; Kalivas and Duffy, 1990; Wolf et al.,
1994; 1995). A second set of rats (n = 30) were given
escalating doses of amphetamine administered at irregular intervals
(i.e., 5 consecutive days alternating with a 2 d drug-free period
over the 4 week treatment period) to more accurately simulate the abuse
pattern of drug addicts (for review, see Robinson and Becker, 1986).
This was done by administering amphetamine each week at an initial dose
of 1 mg/kg and incrementing the dose by 0.5 mg/kg each day to reach a
final dose of 3 mg/kg on the fifth consecutive day followed by a 2 d washout. This regimen was repeated each week for the 4 week treatment
period. Both treatment protocols have been demonstrated to lead to
similar sensitization of behavioral and neurochemical responses after
administration of challenge doses of amphetamine (Paulson et al., 1991;
Paulson and Robinson, 1995; Wolf et al., 1995).
In vivo intracellular recording and Lucifer yellow
injection. After 7-10 and 21-28 days withdrawal from amphetamine
treatment, the animals were anesthetized with 8% chloral hydrate (400 mg/kg, i.p.), with supplemental anesthetic administered via a lateral tail vein catheter. In a subset of rats (n = 10), no
withdrawal time was allowed; in this case, the animals were
anesthetized 1 hr after the last injection of amphetamine or saline. If
a cell was not successfully impaled within 3 hr, an additional dose of amphetamine was administered. In vivo intracellular
recordings were performed as described previously (Grace and Llinas,
1985; Onn and Grace, 1994, 1995, 1999; Onn et al., 1994a,b,c). Briefly, the recording electrodes were pulled on a Flaming-Brown p80/PC electrode puller and filled with 5-10% Lucifer yellow dissolved in 1 M LiCl. The Lucifer yellow-containing
microelectrodes were lowered into the PFC [coordinates:
anteroposterior (AP), 3-3.5 mm anterior to bregma; mediolateral (ML),
0.5-1 mm from the sagittal suture; dorsoventral (DV), 4-6 mm from the
dura] or the nucleus accumbens (coordinates: AP, 1.5-3 mm; ML, 1-2.5
mm; DV, 5-8 mm). Cells were impaled in both control and
amphetamine-treated rats based solely on their location in the PFC or
nucleus accumbens. In each case, cells stained in the PFC were
pyramidal-shaped neurons, and cells stained in the nucleus accumbens
were of the medium spiny type. No selection was made based on their
spontaneous firing pattern or activity state. Hyperpolarizing current
was not applied to the cell during the initial penetration and membrane
stabilization period to minimize potential artifactual labeling of
cells with Lucifer yellow. After achieving stable impalement,
spontaneous basal activity of the cells recorded in the PFC and in the
striatal complex was collected at resting membrane potential for each
neuron studied. Input resistances of the recorded and injected cells were estimated at resting membrane potential by measuring the membrane
voltage deflections produced in response to 0.2-0.4 nA hyperpolarizing
current pulses (150 msec duration). Studies have shown that activation
of cortical afferents can alter dye coupling in postsynaptic neurons
(Hatton and Yang, 1996; O'Donnell and Grace, 1997). Therefore,
stimulation of these afferents was not performed to circumvent possible
secondary alterations in coupling that may occur independent of the
drug treatment effects. The cells were then injected with Lucifer
yellow by applying 1-3 nA constant hyperpolarizing current into the
electrode interrupted by brief (5-10 msec) depolarizing pulses
delivered at 4-7 Hz to prevent clogging of electrodes (Grace and
Llinas, 1985; Onn and Grace, 1994, 1995, 1999; Onn et al., 1994c). Only
cells with a stable resting membrane potential of at least  60 mV and
action potential amplitudes of  55 mV were subject to dye injection. On average, all cells scored for dye coupling in the present study were
injected for at least 4 min (7.6 ± 3.1 min; n = 93). The average time of recordings was 15.1 ± 7.2 min, ranging
from 11 to 45 min. Electrophysiological measures and dye coupling
assessments were done primarily in the 21-28 d withdrawal group. This
was extended to include groups that were recorded with no withdrawal period, after 7-10 d of withdrawal, and after 14 d of withdrawal, to examine the time course of the observed changes, as noted under "The time course of amphetamine withdrawal-induced changes" in the
Results section. The incidence of coupling was found to be similar for
both the daily amphetamine treatment and the escalating treatment
groups within the cortex (daily, 4 of 7; escalating, 7 of 11;
p = 1.0) and the nucleus accumbens (daily, 3 of 6;
escalating, 10 of 15; p = 1.0). Therefore, the results
from both groups were combined.
Staining of tissue containing Lucifer yellow-labeled cells for
calbindin, tyrosine hydroxylase, and nitric oxide synthase immunoreactivity. After completion of the recordings, the deeply anesthetized rats were perfused transcardially with saline followed by
500 ml of 4% buffered paraformaldehyde, pH 7.4. Serial sagittal sections (60 µm in thickness) were cut and collected in 0.1 M phosphate buffer. Tissue slices containing Lucifer
yellow-injected cells were then examined using a Leitz Orthoplan II
epifluorescence microscope equipped with the Leitz I3 filter cube
(excitation: bandpass, 355-425 nm; dichromatic mirror, RKP 580 nm; suppression: lowpass, 580 nm). Dye coupling was defined as the
presence of more than one labeled cell recovered after injection of a
single neuron with Lucifer yellow. Only cells with complete filling of soma and dendritic processes were scored for the presence or absence of
dye coupling. In the case where multiple cells were labeled from a
single injection, the cells were scored as dye-coupled only if the soma
and dendrites of each neuron in the cluster was completely filled. A
given cluster of dye-coupled cells often spanned several sections;
therefore, the total number of cells coupled after injection of a
single neuron (defined as the extent of dye coupling) was determined by
examining serial sections containing the entire cluster of dye-coupled
neurons and dendrites. Sections containing Lucifer yellow-labeled cells
were further labeled for calbindin immunoreactivity (1000×; Sigma, St.
