Directory UMM :Data Elmu:jurnal:B:Biological Psichatry:Vol49.Issue3.2001:
ORIGINAL ARTICLE
Effects of Nicotine Pretreatment on Dopaminergic
and Behavioral Responses to Conditioned Fear
Stress in Rats: Dissociation of Biochemical and
Behavioral Effects
Tony P. George, Marina R. Picciotto, Christopher D. Verrico, and Robert H. Roth
Background: We have examined the effects of nicotine
pretreatment on dopaminergic and behavioral responses
to conditioned fear stress in the rat.
Methods: Rats were pretreated daily with saline or
nicotine for 20 days then challenged with nicotine or
saline on day 21. Animals were trained in a classical
conditioned fear paradigm. Dopamine utilization in the
medial prefrontal cortex and nucleus accumbens shell and
conditioned fear stress–induced immobility responses
were assessed.
Results: Saline pretreated animals rapidly acquired the
conditioned fear stress response as assessed by preferential activation of mesoprefrontal dopamine metabolism
and tone-elicited immobility responses. Repeated, but not
acute, nicotine pretreatment significantly reduced conditioned fear stress–induced dopamine metabolism in the
medial prefrontal cortex and nucleus accumbens shell.
Repeated nicotine pretreatment did not modify the acquisition or expression of conditioned fear stress responses,
however.
Conclusions: The dissimilar effects of repeated nicotine
exposure on the cortical dopamine and behavioral responses to conditioned fear stress suggest that nicotine
differs from other agents with anxiolytic activity that
produce coordinated changes in conditioned fear stress–
induced cortical dopaminergic and behavioral responses.
Furthermore, compared with results of acute footshock
stress, repeated nicotine pretreatment appears to have
differential effects on physical versus psychological stressors. Results are discussed within the clinical context of
stress-related psychopathology syndromes and comorbid
nicotine dependence. Biol Psychiatry 2001;49:300 –306
© 2001 Society of Biological Psychiatry
From the Division of Substance Abuse, Connecticut Mental Health Center (TPG),
and Departments of Psychiatry (TPG, MRP, RHR), Pharmacology (MRP,
CDV, RHR), and Neurobiology (MRP), Yale University School of Medicine,
New Haven, Connecticut.
Address reprint requests to Tony P. George, M.D., Yale University School of
Medicine, Connecticut Mental Health Center, Room S-109, Substance Abuse
Center, 34 Park Street, New Haven CT 06508.
Received January 4, 2000; revised May 2, 2000; accepted May 17, 2000
© 2001 Society of Biological Psychiatry
Key Words: Nicotine, dopamine, prefrontal cortex, nucleus accumbens, conditioned fear stress, immobility
response
Introduction
N
icotine dependence through tobacco smoking has a
prevalence in the general population of ;25%. In
psychiatric populations, smoking rates are much higher;
patients with major depression and anxiety disorders have
rates of nicotine dependence of 40 to 50%, whereas rates
in alcohol and cocaine dependence (60 – 80%) and schizophrenic disorders (58 – 88%) are much higher (Dalack et al
1998; Hughes et al 1986; Ziedonis and George 1997). The
high rates of comorbid nicotine dependence in psychiatric
disorders may relate to 1) self-medication of dysfunctional
affective and cognitive states, 2) enhanced rewarding
properties of nicotine in patients with these disorders, 3)
attenuation of side effects of psychotropic medications
(e.g., neuroleptic-induced parkinsonism; Ziedonis and
George 1997; George et al 1998), 4) a shared susceptibility
for both disorders. As such, nicotine dependence through
habitual tobacco use may attenuate symptoms in individuals with neuropsychiatric disorders, as has been documented in major depression, anxiety disorders, posttraumatic stress disorder (PTSD), Alzheimer’s disease,
Parkinson’s disease, and Tourette’s syndrome (Jarvik
1991). Furthermore, nicotine appears to have stress-reducing effects in both animal models and in human smokers
(Breslau 1995; Brioni et al 1993; George et al 1998).
Dopaminergic (DA) projections from the ventral tegmental area in the midbrain to the prefrontal cortex appear
to have an important role in cognition (Goldman-Rakic
and Selemon 1997; Knable and Weinberger 1997) and
affective responses (Horger and Roth 1996). Dysfunction
of mesoprefrontal DA pathways may be involved in
neuropsychiatric diseases such as schizophrenia and affective disorders (Roth and Elsworth 1995). Mild stressors
including acute footshock stress (George et al 1998, 2000)
and conditioned fear stress (CFS; Goldstein et al 1994;
0006-3223/01/$20.00
PII S0006-3223(00)00928-8
Conditioned Fear Stress in Rats
Morrow et al 1997) produce preferential activation of
mesoprefrontal DA neurons. This may relate to unique
properties of this subset of DA neurons compared with
nigrostriatal and mesoaccumbal DA neurons including a
lack of (or reduced numbers of) terminal field impulsemodulating autoreceptors and complex afferent regulation
by other neurotransmitter systems. Nicotinic acetylcholine
receptors (nAChRs) may play a role in regulation of
mesoprefrontal DA neurons because nicotine stimulates
these neurons acutely and, when given repeatedly, also
alters DA cell activity and neurotransmitter release and
metabolism (George et al 1998, 2000; Nisell et al 1996;
Vezina et al 1992).
We have shown that repeated but not acute nicotine
preexposure reduces the stress responsivity of mesoprefrontal DA neurons and associated stress-induced behavioral responses in rats subjected to acute inescapable
footshock stress, a physical stressor (George et al 2000).
The effect of nicotine was found to be dose dependent,
with maximal responses at low to moderate nicotine doses,
and these effects were dependent on stimulation of
mecamylamine-sensitive nAChRs (George et al 1998) and
activity of the endogenous opioid peptide system (George
et al 2000).
In our study, we tested the effects of nicotine pretreatments on dopaminergic and behavioral responses to CFS,
a psychologic stressor resulting from the pairing of neutral
(conditioned) and aversive (unconditioned) stimuli. Our
results suggest that nicotine pretreatment has differential
effects on mesocorticolimbic dopaminergic and behavioral
responses to CFS, suggesting that nicotine dependence
may produce clinical effects on biochemical and behavioral responses to stress through different mechanisms.