Louis, MO) using the Texas Red indirect fluorescence method as
described previously (Onn and Grace, 1994, 1995, 1999; Onn et al.,
1994c). Tissue containing Lucifer yellow-labeled neurons was
counterstained for calbindin immunoreactivity for two purposes: (1) to
distinguish the nucleus accumbens shell from the calbindin-rich core
region (Onn and Grace, 1995a; Meredith et al., 1996), and (2) to
limit the assessment of coupling to the PFC projection neurons that are
known to be devoid of calbindin immunoreactivity (Kawaguchi, 1995).
Double fluorescence-stained sections were examined using a N2
(rhodamine) filter cube (excitation: bandpass, 530-560 nm; dichromatic
mirror, RKP 580 nm; suppression: lowpass, 580 nm) for Texas Red
fluorescence and using a I3 filter cube for Lucifer yellow fluorescence.
Extensive lesions of the DA system in the striatum have been shown to
cause an upregulation of dye coupling among medium spiny neurons (Onn
and Grace, 1999). To assess whether the response to repeated
amphetamine administration may have been caused in part by drug-induced
degeneration of the DA system, we used immunoperoxidase staining for
tyrosine hydroxylase (TH) (1000×; Eugene Tech, Inc.). This
stain was applied to adjacent forebrain sections or midbrain sections
to assess the extent of DA terminal and cell body loss (Onn et al.,
1986; Onn and Grace, 1999) in the amphetamine-treated brains. DA cell
body counts were conducted on each of three 60-µm-thick coronal
sections of the ventral tegmental area and the substantia nigra at the
following AP coordinates (with respect to bregma): 5.2 mm, 5.6 mm,
and 6.0 mm (Onn and Grace, 1999). Immunoreactivity for nitric oxide
synthase (1500×; Sigma) was performed to examine whether changes in
nitric oxide accompanied the alterations of coupling found in rats
after withdrawal from repeated amphetamine administration. The number
of NOS-immunoreactive cells was determined within a designated area
(1 × 1 mm) in both prelimbic cortex and accumbens on two sagittal
sections at 1.4 and 2.0 mm lateral to the midline sagittal suture. The
specificity of each antibody used in this study was tested by omitting
either the primary or secondary bridging antiserum, resulting in
negative staining. All immunocytochemical markers were assessed in the
14 d withdrawal group, with additional qualitative confirmations
performed in the withdrawal groups used for electrophysiological
recordings and assessment of dye coupling.
Statistics. Data were analyzed by averaging across cells,
and expressed as mean ± SD. Differences between control
and experimental groups were assessed using Student's t
test. A nonparametric Fisher's exact test was used to compare the
incidence of dye coupling, as defined by the percentage of total dye
injections that resulted in labeling of more than one cell per
injection. To fulfill the requirement for independence in the Fisher's
exact test, only a single cell was labeled per structure unilaterally.
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RESULTS |
Effects of withdrawal from repeated amphetamine administration on
PFC projection neurons
Vehicle-treated control rats
A correlative analysis of the morphology and electrophysiology of
single PFC neurons using Lucifer yellow-filled microelectrodes was
conducted in age-matched control rats (i.e., on days 21-28 after
cessation of saline injection). The neurons recorded and stained in
this study exhibited resting membrane potentials of 69.1 ± 8.1 mV (mean ± SD) and input resistances of 52.7 ± 11.6 M .
Approximately half of the cortical neurons recorded (n = 22) fired in a single-spike discharge mode (54%; Fig.
1A,B), with the
remaining cells exhibiting an oscillatory, irregular firing pattern
(24%) or a burst firing pattern (22%; Fig. 1C). Fourteen percent of all cells recorded were found to exhibit spontaneous depolarization plateau potentials (>5 mV in amplitude) that caused the
membrane potential to shift between two stable states of membrane voltages (Cowan and Wilson, 1994). The plateaus exhibited an average amplitude of 12.6 ± 4.7 mV and frequency of 1.3 ± 0.5 Hz
(Fig. 1C). Spontaneous depolarization plateau potentials
were also observed as subthreshold oscillations in nonfiring neurons
and were underlying spontaneous spike discharge in spontaneous firing
cells (Fig. 1C).
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Figure 1.
In vivo intracellular recording
showing examples of the activity patterns of identified PFC projection
neurons from vehicle-treated control rats. A, Recording
from a nonfiring neuron in which single (A1) or doublet
(A2) action potentials were triggered by current
injection-induced depolarization of the membrane. B,
Recording from a spontaneously firing neuron that exhibited
predominantly a single-spike firing pattern. C,
Recording of a burst-firing cell exhibiting clusters of three to five
spikes riding on oscillatory depolarizing plateau potentials. The
depolarizing potentials are typically 10-20 mV in amplitude and occur
irregularly at a frequency of 1.3 Hz in this case. Both regular-spiking
(A, B) and bursting cells (C) were
found to comprise a single morphological class that projects to the
nucleus accumbens.
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Fifteen neurons were labeled by injection with Lucifer yellow. Thirteen
of these neurons were localized to the deep layers of ventral
prefrontal cortices including prelimbic (n = 4),
infralimbic (n = 5), and orbital (n = 4) cortex; in addition, two neurons were localized to the middle layer
of orbital cortex. In each case, only a single neuron was labeled. In
general, these neurons exhibited numerous spiny basilar dendrites that
had branching patterns ranging from sparse and polarized (Fig.