These findings are discussed in the context of the observed
high rates of nicotine dependence in patients with stressexacerbated psychiatric disorders, such as major depression, schizophrenia, and PTSD.
Methods and Materials
Materials
Male Sprague–Dawley rats initially weighing 150 to 175 g were
obtained from CAMM (Rutgers, NJ). The weights of rats at the
conclusion of the experiments was 300 to 350 g. S-(2)-nicotine
bitartarate was obtained from Sigma Chemical (St. Louis).
Treatment Paradigm
Rats were given daily, subcutaneous (SC) injections of saline (1
cc/kg) or nicotine bitartarate (0.15 mg/kg, expressed as the
freebase) for 20 days, and then given a challenge injection with
saline or nicotine on day 21. All injection solutions were freshly
prepared daily, and the pH of the saline and nicotine solutions
BIOL PSYCHIATRY
2001;49:300 –306
301
was adjusted to pH 7.4 with sodium hydroxide. Biochemical
measures were obtained 0.5 hours after challenge injections.
Conditioned Fear Stress Procedure
The procedures for the CFS procedure have been described in
detail elsewhere (Goldstein et al 1994; Morrow et al 1997). On
day 19, rats were placed in sound-attenuated paired chambers
(24 3 30 3 27 cm) with a stainless steel rod floor wired for
footshock for ;0.5 hours (“habituation”) for 30 min. On day 20,
after being given a saline or nicotine (0.15 mg/kg, SC) injection,
rats receiving the conditioning procedure were placed into the
chambers and given 10 tones (2.8 kHz, 5 sec duration) in
combination with an incandescent red signal light (placed behind
the chambers) paired with footshocks (0.8 mA 160 msec duration; Davis and Astrachan 1978) over a 30-min period using a
pulse-shock generator (BRS/LVE Division of Tech Serv, Beltsville, MD) and a pulse stimulator (Grass Medical Instruments,
Quincy, MA) connected to an IBM personal computer (the
“acquisition” period). The intervals between the tones were
randomly selected by the computer to be between 1 and 4 min.
Unstressed control subjects were given tones paired with the
signal light only. On day 21, after receiving challenge injections,
both stressed and unstressed rats were reintroduced to the
chamber and given 10 tones only paired with the signal light (the
“expression” period).
Biochemical Analysis
Immediately after the conclusion of the “expression” period (0.5
hours after challenge injection), rats were sacrificed by decapitation. Samples of mPFC (12.7–1.2 mm from bregma) were
harvested by block dissection (1.5 mm thick), and samples of the
shell portion of NAS (11.2– 0.2 mm from bregma) and dorsolateral striatum (20.2–1.2 mm from bregma) were obtained by
tissue punch (Figure 1; Paxinos and Watson 1986) and were 2
mm in diameter and 1 mm in thickness. All tissue samples were
stored frozen at 270°C until prepared for extraction.
Dopamine (DA) and its major metabolite, dihydroxyphenylacetic acid (DOPAC), were quantitated using high-performance
liquid chromatography with electrochemical detection (HPLCED) with a glassy carbon electrode set at 10.7 V and an
Ag/AgCl reference electrode. Tissue was sonicated before the
extraction procedure, which involved alumina extraction before
HPLC analysis as described previously (Elsworth et al 1996). A
reverse phase 3-m C18 HPLC column (Ranin Instruments,
Woburn, MA) was utilized. The mobile phase, delivered at 0.65
mL/min, was comprised of sodium citrate (30 mmol/L), sodium
dihydrogen phosphate (14 mmol/L), sodium octanesulphonate
(2.3 mmol/L), EDTA (0.025 mmol/L), acetonitrile (6.5%), tetrahydrofuran (0.6%), and diethylamine (0.1%), adjusted to pH 3.10
with concentrated phosphoric acid. Dihydroxybenzamine was
used as an internal standard and used to calculate percentage
recovery of DOPAC and DA. Results are expressed as the ratio
of DOPAC to DA, with tissue levels calculated in ng/mg protein.
Protein determination was performed using the method of Lowry
et al (1957) with bovine serum albumin as standard.
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2001;49:300 –306
T.P. George et al
Figure 1. Coronal sections of regions sampled for tissue analysis
of dopamine and DOPAC. Slices are adapted from the atlas of
Paxinos and Watson (1986).
Behavioral Ratings
All footshock sessions were recorded by videotaping. The
percentage of freezing (% immobility) behavior each minute
after presentation of the 10 sequential tones (acquisition and
expression) was scored by blinded examiners (TPG, CDV) who
manually rated the taped sessions post hoc. There was excellent
interrater reliability (k 5 .80) in the scoring of immobility
(George et al 1998, 2000).
Figure 2. Effects of nicotine (NIC) pretreatments on dopaminergic responses to conditioned fear stress in medial prefrontal
cortex (mPFC) and shell subdivision of the nucleus accumbens
[NAS(shell)]. Treatment group numbers were saline (SAL)/SAL
SHAM (7), SAL/NIC/SHAM (7), NIC/NIC/SHAM (6), NIC/
SAL/SHAM (6), SAL/SAL/STRESS (8), SAL/NIC/STRESS
(8), NIC/NIC/STRESS (8), NIC/SAL/STRESS (7). DA, dopamine; DOPAC, dihydroxyphenylacetic acid; *p , .05 vs. saline
control subjects; **p , .05 vs. stress control subjects.
Statistics
SuperANOVA (Abacus Concepts, Berkeley, CA) was used for
statistical analysis. For biochemical analyses, comparisons were
performed using one- and three-factor analysis of variance
(ANOVA) with pretreatment, challenge, and conditioned fear
stress as the independent variables. For analysis of tone presentation effects on immobility responses, two-factor ANOVA with
repeated measures (one within and one between-groups comparison) was used. Post hoc comparisons were performed using
Fisher’s least significant difference procedure; differences were
considered significant when p , .05.