2A,C) to densely radiating (Fig. 2E) patterns. Despite the variable
appearance of their somatodendritic morphology, these neurons exhibited
characteristics consistent with pyramidal neurons in the PFC. Nine of
these 15 labeled neurons exhibited: (1) a regular, rhythmic single
spike discharge pattern at ~3-5 Hz, (2) the presence of depolarizing afterpotentials after the fast repolarization phase of the action potential, and (3) a prominent spike afterhyperpolarization (Fig. 1A). The remaining six neurons exhibited oscillatory
irregular or burst firing patterns (Fig. 1B,C), with
burst firing defined as the presence of spontaneously occurring doublet
or triplet spikes riding on a plateau membrane depolarization. These
characteristics are similar to those of cortical pyramidal projection
neurons described previously (McCormick et al., 1985; Cowan and Wilson, 1994; Steriade et al., 1996; Yang et al., 1996).
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Figure 2.
Photomicrographs showing Lucifer yellow-labeled
PFC pyramidal neurons injected during in vivo
intracellular recording from the PFC of drug-naive rats. In addition to
the presence of truncated apical dendrites, the labeled cortical cells
are identified as corticostriatal projection neurons based on the
following features: (1) the axon of the Lucifer yellow-stained cell had
a trajectory (arrows in D from a section
adjacent to A) that could be traced into the striatal
complex, and (2) the cell (A, open arrow, Lucifer yellow
fluorescence) did not contain calbindin immunoreactivity that is known
to stain cortical GABAergic interneurons (B, arrowheads;
same section showing Texas Red fluorescence for
calbindin-D28k). Open arrow in
B marks the site of the Lucifer yellow-stained cell
shown in A. C (high power of
A) shows numerous dendritic spines on basilar dendrites
of this pyramidal projection neuron. Arrowhead indicates
a pair of calbindin-positive interneurons stained by Texas Red
fluorescence and examined using the Lucifer yellow filter cube.
E, F, Another Lucifer yellow-filled PFC
neuron that had a stellate-like somatodendritic morphology
(E) with dense dendritic branching in which the
apical dendrite (E, open arrow) was not immediately
apparent. Nevertheless, the axon of this cell could also be traced into
the striatal complex (F, arrows). The primary axon was
found to branch into two equal-diameter axons before entering the
corpus callosum (asterisks); only one branch is observed
to enter the ipsilateral striatal complex. bv, Blood
vessel (in A-D indicating the same structure);
vs, ventral striatum; cc, corpus
callosum. Scale bars: A, B, 80 µm;
C-F, 40 µm.
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In 9 of 15 of the cortical cells labeled with Lucifer yellow, the axon
was traced into the ipsilateral striatal complex (Fig. 2D,F). In addition, none of the neurons
stained exhibited double labeling for calbindin immunoreactivity (Fig.
2B,C), which is known to label a subclass of
GABAergic interneurons in the cortex (Kawaguchi, 1995). In five of nine
cases, the axon was observed to bifurcate before entering the corpus
callosum along its course to the striatal complex (Fig.
2F). These morphological characteristics corresponded
to the corticostriatal pyramidal neurons described recently in the
prelimbic cortex (Levesque and Parent, 1998) and in the medial
agranular cortex (Wilson, 1987).
Amphetamine-treated rats
The cells recorded and injected in rats that had been withdrawn
from amphetamine for 21-28 d displayed resting membrane potentials of
71.3 ± 10.4 mV (p < 0.4; NS) and input
resistances of 54.3 ± 4.7 M (p < 0.6;
NS), which were not significantly different from those recorded in
vehicle-treated controls. In contrast to neurons recorded in the
control animals, limbic cortical neurons recorded in
amphetamine-treated rats exhibited predominantly a burst-discharge
pattern (55% firing spikes in bursts; Fig.
3), with 30% of the remaining neurons
discharging in a single-spiking pattern and 15% that were nonfiring.
In addition, neurons recorded in amphetamine-withdrawn rats exhibited a
trend toward elevated firing rates, although the firing frequency in
the withdrawn rats was highly variable (2.7 ± 3.2 Hz vs 3.9 ± 4.5 Hz; p < 0.07). Moreover, 40% (8 of 20) of all
cortical cells recorded in amphetamine-treated rats were found to
display spontaneous membrane depolarizing plateau potentials (Fig.
3B,C), as compared to 14% in controls
(p < 0.05). These membrane oscillations were
found to occur at approximately the same frequency (1.2 ± 0.4 Hz)
but with a larger amplitude (17.5 ± 3.2 mV) than those recorded
in PFC neurons of vehicle-treated rats (1.3 ± 0.5 Hz, NS, and
12.6 ± 2.4 mV, p < 0.05, respectively).
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Figure 3.
In vivo intracellular recording
showing examples of membrane oscillations and the activity patterns of
identified PFC projection neurons from amphetamine-withdrawn rats. In
the withdrawn rats, limbic cortical neurons exhibited primarily a
burst-discharge pattern. A, An example of a
spontaneously burst-firing neuron identified in layer 5 infralimbic
cortex in a rat after 14 d of withdrawal from repeated exposure to
amphetamine for 4 weeks. This neuron had a resting membrane potential
of 79 mV. This neuron fired spontaneous spikes at 7 Hz within bursts
with two to four spikes riding on membrane depolarization plateau
potentials. B, In ~40% of PFC neurons recorded in
amphetamine-withdrawn rats, the cell membrane oscillated in a rhythmic
manner at ~1 Hz. C, Histogram showing the distribution
of membrane potential with respect to the percentage of time that the
membrane existed at each potential. This histogram reveals the presence
of two peaks, with equal time spent in the up (59 mV) and down (70
mV) states.
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Of the cells recorded in amphetamine-treated rats, 18 were labeled with
Lucifer yellow at the completion of the recording period. Of these, 10 exhibited a burst firing pattern, and eight were regular-spiking
neurons before dye injection. Eleven of these 18 cells (60%) injected
with Lucifer yellow exhibited dye coupling, defined as the labeling of
more than one cell per single cell injected (Fig.
4), presumably as a consequence of dye
transfer via gap junctions (Stewart, 1978; MacVicar and Dudek, 1981;
Grace and Bunney, 1983; Cepeda et al., 1989; Hatton and Micevych, 1992; O'Donnell and Grace, 1993; Peinado et al., 1993; Onn and Grace, 1994,
1995, 1999; Rorig et al., 1995; Rorig and Sutor, 1996). This
represented a significantly higher incidence of coupling between PFC
pyramidal cells in rats that had been withdrawn from repeated
amphetamine when compared to that observed in controls (11 of 18 vs 0 of 15 in controls; p = 0.03; Fisher's exact test). These eleven sets of coupled cells were located in the middle to deep
layers (i.e., layers 5 and 6) of the PFC, including orbital, infralimbic, and prelimbic cortices (Fig. 4A-C).
Typically, axons of cells engaged in coupling failed to exhibit a
substantial level of staining, possibly because of shunting of dye
among the coupled cells. Nevertheless, in four of the neurons that did
not exhibit dye coupling, the axons could be traced into the accumbens
(data not shown), which was similar to that observed in control rats (Fig. 2). As in the control cases, coupled cells did not exhibit staining for calbindin-D28k protein (data
not shown) that is known to label a subclass of GABAergic interneurons
in the cortex (Kawaguchi, 1995).
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Figure 4.
Photomicrographs showing examples of dye-coupled
cells in the deep layer of PFC (A-C) and in the
ventral striatum (D, E) of rats recorded 21-28 d after
withdrawal from amphetamine. In each case, a single cell was injected
with the dye during in vivo intracellular recording from
amphetamine-withdrawn rats. Coupled cells were recovered in the
prelimbic (A), infralimbic
(B), and orbital (C)
cortices that exhibit properties consistent with PFC pyramidal neurons.
Arrows with numbers indicate the number
of cell bodies in each cluster of coupled cells, whereas the
open arrows label their apical dendrites. Superficial
layers in A-C are toward the top of the
photomicrographs. D, A pair of spiny neurons
(arrows) in the ventrolateral striatum of an
amphetamine-treated rats after injecting a single neuron. Double
staining of the coupled neurons for calbindin-D28k
immunoreactivity (marked by Texas Red fluorescence) reveals that the
coupled cells lie at the border of a striosome (as marked by negative
neuropil staining for calbindin immunoreactivity). The background level
of Texas Red fluorescence does not appear as intense because of the use
of the I3 filter cube to view the Lucifer yellow fluorescence, but
nevertheless still reveals the border of this striosome.
E, A cluster of Lucifer yellow-labeled spiny cells
(arrows) in the ventromedial striatum (at the juncture
of the accumbens core region) of an amphetamine-withdrawn rat. The
Lucifer yellow was subsequently converted into a peroxidase stain
(brown) using antibodies to Lucifer yellow to better
reveal the dendrodendritic contacts between the coupled cells. In all
cases, the coupling was restricted to cells of the same morphological
cell class. Open arrows mark axonal collaterals in
contact with dendritic processes. The cell marked by arrow
1 was located on the adjacent section to F (data
not shown). Scale bars: A-C, 30 µm; D,
40 µm; E is at the same scale as
D.
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Effects of withdrawal from repeated amphetamine treatment on
ventral striatal spiny neurons
Vehicle-treated control rats
In recordings from the ventral striatal region (i.e., ventral
posterior striatum, nucleus accumbens core, and shell regions), injection of single cells with Lucifer yellow (n = 14)
resulted in the labeling of spiny neurons, with eight located within
the core (Fig. 5A) and six
localized to the shell region (Fig. 5B) of the nucleus
accumbens. The shell and core regions were delineated by the absence of
calbindin-stained neuropil in the shell when compared with the core and
other striatal regions (Onn and Grace, 1995a, 1999). Unlike
those stained in the dorsal striatum (Onn and Grace, 1994, 1995; Onn et
al., 1994c), reconstructions from multiple sections of spiny neurons
stained in the shell region revealed smaller somatal size (13.7 ± 1.7 vs 16.2 ± 2.1 µm in diameter for the spiny neurons in the
core and ventral striatum, respectively; p < 0.05) and
fewer dendritic branches (4.3 ± 1.6 vs 6.2 ± 1.0 per cell
in the core or ventral striatum, respectively; p < 0.05) that tended to extend preferentially along the anteroposterior plane. Moreover, axons of spiny neurons in the shell were often observed to arise from one of the primary dendrites (Fig.
5B1) and project toward the ventral pallidum where they
arborized into an extensive collateral system (Fig. 5B4,B5).
Consistent with our previous observations made in vitro
(O'Donnell and Grace, 1993), the accumbens core and shell neurons
exhibit similar passive membrane properties. Thus, the averaged resting
membrane potential in the accumbens neurons recorded and injected in
control rats was 73.1 ± 7.4 mV. In addition, neurons in the
shell exhibited higher levels of spontaneous activity (8.7 ± 4.2 Hz; p < 0.05; Fig. 6)
and a trend toward higher input resistances (57.2 ± 15.7 M;
p < 0.06; NS) when compared to spiny neurons
identified in the accumbens core (2.9 ± 4.5 Hz; 41.8 ± 12.2 M). Only 9% (2 of 23 cells, with nine of these cells recorded using
neurobiotin-filled microelectrodes) of the spiny neurons recorded in
the accumbens of control rats displayed spontaneous depolarizing
plateau potentials (frequency, 0.9 ± 0.6 Hz; amplitude, 13.5 ± 5.2 mV). This occurred independent of whether the cell was not
firing (Fig. 6A) or was firing action potentials
spontaneously (Fig. 6C). It should be noted that the
proportion of neurons exhibiting this bistable state was substantially
lower than what we had reported previously (O'Donnell and Grace,
1995). This is likely attributable to a number of factors. For example,
membrane oscillations are known to be sensitive to the level of
anesthesia (Leung and Yim, 1993; Wilson and Kawaguchi, 1996), and for
this reason, in our previous study we used constant infusion of chloral
hydrate anesthesia to keep the rats at a moderate level of anesthesia.
However, because of the need to perform long-term injections of Lucifer
yellow, the rats in this case were kept for several hours at a deep
level of anesthesia, as monitored by the hindlimb withdrawal
reflex.