Results
Effects of Nicotine Pretreatment on Conditioned
Fear Stress–Induced Mesoprefrontal and NAS DA
Utilization
mPFC. In mPFC (Figure 2, upper panel), there were
significant effects of nicotine pretreatment [F(1,43) 5
17.88, p , .01] and stress [F(1,43) 5 10.23, p , .01], but
not nicotine challenge [F(1,43) 5 1.04, p 5 .31] on DA
utilization and a nearly significant pretreatment 3 challenge 3 stress interaction [F(1,43) 5 3.38, p 5 .07].
Acute nicotine augmented mPFC DA utilization (p ,
.01), whereas repeated nicotine pretreatment (0.15 mg/kg,
SC) produced tolerance to saline or nicotine challenge
(Figure 2, upper panel). Conditioned fear stress produced
a significant increase in mPFC DA utilization (p , .01),
and this was significantly reduced by repeated (p , .05),
but not acute (p 5 .56) nicotine pretreatment. Furthermore, when the challenge injection after repeated nicotine
administration was saline, the mesoprefrontal DA responses to footshock stress were also reduced (p , .01).
There were no significant differences in tissue DA
levels between the various pretreatment groups (data not
shown), suggesting that changes in the DOPAC/DA ratio
were metabolite driven (i.e., enhanced formation of
DOPAC). The 21-day saline or nicotine pretreatments
Conditioned Fear Stress in Rats
BIOL PSYCHIATRY
2001;49:300 –306
303
used in our present study produced similar effects on
mesoprefrontal DA utilization compared with our previously established pretreatment paradigm using 5-day saline or nicotine pretreatments (George et al 1998, 2000).
SHELL SUBDIVISION OF NAS (NASsh). In the NASsh
(Figure 2, lower panel), there were significant effects of
nicotine pretreatment [F(1,43) 5 33.83, p , .01] and
nicotine challenge [F(1,43) 5 10.49, p , .01], but not
stress [F(1,43) 5 1.33, p 5 .26] on DA utilization, and a
significant pretreatment 3 challenge 3 stress interaction
[F(1,43) 5 5.33, p 5 .03].
Acute nicotine challenge augmented NASsh DA utilization (p , .01), whereas repeated nicotine pretreatment
(0.15 mg/kg, SC) produced tolerance to saline or nicotine
challenge. Nicotine withdrawal for 24 hours (NIC/SAL)
under nonstress conditions led to a reduction in NASsh DA
utilization compared with SAL/SAL control subjects (p ,
.05). Stress produced a significant increase in NASsh DA
utilization (p , .01), and, similar to the mPFC, this was
significantly reduced by repeated nicotine whether the
challenge was with nicotine (p , .01) or saline (p , .01).
Similar to the mPFC, acute nicotine pretreatment did not
alter CSF-induced NASsh responses (p 5 .41). There were
no differences in tissue DA levels in NASsh among the
various pretreatment groups.
There were no significant effects of nicotine pretreatments or CFS on DA utilization in the core subdivision of
the NAS or the dorsolateral striatum (data not shown),
consistent with our previous results with acute footshock
stress (George et al 1998, 2000).
Effects of Nicotine Pretreatments on Conditioned
Fear Responses
ACQUISITION OF CFS (FIGURE 3, UPPER PANEL). In
unstressed rats, there were no differences in immobility
responses between the treatment groups during tone presentation. Tones paired with footshock produced a rapid
increase in immobility with a significant effect of tone–
shock presentation [F(9,225) 5 23.10, p , .01) and a
tones 3 pretreatment interaction [F(27,225) 5 1.58, p 5
.04], but not nicotine pretreatment [F(3,225) 5 2.71,
p 5 .07]. Post hoc analysis revealed differences between
saline pretreated control subjects (SAL/SAL) and repeated
nicotine pretreated animals at Tone 6 (NIC/NIC; p , .05)
and Tone 8 (NIC/NIC and NIC/SAL; p , .05).
EXPRESSION OF CFS (FIGURE 3, LOWER PANEL). In
unstressed rats, there were no differences in immobility
between the treatment groups during tone presentation.
Before the presentation of the first tone during the expression session, animals pretreated with nicotine versus saline
Figure 3. Effects of nicotine (NIC) pretreatments on immobility
responses to the acquisition and expression of conditioned fear
(CF) stress. Treatment group numbers were as in Figure 2. SAL,
saline; *p , .05 vs. saline control subjects; **p , .05 vs. saline
control subjects.
and trained in the CFS procedure displayed a modest
increase in freezing behavior (data not shown) and subsequently displayed robust immobility responses when the
first tone was presented. This is consistent with data in
mice suggesting that repeated nicotine administration
enhances contextual, but not cued, fear conditioning when
given during both CFS training and testing phases (Gould
and Wehner 1999).
The presentation of tones alone produced a significant
but modest effect on reduction of the expression of
CFS-induced immobility responses [F(9,216) 5 2.74, p ,
.01], and there was a reduction of tone-induced immobility
with successive tone presentation, consistent with partial
extinction of the conditioned fear response. There were no
significant effects of nicotine pretreatment [F(3,216) 5
0.93, p 5 .44] and no pretreatment 3 tones interaction
[F(27,216) 5 0.74, p 5 .82]. There were few significant
post hoc differences among the various pretreatment group
across the series of tones presented. The NIC/NIC and
NIC/SAL groups demonstrated a decrease in immobility
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2001;49:300 –306
responses compared with SAL/SAL control subjects at the
Tone 6 presentation only (Figure 3, lower panel).