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Figure 5.
Photomicrographs showing Lucifer yellow-labeled
spiny cells identified in the accumbens core (A)
and shell (B) region. In control rats, most
injections of individual cells with Lucifer yellow resulted in
single-cell labeling of spiny neurons. A, A single
Lucifer yellow-labeled spiny neuron (A1, arrow) that was
converted into a peroxidase stain using Lucifer yellow antibodies
(A2). This cell was subsequently identified in the
accumbens core region (A3, arrow), as delineated by the
calbindin-positive neuropil (presence of Texas Red fluorescence). Spiny
neurons in the core region tended to have more widespread dendritic
processes than those recovered in the shell region. B, A
single Lucifer yellow-labeled spiny neuron (B1, arrow)
in which the fluorescence was converted into a peroxidase stain
(B2, arrow). This neuron was located in the accumbens
shell region as outlined by the negative staining for calbindin
immunoreactivity (B3, absence of Texas Red
fluorescence). The axon of this cell (B1, asterisk) was
traced into the ventral pallidum where numerous collaterals were
emitted (B4, low power; B5, high power).
The three highlighted arrows with
asterisks in B4 show collaterals of the
primary axon. The solid arrows in B4 and
B5 mark the same axonal collateral. bv,
Blood vessel (in B4 and B5 indicating the
same structure); VS, ventral striatum;
VP, ventral pallidum; ac, anterior
commissure; cc, corpus callosum. Scale bars, 30 µm.
A2 and A3 are at the same scale as
A1; B3 is at the same scale as
B2.
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Figure 6.
In vivo intracellular recordings
from identified accumbens spiny neurons showing spontaneous firing
activity in vehicle-treated control rats. A,
Approximately half of the accumbens core neurons recorded in control
rats are in a state of quiescence, demonstrating only subthreshold
membrane depolarizations. These membrane depolarizations can occur with
an oscillatory bistable (A) or irregular
(B) pattern. B, A second
population of neurons exhibited low levels of spontaneous spike
discharge with the spikes occurring at irregular intervals.
C, An accumbens core spiny neuron that exhibited an
oscillatory firing pattern is characterized by the occurrence of
multiple spikes riding on depolarizing plateau potentials. This
represents the bistable membrane potential composed of an up state at
63 mV and a down state at 78 mV, respectively.
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Consistent with our previous observations made in vivo (Onn
and Grace, 1994, 1995) and in vitro (O'Donnell and Grace,
1993), low incidences of dye coupling were found among neurons stained in the ventral striatum of control rats (2 of 14; Fig. 5), with two
cases of coupling being identified in the accumbens core region and in
the ventrolateral striatum (data not shown). Cases of dye coupling were
not observed for cells injected in the accumbens shell region
(n = 6), including the anterior sector (three of six
cases; Fig. 5B). Similar low incidences of intercellular
coupling were observed when single accumbens neurons were injected with neurobiotin, in that only one of nine cases resulted in the labeling of
more than one cell.
Amphetamine-treated rats
The cells recorded and injected in the accumbens in rats that had
been withdrawn from amphetamine exhibited an averaged resting membrane
potential of 74.7 ± 11.7 mV, which was not significantly different from that observed in the control rats. However, after withdrawal from amphetamine, staining of neurons in the ventral striatum revealed significantly higher levels of dye coupling (controls, 2 of 14 vs withdrawal, 13 of 21; p = 0.007;
Fisher's exact test). Sets of coupled cells in the ventrolateral
striatum were often found to cross matrix and striosome compartments,
which could be distinguished by calbindin neuropil staining (Fig.
4D; Onn and Grace, 1995a, 1999). In addition
to an increase in the incidence of coupling, there was also a
significant increase in the extent of coupling, defined as the average
number of cells labeled per injection (vehicle, 2.0 vs amphetamine,
3.4; p < 0.01). All cases of coupling that extended
beyond three cells (n = 5) were found only in the
treatment-withdrawal group and were located in the fundus of the
striatum and medial posterior striatum (core region) (Fig.
4E). As observed previously, even in those cases in
which injection of a single cell resulted in dye coupling among five
cells, the coupling was nonetheless restricted to a single morphological cell class; i.e., medium spiny cells (Fig.
4E). Thus, repeated treatment with amphetamine
resulted in an increase in both the incidence and the extent of
coupling observed in the ventral striatal complex. Moreover, in
amphetamine-withdrawn rats significantly higher numbers (39%; 8 of 21;
p = 0.03; Fisher's exact test) of accumbens spiny
neurons were observed to display slow membrane oscillations (Fig.
7B1,B2). Both the frequency of the depolarizing events (amphetamine, 0.9 ± 0.6 Hz; control,
1.1 ± 0.5 Hz; NS) and their amplitude (amphetamine, 18.1 ± 7.6 mV; control, 13.5 ± 5.2 mV; NS) were not significantly
different from that observed in vehicle-treated controls (Fig.
6C).
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Figure 7.
In amphetamine-withdrawn rats, significantly more
neurons in the nucleus accumbens were found to exhibit bistable
membrane potentials. A, Histogram showing the
distribution of membrane potential with respect to the percentage of
time that the membrane existed at each potential. This distribution
reveals the presence of two distinct peaks, corresponding to the up
(66 mV) and down (84 mV) states. In the inset, the
membrane potentials corresponding to up and down states are delineated.
B, These bistable membrane potentials are recorded for
extended periods of time and are not affected by the injection of
Lucifer yellow into the neuron. B1, Bistable state of a
neuron soon after penetration with a Lucifer yellow-containing
electrode. B2, The same neuron recorded 25 min later and
after intracellular injection with Lucifer yellow.
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Time course of amphetamine-induced changes in
cellular coupling
The observed increases in the incidence of dye coupling in both
PFC and nucleus accumbens on days 21-28 after withdrawal were also
found in rats on days 7-10 after withdrawal. The incidence of dye
coupling observed after 7-10 d of withdrawal (accumbens, five of
seven; cortex, four of seven) was not different from that observed
after 21-28 d of withdrawal (accumbens, 13 of 21; cortex, 11 of 18;
p = 1.0; Fisher's exact test; NS). In contrast, in a subset of rats treated with amphetamine and recorded while the rats
were still on the drug (i.e., no amphetamine-withdrawal time allowed),
the level of coupling was not different from that observed in
vehicle-treated control rats (p = 1.0 for both
comparisons; NS). Thus, even though each cell exhibited complete dye
filling, none of the cells injected in the PFC (zero of six) or in the nucleus accumbens (zero of five) of nonwithdrawn rats exhibited dye
coupling, which was significantly different from that observed in the
amphetamine-withdrawn rats (p = 0.02 and
p = 0.04, respectively).
Effects of repeated amphetamine on dopaminergic terminals in
accumbens and prefrontal cortex
Several studies have shown that prolonged treatment with
amphetamine can cause destruction of DA-containing afferents to
subcortical structures such as the striatum (Ellison and Switzer, 1993;
Eisch and Marshall, 1998) (see also Castner and Goldman-Rakic,
1999). In light of our recent finding that severe loss of DA
fibers produced by injection of the neurotoxin 6-hydroxydopamine
leads to an increase in intercellular coupling among striatal
neurons (Onn and Grace, 1999), we examined whether there was evidence
of extensive loss of catecholamine-containing afferent fibers using
antibodies specific to TH. We found no substantial alterations in TH
immunoreactivity in the PFC and accumbens of the amphetamine-treated
rats or in rats after withdrawal from amphetamine (Fig.
8B,D), although only cells stained in the latter group displayed high levels of
intercellular coupling. Clearly, any amphetamine-induced
damage, if present, was indiscernible from controls, especially when
compared to that required for the 6-hydroxydopamine-induced
increase in coupling (Onn and Grace, 1999). Indeed,
substantially high levels of TH staining were noted in the limbic
cortices of amphetamine-treated rats (Fig. 8B,D), in
contrast to the relatively sparse TH fiber staining in the control rat
cortices (Fig. 8A,C). In particular, numerous
TH-immunoreactive somata were revealed in the amphetamine-treated cortices. Therefore, the amphetamine treatment paradigm used in the
present study, which is known from other studies to produce sensitized
behaviors (Wolf et al., 1994, 1995), did not cause any damage to the DA
terminals as measured by TH immunoreactivity in these brain regions
where high levels of intercellular coupling were observed. This
observation was further confirmed by DA cell body counts in
both the ventral tegmental area and substantia nigra (Fig. 8E), in
that similar numbers of TH- stained DA cells were observed in
amphetamine-treated rats (375 ± 27; n = 3) when compared to vehicle-treated controls (386 ± 34; n = 3; p < 0.7; NS).
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Figure 8.
Photomicrographs illustrating TH immunostaining of
a sagittal section containing the PFC (A, B) and the
nucleus accumbens (C, D) in a control rat (A,
C) and a rat 14 d after withdrawal from amphetamine
treatment (B, D). In the PFC of the
amphetamine-withdrawn rat (B), the levels of TH
immunoreactivity are substantially similar to that found in the control
rat (A). In addition, there appears to be an
increase in TH staining in the cortex of amphetamine-withdrawn rats, as
revealed by the presence of numerous TH-immunoreactive somata
(B, D, arrows) that are less frequently observed in the
control cortices. Similar levels of TH staining were also noted in the
ventral striatum, as shown in an amphetamine-withdrawn rat
(D) when compared to a vehicle-treated control
(C). E, An example of
TH-immunoperoxidase stain of DA cell bodies in VTA and SN, at bregma
5.6 mm. VS, Ventral striatum; PFC,
prefrontal cortex; VTA, ventral tegmental area;
SN, substantia nigra; cc, corpus
callosum; bv, blood vessel. Scale bar ( in
A), 200 µm.
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Decreased nitric oxide synthase immunoreactivity in PFC-accumbens
systems after withdrawal from repeated amphetamine
Several classes of GABAergic interneurons are known to be present
in both cortical and subcortical regions, where they exert potent
modulatory actions over the principal cell classes; i.e., the
projection neurons (Bennett and Bolam, 1994; Kita, 1996). One subclass
of GABAergic interneuron is reported to contain nitric oxide synthase
and is known to release nitric oxide in response to NMDA receptor
stimulation (Bredt and Synder, 1992). Given that nitric oxide has been
reported to influence dye coupling (Hatton and Yang, 1996; Rorig and
Sutor, 1996; O'Donnell and Grace, 1997), we examined whether there was
an alteration in NOS immunoreactivity in rats after withdrawal from
repeated amphetamine. When compared to control rats, in rats after
14 d of withdrawal from amphetamine treatment there was a
significant decrease in NOS immunoreactivity in the neuropil of both
the PFC and nucleus accumbens (Fig. 9). This was accompanied by a significant decrease in the number of neurons
displaying NOS immunoreactivity in both brain regions of the
amphetamine-withdrawn group (Table 1).
The decreased NOS-immunoreactive neurons and processes were also
confirmed at 7 and 28 d of withdrawal (n = 3 for
each time point).
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Figure 9.
Photomicrographs illustrating nitric oxide
synthase immunoreactivity in the PFC (A, B) and the
nucleus accumbens (C, D) in control rats (A,
C) and in rats at 14 d after withdrawal from amphetamine
(B, D). A, B, Comparisons of
peroxidase-stained NOS-positive neurons in the PFC of control
(A) and amphetamine-withdrawn
(B) rats in a sagittal plane. Lower levels of NOS
immunoreactivity were observed in the deep layer of PFC of
amphetamine-withdrawn rats. C, D, Comparisons of Texas
Red-labeled NOS-containing neurons in accumbens at an equivalent AP
level in a coronal plane. Significantly fewer NOS-containing neurons
were noted in these brain regions in amphetamine-treated rats
(D) as compared to those observed in paired
controls (C; Table 1). Arrows indicate
NOS-positive neurons; PFC, prefrontal cortex;
VS, ventral striatum; ac, anterior
commissure. Scale bar (in A), 250 µm.