Discussion
The results of our study suggest that repeated nicotine
pretreatment differentially affects mesocorticolimbic dopaminergic (DA) and behavioral responses to conditioned
fear stress (CFS). Similar to our previous studies, repeated,
but not acute, nicotine administration attenuated CSFinduced mesoprefrontal DA metabolism (George et al
1998). In contrast to our previous findings, the uncoupling
of the effects of repeated nicotine administration on
conditioned fear stress-induced cortical DA and immobility responses suggests that the situation with a conditioned
fear stressor is more complex. Different neuroanatomic
pathways appear to subserve immobility responses to
acute footshock and conditioned fear stressors. Acute
footshock (physical) stress appears to recruit nociceptive
pathways and the involvement of endogenous opioid
peptide systems (George et al 2000), whereas conditioned
fear (psychologic) stress utilizes circuits involving the
amygdala (i.e., basolateral and central nuclei), hippocampus, and medial prefrontal cortex (Armony and LeDoux
1997; Morgan and LeDoux 1995). Several studies have
suggested that augmentation of mesocortical DA function
is linked to conditioned fear immobility responses and that
agents that reduce mPFC dopaminergic responses to CFS
(i.e., diazepam and the NMDA/glycine site antagonist
(1)-HA-966) also reduce CFS immobility responses (i.e.,
block the acquisition of or enhance the extinction of
immobility responses (Goldstein et al 1994; Ida et al 1989;
Morrow et al 1997, 1999b; Yoshioka et al 1996). This
coordinated effect of putative anxiolytic agents on dopaminergic and behavioral responses to CFS is not seen in
the present experiments involving nicotine pretreatment.
Our findings are consistent with those of Gould and
Wehner (1999), who observed that repeated nicotine
pretreatment (during both training and testing phases of
the fear conditioning procedure) did not alter the expression of conditioned fear responses in C57BL/6 mice at the
nicotine doses used in our study. This group also found
that nicotine pretreatment (0.1– 0.3 mg/kg) during training
and testing phases of the CFS procedure enhanced contextual fear conditioning, which we observed to be elevated during the testing (expression phase) before tone
presentation. Contextual fear conditioning is known to be
dependent on hippocampal function, whereas cued conditioned fear responses are thought to be mediated through
the amygdala (Phillips and LeDoux 1992). This is consistent with the differential effects of repeated nicotine
administration on contextual versus cued conditioned fear
responses observed in the study of Gould and Wehner
T.P. George et al
(1999) and in our study. In addition, we used a higher
footshock intensity (0.8 mA) compared with previous
conditioned fear studies in this laboratory (0.3– 0.4 mA;
Goldstein et al 1994; Morrow et al 1999a) to produce more
consistent activation of the mesoprefrontal DA system
(George et al 1998, 2000). Thus, the degree of extinction
of immobility responses achieved with presentation of the
10 tones during the 30 minute expression phase (20 –30%)
in our present study was less than in our previous studies
(40 – 60%).
The finding that repeated nicotine modulates stressinduced DA metabolism in both the medial prefrontal
cortex and the shell subdivision of the nucleus accumbens,
both of which are A10 DA neuron-innervated terminal
fields with preferential responsiveness to mild stressors,
suggests that these previously observed findings in the
mesoprefrontal DA projection extend to the shell portion
of the NAS (Morrow et al 1999). In contrast, there were no
effects of repeated nicotine or CFS on DA utilization in
the core subdivision of NAS or dorsolateral striatum,
suggesting that A9 DA neurons are not involved in this
process. There is both physical (Clarke and Pert 1985) and
functional (George et al 1998, 2000; Nisell et al 1996;
Vezina et al 1992) evidence for the presence of nicotinic
acetylcholine receptors (nAChRs) on both mesolimbic and
mesoprefrontal DA neurons, and the similar effects of
nicotine of both mesocortical and shell subdivision mesoaccumbal DA pathways probably relates to common
afferent regulation of these DA systems by nicotinic
cholinergic systems, either directly or indirectly.
CFS may be phenomenologically similar to some of the
psychopathologic sequelae of PTSD (Yehuda and Antelmann 1993). There are few studies that have characterized
the prevalence rates of nicotine dependence in PTSD or
the modulation of stress-exacerbated symptomatology in
this disorder. One study found that Vietnam combat
veterans with PTSD have rates of cigarette smoking
similar to those of veterans without PTSD (53 vs. 45%),
but that PTSD veterans were heavier smokers and that
heavy smoking was correlated with avoidance and numbing and with hyperarousal symptoms (Beckham et al
1997). These symptoms may resemble features of behavioral responses seen with CFS. It is notable that in the
fear-potentiated startle paradigm, which resembles CFS
(Davis 1992), nicotine reduced fear-potentiated startle
responses without affecting baseline startle. Studies by
several groups (Jarvik et al 1989; Pomerleau and Pomerleau 1987; Rose et al 1983; Schachter 1978; Warburton
1992) have suggested that cigarette smoking increases
during periods of stress, especially with respect to psychologic stressors, and smoking severity is known to be
robustly linked to the number of life stressors in childhood
(Anda et al 1999). Nonetheless, a controlled demonstra-
Conditioned Fear Stress in Rats
tion that psychologic stress increases nicotine use has not
been reported (Pomerleau and Pomerleau 1991). The issue
of whether nicotine exerts anxiolytic effects or produces
withdrawal attenuation is still unresolved (Parrott 1995;
Silverstein 1982) and requires further study in nicotinenaive human subjects.
Taken together, our findings with respect to a lack of an
effect of repeated nicotine administration (presumably
similar to habitual smoking) in modulating behavioral
responses to CFS, but reducing cortical DA responses,
suggests a complex relationship between psychologic
stress and the effects of repeated nicotine administration. It
is possible that 1) the CFS-reducing effects of repeated
nicotine pretreatment correlate with blunted cortical DA
responses, but not behavior responses; 2) the stressreducing effects of repeated nicotine are not sensitively
measured by conditioned fear immobility; and 3) conditioned fear immobility responses are not closely linked to
stress-induced evoked changes cortical DA function. Our
previous work has demonstrated that the repeated effects
of nicotine on acute footshock (physical) stress are mediated by endogenous opioid peptide systems (George et al
2000), and thus behavioral responses to conditioned fear
stress (a psychological stressor) may not be mediated by
these systems. Furthermore, the relationship between nicotine dependence and stress-responsive psychiatric disorders such as PTSD, depression, and schizophrenia, as well
as the effects of nicotine dependence on the course of
these disorders, requires further evaluation.
Supported in part by grants from the Lucille P. Markey Foundation, the
National Alliance for Research on Schizophrenia and Depression, and
Grants Nos. K12-DA-00167 (TPG), K02-DA-00436 (MRP), R37-MH14092 (RHR), and P50-DA-84733.
The authors thank Barbara J. Caldarone, Ph.D., and Therese A.