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Table 1.
Effect of repeated administration of amphetamine on
neuronal nitric oxide synthase immunoreactivity in the rat prefrontal
cortex and nucleus accumbens
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DISCUSSION |
Using in vivo intracellular recordings in rats after
repeated amphetamine administration and withdrawal, we observed
alterations in the corticoaccumbens network interactions that are
persistent in nature. These included: (1) increased intercellular
coupling among neurons in the PFC and nucleus accumbens, (2) increased bistable membrane activity, and (3) an apparent compensatory
downregulation of NOS. In contrast, in rats that received identical
amphetamine treatment but were recorded during the treatment period,
none of these changes were observed. Therefore, withdrawal from
amphetamine unmasked alterations in the corticoaccumbens system that
reflect long-term adaptations in limbic system function.
Amphetamine withdrawal alters the neurophysiology of the
corticoaccumbens system
Within the neocortex, withdrawal from amphetamine
caused increases in indices of neuronal activity, including an increase in the amount of burst discharge, a larger number of neurons displaying bistable states, and an increase in intercellular coupling. The Lucifer
yellow-stained neurons consisted of corticoaccumbens-projecting pyramidal neurons, given: (1) the absence of calbindin, which is a
marker for interneuronal cell classes in the PFC
(Kawaguchi, 1995), (2) their pyramidally shaped soma with
an identifiable apical dendrite, and (3) the ability to follow their
axons into the nucleus accumbens in 9 of 15 cases in control rats and 4 of 18 cases in amphetamine-withdrawn rats. These identified PFC
projection neurons were both morphologically (Wilson, 1987; Yang et
al., 1996; Levesque and Parent, 1998) and electrophysiologically (Cowan and Wilson, 1994) similar to pyramidal neurons identified as the corticostriatal projection neurons. Although there was a trend toward
increased corticoaccumbens neuron firing rates in amphetamine-withdrawn rats, the high degree of variability after amphetamine appears to have
prevented the differences from reaching statistical significance. One
possible cause of this variability could be an activation of normally
quiescent neurons which, because of their slow firing rates, would
dilute the impact of increased firing in the spontaneously discharging
population of cells (Hollerman and Grace, 1990). The increase in
bursting discharge observed here is consistent with glutamate-induced
activation of neuronal firing in several brain regions, including the
nucleus accumbens, dorsal striatum, and PFC (Herrling et al., 1983;
Cherubini et al., 1987, 1988; Cepeda et al., 1991; Hu and White,
1996). The increase in corticoaccumbens drive may also account for the
observed increase in intercellular coupling, given the reported
increases in intercellular coupling observed after activation of
glutamatergic afferents to the supraoptic nucleus (Hatton and Yang,
1996) and striatum (O'Donnell and Grace, 1997).
In concert with the increase in neuronal activity in the PFC, we also
observed an activation of neurons within the nucleus accumbens in
amphetamine-withdrawn rats. This activation consisted primarily of an
increase in the proportion of accumbens neurons exhibiting bistable
membrane oscillations, which is a characteristic of these neurons (Yim
and Morgenson, 1988; O'Donnell and Grace, 1995). At least a part of
the increase in membrane oscillations may be attributable to the
depolarization produced by enhanced excitatory transmission from
corticoaccumbens afferents. We also have shown previously that the
membrane depolarizing plateaus ("up state") of accumbens neurons
were derived from afferents originating within the fornix, presumably
representing hippocampal subiculum neuronal activity (O'Donnell and
Grace, 1995). Therefore, an increase in hippocampal drive would be
expected to augment oscillatory activity within the accumbens. Given
the presence of reciprocal connections between the PFC and hippocampal
formation (Jay et al., 1992; Carr and Sesack, 1996), an increase in
hippocampal drive may also account for the enhanced oscillatory
membrane activity in the PFC.
Amphetamine withdrawal alters intercellular coupling and
network interactions in the corticoaccumbens system
After withdrawal from repeated amphetamine, PFC and accumbens
spiny neurons exhibited high levels of intercellular coupling. Indeed,
others have shown that chronic cocaine administration will induce
changes in connexin expression in the accumbens and persistent
increases in connexin 32 immunoreactivity in hippocampal CA1 neurons
(Bennett et al., 1999). Although our previous studies showed that
long-term increases in coupling can occur in vivo in adult
striatal neurons after severe DA depletions by the neurotoxin 6-hydroxydopamine (Onn and Grace, 1999), DA depletion was not likely to
contribute to the changes observed after amphetamine withdrawal. Given
that there was no evidence for a decrease in TH-immunolabeled DA-containing cells in the ventral tegmental area
(VTA) or fibers in these forebrain areas in the amphetamine-treated rats when compared to the vehicle-treated paired controls, the increased incidence of coupling in amphetamine-withdrawn rats was not a
consequence of amphetamine-induced DA terminal neurotoxicity (Ellison
and Switzer, 1993; Eisch and Marshall, 1998). Nonetheless, several studies have shown that disruption of DA transmission can
result in marked changes in coupling in the striatum (Cepeda et al.,
1989; Onn and Grace, 1999). Therefore, it is possible that a dampening
in DA transmission (Robertson et al., 1991; Segal and Kuczenski, 1992;
Paulson and Robinson, 1995) was a factor in the activation of coupling
in amphetamine-withdrawn rats. This hypothesis is consistent with
several studies showing a reduction in DA release after psychostimulant
withdrawal (Acquas et al., 1991; Robertson et al., 1991; Segal and
Kuczenski, 1992). The amphetamine withdrawal-induced enhancement of
corticoaccumbens activity (Keys et al., 1998; Robinson and Kolb, 1998)
and striatal glutamate release (Smith et al., 1995; Reid et al., 1997;
Rawls and McGinty, 1998) may play a role in this response. A
compromised DA system may activate corticostriatal transmission through
at least two mechanisms: (1) an increase in activity within the
cortical neurons projecting to striatal/accumbens sites, as
demonstrated in the present study and/or (2) a decreased DA inhibition
of corticostriatal fibers (Calabresi et al., 1988; O'Donnell and
Grace, 1994; Levine et al., 1996; Nicola et al., 1996; Onn and Grace,
1998). Therefore, a common mechanism of treatment-induced alteration in
coupling may be the activation of corticostriatal glutamatergic transmission.