Kosten, Ph.D., for their helpful comments on the manuscript, and Li Xu
for technical assistance.
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23:247–254.
Effects of Nicotine Pretreatment on Dopaminergic
and Behavioral Responses to Conditioned Fear
Stress in Rats: Dissociation of Biochemical and
Behavioral Effects
Tony P. George, Marina R. Picciotto, Christopher D. Verrico, and Robert H. Roth
Background: We have examined the effects of nicotine
pretreatment on dopaminergic and behavioral responses
to conditioned fear stress in the rat.
Methods: Rats were pretreated daily with saline or
nicotine for 20 days then challenged with nicotine or
saline on day 21. Animals were trained in a classical
conditioned fear paradigm. Dopamine utilization in the
medial prefrontal cortex and nucleus accumbens shell and
conditioned fear stress–induced immobility responses
were assessed.
Results: Saline pretreated animals rapidly acquired the
conditioned fear stress response as assessed by preferential activation of mesoprefrontal dopamine metabolism
and tone-elicited immobility responses. Repeated, but not
acute, nicotine pretreatment significantly reduced conditioned fear stress–induced dopamine metabolism in the
medial prefrontal cortex and nucleus accumbens shell.
Repeated nicotine pretreatment did not modify the acquisition or expression of conditioned fear stress responses,
however.
Conclusions: The dissimilar effects of repeated nicotine
exposure on the cortical dopamine and behavioral responses to conditioned fear stress suggest that nicotine
differs from other agents with anxiolytic activity that
produce coordinated changes in conditioned fear stress–
induced cortical dopaminergic and behavioral responses.
Furthermore, compared with results of acute footshock
stress, repeated nicotine pretreatment appears to have
differential effects on physical versus psychological stressors. Results are discussed within the clinical context of
stress-related psychopathology syndromes and comorbid
nicotine dependence. Biol Psychiatry 2001;49:300 –306
© 2001 Society of Biological Psychiatry
From the Division of Substance Abuse, Connecticut Mental Health Center (TPG),
and Departments of Psychiatry (TPG, MRP, RHR), Pharmacology (MRP,
CDV, RHR), and Neurobiology (MRP), Yale University School of Medicine,
New Haven, Connecticut.
Address reprint requests to Tony P. George, M.D., Yale University School of
Medicine, Connecticut Mental Health Center, Room S-109, Substance Abuse
Center, 34 Park Street, New Haven CT 06508.
Received January 4, 2000; revised May 2, 2000; accepted May 17, 2000
© 2001 Society of Biological Psychiatry
Key Words: Nicotine, dopamine, prefrontal cortex, nucleus accumbens, conditioned fear stress, immobility
response
Introduction
N
icotine dependence through tobacco smoking has a
prevalence in the general population of ;25%. In
psychiatric populations, smoking rates are much higher;
patients with major depression and anxiety disorders have
rates of nicotine dependence of 40 to 50%, whereas rates
in alcohol and cocaine dependence (60 – 80%) and schizophrenic disorders (58 – 88%) are much higher (Dalack et al
1998; Hughes et al 1986; Ziedonis and George 1997). The
high rates of comorbid nicotine dependence in psychiatric
disorders may relate to 1) self-medication of dysfunctional
affective and cognitive states, 2) enhanced rewarding
properties of nicotine in patients with these disorders, 3)
attenuation of side effects of psychotropic medications
(e.g., neuroleptic-induced parkinsonism; Ziedonis and
George 1997; George et al 1998), 4) a shared susceptibility
for both disorders. As such, nicotine dependence through
habitual tobacco use may attenuate symptoms in individuals with neuropsychiatric disorders, as has been documented in major depression, anxiety disorders, posttraumatic stress disorder (PTSD), Alzheimer’s disease,
Parkinson’s disease, and Tourette’s syndrome (Jarvik
1991). Furthermore, nicotine appears to have stress-reducing effects in both animal models and in human smokers
(Breslau 1995; Brioni et al 1993; George et al 1998).
Dopaminergic (DA) projections from the ventral tegmental area in the midbrain to the prefrontal cortex appear
to have an important role in cognition (Goldman-Rakic
and Selemon 1997; Knable and Weinberger 1997) and
affective responses (Horger and Roth 1996). Dysfunction
of mesoprefrontal DA pathways may be involved in
neuropsychiatric diseases such as schizophrenia and affective disorders (Roth and Elsworth 1995). Mild stressors
including acute footshock stress (George et al 1998, 2000)
and conditioned fear stress (CFS; Goldstein et al 1994;
0006-3223/01/$20.00
PII S0006-3223(00)00928-8
Conditioned Fear Stress in Rats
Morrow et al 1997) produce preferential activation of
mesoprefrontal DA neurons. This may relate to unique
properties of this subset of DA neurons compared with
nigrostriatal and mesoaccumbal DA neurons including a
lack of (or reduced numbers of) terminal field impulsemodulating autoreceptors and complex afferent regulation
by other neurotransmitter systems. Nicotinic acetylcholine
receptors (nAChRs) may play a role in regulation of
mesoprefrontal DA neurons because nicotine stimulates
these neurons acutely and, when given repeatedly, also
alters DA cell activity and neurotransmitter release and
metabolism (George et al 1998, 2000; Nisell et al 1996;
Vezina et al 1992).
We have shown that repeated but not acute nicotine
preexposure reduces the stress responsivity of mesoprefrontal DA neurons and associated stress-induced behavioral responses in rats subjected to acute inescapable
footshock stress, a physical stressor (George et al 2000).
The effect of nicotine was found to be dose dependent,
with maximal responses at low to moderate nicotine doses,
and these effects were dependent on stimulation of
mecamylamine-sensitive nAChRs (George et al 1998) and
activity of the endogenous opioid peptide system (George
et al 2000).
In our study, we tested the effects of nicotine pretreatments on dopaminergic and behavioral responses to CFS,
a psychologic stressor resulting from the pairing of neutral
(conditioned) and aversive (unconditioned) stimuli. Our
results suggest that nicotine pretreatment has differential
effects on mesocorticolimbic dopaminergic and behavioral
responses to CFS, suggesting that nicotine dependence
may produce clinical effects on biochemical and behavioral responses to stress through different mechanisms.