Withdrawal from amphetamine treatment also resulted in a decrease in
the number of neurons exhibiting NOS staining in the accumbens of
amphetamine-withdrawn rats. Glutamate is known to elicit nitric oxide
release in the striatum via stimulation of NOS-containing interneurons
(Bredt and Synder, 1992). This modulation of coupling by NO has also
been shown to occur in several brain regions, e.g., cortex (Rorig and
Sutor, 1996), hypothalamus (Hatton and Yang, 1996; Yang and Hatton,
1999), and nucleus accumbens (O'Donnell and Grace, 1997). However, the
mechanism underlying this NOS downregulation is not clear. One
possibility is that the decrease in NOS is a compensatory change in
response to the heightened drive by the cortex, to keep the level of
coupling in the nucleus accumbens in balance. Alternatively, if there
is an alteration in gap junction connexin composition (Bennett et al.,
1999) or in their phosphorylation state (Matsumoto et al., 1991; Kwak
et al., 1995) after amphetamine, the connexins may show a heightened
sensitivity to NOS. Thus, in one scenario, during amphetamine treatment
there could be a downregulation of corticoaccumbens activity,
necessitating an alteration in the connexins to maintain coupling at a
"normal" level. Consequently, the activation of corticoaccumbens
drive after amphetamine withdrawal may induce a compensatory
downregulation in NOS. Thus, it is apparent that, at least with respect
to dye coupling, the compensatory processes that occur after withdrawal
are insufficient to restore the system to normal.
Implications of amphetamine withdrawal-induced changes in the
corticoaccumbens system to drug abuse
We have shown that there are several alterations in the physiology
of the corticoaccumbens system that persist for at least 28 d
after amphetamine withdrawal. A number of studies have shown persistent
changes in the forebrain after withdrawal from repeated psychostimulants (Kamata and Rebec, 1983; Koob and Bloom, 1988; Henry
and White, 1991, 1995; Paulson et al., 1991; Grace, 1995; White et al.,
1995; Kuhar and Pilotte, 1996; Keys et al., 1998; Robinson and Kolb,
1998; Bennett et al., 1999). However, the mechanism underlying these
persistent changes is not understood. One possibility may
be related to the homeostatic compensations that take place in this
system during amphetamine treatment and withdrawal. Thus, during
psychostimulant administration, there is evidence for an alteration in
connexin content of gap junctions within limbic regions
(Bennett et al., 1999). Given that the alteration in dye coupling is
not observed until after amphetamine is withdrawn, it appears that the
change in connexin may be part of a compensatory process to maintain
coupling at a normal state during amphetamine treatment. After
amphetamine withdrawal, there is in addition another alteration layered
into the system; i.e., a decrease in NOS-positive neurons in the PFC
and nucleus accumbens. One possibility is that this is the means by
which the system compensates for the alterations in gap junction
composition, because a return of the gap junctions to their original
configuration may require substantial amounts of time to occur. The
consequence of these multiple layers of adaptation is that the
amphetamine-withdrawn system is now in a new steady-state, because the
compensatory changes produced by amphetamine withdrawal do not return
the system to the pre-amphetamine state (Grace, 1995). Such a condition
may be similar to those adaptive cellular processes that occur in association with each phase of drug addiction as advanced by our laboratory (Grace, 1995) and by others (Koob, 1992; Bonei and Williams,
1996; Nestler and Aghajanian, 1997). Therefore, repeated amphetamine
administration may drive the system into a state from which it cannot return.
What is the consequence of these alterations? One possibility is that
the increase in coupling may be responsible for the observed synchrony
in responses reported to occur in accumbens neurons in
self-administering rats after repeated exposure to psychostimulants
(Peoples et al., 1998). Moreover, at a functional level, in
studies using the gap junction inhibitor carbenoxolone, we found that
inhibition of gap junctions blocked the stereotyped motor behavior
elicited by apomorphine in the dorsal striatum (Grace and Moore, 1996),
possibly by reversing the apomorphine-induced increase in dye coupling
in this region (Onn and Grace, 1994). Although speculative, one
possibility is that increased intercellular communication in the
ventral striatum may lead to a type of limbic perseveration, such as
the persistent drug-seeking behavioral patterns demonstrated by drug
addicts even after long-term drug abstinence (Robinson and Berridge,
1993). Such a process could be related to the permanent changes that
occur in the brains of drug addicts (Kuhar and Pilotte, 1996; Gatley
and Volkow, 1998) that cause them to persist in drug-seeking behavior
and make them prone to relapse.
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FOOTNOTES |
Received Sept. 21, 1999; revised Dec. 23, 1999; accepted Dec. 29, 1999.
This work was supported by United States Public Health Service Grants
MH 01055, MH 292670, MH57440, and MH 45156. We thank Dr. Susan Sesack
for insightful discussions on quantification of anatomy. We also thank
Nicole MacMurdo for providing excellent histology assistance and Brian
Lowry for providing excellent computer programs for data acquisition
and analysis.
Correspondence should be addressed to Dr. Shao-Pii Onn, 464 Crawford
Hall, Department of Neuroscience, University of Pittsburgh, Pittsburgh,
PA 15260. E-mail: onn{at}bns.pitt.edu.
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TOP
ABSTRACT
INTRODUCTION