These findings are discussed in the context of the observed
high rates of nicotine dependence in patients with stressexacerbated psychiatric disorders, such as major depression, schizophrenia, and PTSD.
Methods and Materials
Materials
Male Sprague–Dawley rats initially weighing 150 to 175 g were
obtained from CAMM (Rutgers, NJ). The weights of rats at the
conclusion of the experiments was 300 to 350 g. S-(2)-nicotine
bitartarate was obtained from Sigma Chemical (St. Louis).
Treatment Paradigm
Rats were given daily, subcutaneous (SC) injections of saline (1
cc/kg) or nicotine bitartarate (0.15 mg/kg, expressed as the
freebase) for 20 days, and then given a challenge injection with
saline or nicotine on day 21. All injection solutions were freshly
prepared daily, and the pH of the saline and nicotine solutions
BIOL PSYCHIATRY
2001;49:300 –306
301
was adjusted to pH 7.4 with sodium hydroxide. Biochemical
measures were obtained 0.5 hours after challenge injections.
Conditioned Fear Stress Procedure
The procedures for the CFS procedure have been described in
detail elsewhere (Goldstein et al 1994; Morrow et al 1997). On
day 19, rats were placed in sound-attenuated paired chambers
(24 3 30 3 27 cm) with a stainless steel rod floor wired for
footshock for ;0.5 hours (“habituation”) for 30 min. On day 20,
after being given a saline or nicotine (0.15 mg/kg, SC) injection,
rats receiving the conditioning procedure were placed into the
chambers and given 10 tones (2.8 kHz, 5 sec duration) in
combination with an incandescent red signal light (placed behind
the chambers) paired with footshocks (0.8 mA 160 msec duration; Davis and Astrachan 1978) over a 30-min period using a
pulse-shock generator (BRS/LVE Division of Tech Serv, Beltsville, MD) and a pulse stimulator (Grass Medical Instruments,
Quincy, MA) connected to an IBM personal computer (the
“acquisition” period). The intervals between the tones were
randomly selected by the computer to be between 1 and 4 min.
Unstressed control subjects were given tones paired with the
signal light only. On day 21, after receiving challenge injections,
both stressed and unstressed rats were reintroduced to the
chamber and given 10 tones only paired with the signal light (the
“expression” period).
Biochemical Analysis
Immediately after the conclusion of the “expression” period (0.5
hours after challenge injection), rats were sacrificed by decapitation. Samples of mPFC (12.7–1.2 mm from bregma) were
harvested by block dissection (1.5 mm thick), and samples of the
shell portion of NAS (11.2– 0.2 mm from bregma) and dorsolateral striatum (20.2–1.2 mm from bregma) were obtained by
tissue punch (Figure 1; Paxinos and Watson 1986) and were 2
mm in diameter and 1 mm in thickness. All tissue samples were
stored frozen at 270°C until prepared for extraction.
Dopamine (DA) and its major metabolite, dihydroxyphenylacetic acid (DOPAC), were quantitated using high-performance
liquid chromatography with electrochemical detection (HPLCED) with a glassy carbon electrode set at 10.7 V and an
Ag/AgCl reference electrode. Tissue was sonicated before the
extraction procedure, which involved alumina extraction before
HPLC analysis as described previously (Elsworth et al 1996). A
reverse phase 3-m C18 HPLC column (Ranin Instruments,
Woburn, MA) was utilized. The mobile phase, delivered at 0.65
mL/min, was comprised of sodium citrate (30 mmol/L), sodium
dihydrogen phosphate (14 mmol/L), sodium octanesulphonate
(2.3 mmol/L), EDTA (0.025 mmol/L), acetonitrile (6.5%), tetrahydrofuran (0.6%), and diethylamine (0.1%), adjusted to pH 3.10
with concentrated phosphoric acid. Dihydroxybenzamine was
used as an internal standard and used to calculate percentage
recovery of DOPAC and DA. Results are expressed as the ratio
of DOPAC to DA, with tissue levels calculated in ng/mg protein.
Protein determination was performed using the method of Lowry
et al (1957) with bovine serum albumin as standard.
302
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2001;49:300 –306
T.P. George et al
Figure 1. Coronal sections of regions sampled for tissue analysis
of dopamine and DOPAC. Slices are adapted from the atlas of
Paxinos and Watson (1986).
Behavioral Ratings
All footshock sessions were recorded by videotaping. The
percentage of freezing (% immobility) behavior each minute
after presentation of the 10 sequential tones (acquisition and
expression) was scored by blinded examiners (TPG, CDV) who
manually rated the taped sessions post hoc. There was excellent
interrater reliability (k 5 .80) in the scoring of immobility
(George et al 1998, 2000).
Figure 2. Effects of nicotine (NIC) pretreatments on dopaminergic responses to conditioned fear stress in medial prefrontal
cortex (mPFC) and shell subdivision of the nucleus accumbens
[NAS(shell)]. Treatment group numbers were saline (SAL)/SAL
SHAM (7), SAL/NIC/SHAM (7), NIC/NIC/SHAM (6), NIC/
SAL/SHAM (6), SAL/SAL/STRESS (8), SAL/NIC/STRESS
(8), NIC/NIC/STRESS (8), NIC/SAL/STRESS (7). DA, dopamine; DOPAC, dihydroxyphenylacetic acid; *p , .05 vs. saline
control subjects; **p , .05 vs. stress control subjects.
Statistics
SuperANOVA (Abacus Concepts, Berkeley, CA) was used for
statistical analysis. For biochemical analyses, comparisons were
performed using one- and three-factor analysis of variance
(ANOVA) with pretreatment, challenge, and conditioned fear
stress as the independent variables. For analysis of tone presentation effects on immobility responses, two-factor ANOVA with
repeated measures (one within and one between-groups comparison) was used. Post hoc comparisons were performed using
Fisher’s least significant difference procedure; differences were
considered significant when p , .05.
Results
Effects of Nicotine Pretreatment on Conditioned
Fear Stress–Induced Mesoprefrontal and NAS DA
Utilization
mPFC. In mPFC (Figure 2, upper panel), there were
significant effects of nicotine pretreatment [F(1,43) 5
17.88, p , .01] and stress [F(1,43) 5 10.23, p , .01], but
not nicotine challenge [F(1,43) 5 1.04, p 5 .31] on DA
utilization and a nearly significant pretreatment 3 challenge 3 stress interaction [F(1,43) 5 3.38, p 5 .07].
Acute nicotine augmented mPFC DA utilization (p ,
.01), whereas repeated nicotine pretreatment (0.15 mg/kg,
SC) produced tolerance to saline or nicotine challenge
(Figure 2, upper panel). Conditioned fear stress produced
a significant increase in mPFC DA utilization (p , .01),
and this was significantly reduced by repeated (p , .05),
but not acute (p 5 .56) nicotine pretreatment. Furthermore, when the challenge injection after repeated nicotine
administration was saline, the mesoprefrontal DA responses to footshock stress were also reduced (p , .01).
There were no significant differences in tissue DA
levels between the various pretreatment groups (data not
shown), suggesting that changes in the DOPAC/DA ratio
were metabolite driven (i.e., enhanced formation of
DOPAC). The 21-day saline or nicotine pretreatments
Conditioned Fear Stress in Rats
BIOL PSYCHIATRY
2001;49:300 –306
303
used in our present study produced similar effects on
mesoprefrontal DA utilization compared with our previously established pretreatment paradigm using 5-day saline or nicotine pretreatments (George et al 1998, 2000).
SHELL SUBDIVISION OF NAS (NASsh). In the NASsh
(Figure 2, lower panel), there were significant effects of
nicotine pretreatment [F(1,43) 5 33.83, p , .01] and
nicotine challenge [F(1,43) 5 10.49, p , .01], but not
stress [F(1,43) 5 1.33, p 5 .26] on DA utilization, and a
significant pretreatment 3 challenge 3 stress interaction
[F(1,43) 5 5.33, p 5 .03].
Acute nicotine challenge augmented NASsh DA utilization (p , .01), whereas repeated nicotine pretreatment
(0.15 mg/kg, SC) produced tolerance to saline or nicotine
challenge. Nicotine withdrawal for 24 hours (NIC/SAL)
under nonstress conditions led to a reduction in NASsh DA
utilization compared with SAL/SAL control subjects (p ,
.05). Stress produced a significant increase in NASsh DA
utilization (p , .01), and, similar to the mPFC, this was
significantly reduced by repeated nicotine whether the
challenge was with nicotine (p , .01) or saline (p , .01).
Similar to the mPFC, acute nicotine pretreatment did not
alter CSF-induced NASsh responses (p 5 .41). There were
no differences in tissue DA levels in NASsh among the
various pretreatment groups.
There were no significant effects of nicotine pretreatments or CFS on DA utilization in the core subdivision of
the NAS or the dorsolateral striatum (data not shown),
consistent with our previous results with acute footshock
stress (George et al 1998, 2000).
Effects of Nicotine Pretreatments on Conditioned
Fear Responses
ACQUISITION OF CFS (FIGURE 3, UPPER PANEL). In
unstressed rats, there were no differences in immobility
responses between the treatment groups during tone presentation. Tones paired with footshock produced a rapid
increase in immobility with a significant effect of tone–
shock presentation [F(9,225) 5 23.10, p , .01) and a
tones 3 pretreatment interaction [F(27,225) 5 1.58, p 5
.04], but not nicotine pretreatment [F(3,225) 5 2.71,
p 5 .07]. Post hoc analysis revealed differences between
saline pretreated control subjects (SAL/SAL) and repeated
nicotine pretreated animals at Tone 6 (NIC/NIC; p , .05)
and Tone 8 (NIC/NIC and NIC/SAL; p , .05).
EXPRESSION OF CFS (FIGURE 3, LOWER PANEL). In
unstressed rats, there were no differences in immobility
between the treatment groups during tone presentation.
Before the presentation of the first tone during the expression session, animals pretreated with nicotine versus saline
Figure 3. Effects of nicotine (NIC) pretreatments on immobility
responses to the acquisition and expression of conditioned fear
(CF) stress. Treatment group numbers were as in Figure 2. SAL,
saline; *p , .05 vs. saline control subjects; **p , .05 vs. saline
control subjects.
and trained in the CFS procedure displayed a modest
increase in freezing behavior (data not shown) and subsequently displayed robust immobility responses when the
first tone was presented. This is consistent with data in
mice suggesting that repeated nicotine administration
enhances contextual, but not cued, fear conditioning when
given during both CFS training and testing phases (Gould
and Wehner 1999).
The presentation of tones alone produced a significant
but modest effect on reduction of the expression of
CFS-induced immobility responses [F(9,216) 5 2.74, p ,
.01], and there was a reduction of tone-induced immobility
with successive tone presentation, consistent with partial
extinction of the conditioned fear response. There were no
significant effects of nicotine pretreatment [F(3,216) 5
0.93, p 5 .44] and no pretreatment 3 tones interaction
[F(27,216) 5 0.74, p 5 .82]. There were few significant
post hoc differences among the various pretreatment group
across the series of tones presented. The NIC/NIC and
NIC/SAL groups demonstrated a decrease in immobility
304
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2001;49:300 –306
responses compared with SAL/SAL control subjects at the
Tone 6 presentation only (Figure 3, lower panel).
Discussion
The results of our study suggest that repeated nicotine
pretreatment differentially affects mesocorticolimbic dopaminergic (DA) and behavioral responses to conditioned
fear stress (CFS). Similar to our previous studies, repeated,
but not acute, nicotine administration attenuated CSFinduced mesoprefrontal DA metabolism (George et al
1998). In contrast to our previous findings, the uncoupling
of the effects of repeated nicotine administration on
conditioned fear stress-induced cortical DA and immobility responses suggests that the situation with a conditioned
fear stressor is more complex. Different neuroanatomic
pathways appear to subserve immobility responses to
acute footshock and conditioned fear stressors. Acute
footshock (physical) stress appears to recruit nociceptive
pathways and the involvement of endogenous opioid
peptide systems (George et al 2000), whereas conditioned
fear (psychologic) stress utilizes circuits involving the
amygdala (i.e., basolateral and central nuclei), hippocampus, and medial prefrontal cortex (Armony and LeDoux
1997; Morgan and LeDoux 1995). Several studies have
suggested that augmentation of mesocortical DA function
is linked to conditioned fear immobility responses and that
agents that reduce mPFC dopaminergic responses to CFS
(i.e., diazepam and the NMDA/glycine site antagonist
(1)-HA-966) also reduce CFS immobility responses (i.e.,
block the acquisition of or enhance the extinction of
immobility responses (Goldstein et al 1994; Ida et al 1989;
Morrow et al 1997, 1999b; Yoshioka et al 1996). This
coordinated effect of putative anxiolytic agents on dopaminergic and behavioral responses to CFS is not seen in
the present experiments involving nicotine pretreatment.
Our findings are consistent with those of Gould and
Wehner (1999), who observed that repeated nicotine
pretreatment (during both training and testing phases of
the fear conditioning procedure) did not alter the expression of conditioned fear responses in C57BL/6 mice at the
nicotine doses used in our study. This group also found
that nicotine pretreatment (0.1– 0.3 mg/kg) during training
and testing phases of the CFS procedure enhanced contextual fear conditioning, which we observed to be elevated during the testing (expression phase) before tone
presentation. Contextual fear conditioning is known to be
dependent on hippocampal function, whereas cued conditioned fear responses are thought to be mediated through
the amygdala (Phillips and LeDoux 1992). This is consistent with the differential effects of repeated nicotine
administration on contextual versus cued conditioned fear
responses observed in the study of Gould and Wehner
T.P. George et al
(1999) and in our study. In addition, we used a higher
footshock intensity (0.8 mA) compared with previous
conditioned fear studies in this laboratory (0.3– 0.4 mA;
Goldstein et al 1994; Morrow et al 1999a) to produce more
consistent activation of the mesoprefrontal DA system
(George et al 1998, 2000). Thus, the degree of extinction
of immobility responses achieved with presentation of the
10 tones during the 30 minute expression phase (20 –30%)
in our present study was less than in our previous studies
(40 – 60%).
The finding that repeated nicotine modulates stressinduced DA metabolism in both the medial prefrontal
cortex and the shell subdivision of the nucleus accumbens,
both of which are A10 DA neuron-innervated terminal
fields with preferential responsiveness to mild stressors,
suggests that these previously observed findings in the
mesoprefrontal DA projection extend to the shell portion
of the NAS (Morrow et al 1999). In contrast, there were no
effects of repeated nicotine or CFS on DA utilization in
the core subdivision of NAS or dorsolateral striatum,
suggesting that A9 DA neurons are not involved in this
process. There is both physical (Clarke and Pert 1985) and
functional (George et al 1998, 2000; Nisell et al 1996;
Vezina et al 1992) evidence for the presence of nicotinic
acetylcholine receptors (nAChRs) on both mesolimbic and
mesoprefrontal DA neurons, and the similar effects of
nicotine of both mesocortical and shell subdivision mesoaccumbal DA pathways probably relates to common
afferent regulation of these DA systems by nicotinic
cholinergic systems, either directly or indirectly.
CFS may be phenomenologically similar to some of the
psychopathologic sequelae of PTSD (Yehuda and Antelmann 1993). There are few studies that have characterized
the prevalence rates of nicotine dependence in PTSD or
the modulation of stress-exacerbated symptomatology in
this disorder. One study found that Vietnam combat
veterans with PTSD have rates of cigarette smoking
similar to those of veterans without PTSD (53 vs. 45%),
but that PTSD veterans were heavier smokers and that
heavy smoking was correlated with avoidance and numbing and with hyperarousal symptoms (Beckham et al
1997). These symptoms may resemble features of behavioral responses seen with CFS. It is notable that in the
fear-potentiated startle paradigm, which resembles CFS
(Davis 1992), nicotine reduced fear-potentiated startle
responses without affecting baseline startle. Studies by
several groups (Jarvik et al 1989; Pomerleau and Pomerleau 1987; Rose et al 1983; Schachter 1978; Warburton
1992) have suggested that cigarette smoking increases
during periods of stress, especially with respect to psychologic stressors, and smoking severity is known to be
robustly linked to the number of life stressors in childhood
(Anda et al 1999). Nonetheless, a controlled demonstra-
Conditioned Fear Stress in Rats
tion that psychologic stress increases nicotine use has not
been reported (Pomerleau and Pomerleau 1991). The issue
of whether nicotine exerts anxiolytic effects or produces
withdrawal attenuation is still unresolved (Parrott 1995;
Silverstein 1982) and requires further study in nicotinenaive human subjects.
Taken together, our findings with respect to a lack of an
effect of repeated nicotine administration (presumably
similar to habitual smoking) in modulating behavioral
responses to CFS, but reducing cortical DA responses,
suggests a complex relationship between psychologic
stress and the effects of repeated nicotine administration. It
is possible that 1) the CFS-reducing effects of repeated
nicotine pretreatment correlate with blunted cortical DA
responses, but not behavior responses; 2) the stressreducing effects of repeated nicotine are not sensitively
measured by conditioned fear immobility; and 3) conditioned fear immobility responses are not closely linked to
stress-induced evoked changes cortical DA function. Our
previous work has demonstrated that the repeated effects
of nicotine on acute footshock (physical) stress are mediated by endogenous opioid peptide systems (George et al
2000), and thus behavioral responses to conditioned fear
stress (a psychological stressor) may not be mediated by
these systems. Furthermore, the relationship between nicotine dependence and stress-responsive psychiatric disorders such as PTSD, depression, and schizophrenia, as well
as the effects of nicotine dependence on the course of
these disorders, requires further evaluation.
Supported in part by grants from the Lucille P. Markey Foundation, the
National Alliance for Research on Schizophrenia and Depression, and
Grants Nos. K12-DA-00167 (TPG), K02-DA-00436 (MRP), R37-MH14092 (RHR), and P50-DA-84733.
The authors thank Barbara J. Caldarone, Ph.D., and Therese A.
Kosten, Ph.D., for their helpful comments on the manuscript, and Li Xu
for technical assistance.
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