Department of Psychology,
East Tennessee State University, Johnson City,
USA
Ohmura Y,
Jutkiewicz EM, Zhang A, Domino EF. Dopamine
D1/5 and D2/3 agonists differentially attenuate
somatic signs of nicotine withdrawal in
rats.Pharmacol Biochem Behav.
2011;99(4):552-556
Tizabi Y et
al Nicotine blocks quinpirole-induced
behavior in rats: psychiatric implications
Psychopharmacology 1999;145:433-441
ABSTRACT
This study was designed to analyze the
effects of nicotine on yawning behavior and
neurotrophin content in the hippocampus and
frontal cortex of D2-receptor primed female
adult Sprague-Dawley rats. Animals were
neonatally treated with quinpirole, a dopamine
(DA) D2/D3 agonist, from postnatal day
1&endash;21 (P1&endash;21) and raised to P60 and
administered nicotine tartarate (0.3 mg/kg free
base) or saline twice daily for 14 days.
One day after nicotine treatment had ceased,
the number of yawns was recorded for 1 h in
response to an acute injection of quinpirole
(i.p., 100 lg/kg). Yawning is a D2-receptor
mediated event. D2-primed rats demonstrated a
significant increase in yawning in response to
acute quinpirole compared with that of controls,
but nicotine did not alleviate this effect.
Neonatal quinpirole treatment produced a
significant decrease of nerve growth factor
(NGF) and brain-derived neurotrophic factor
(BDNF) in the hippocampus that was alleviated by
adulthood nicotine treatment. Interestingly,
nicotine treatment to controls produced a
significant increase of NGF in the frontal
cortex, but a significant decrease of both NGF
and BDNF in the hippocampus and BDNF in the
frontal cortex.
The decreases shown in NGF and BDNF is
contrary to past findings that have shown
nicotine to produce significant increases of
hippocampal NGF and BDNF, but these past studies
utilized male rats or mice or were performed in
vitro. This study shows that nicotine has
complex interactions with NGF and BDNF in
D2-primed and control animals, and emphasizes
the importance of gender differences when
analyzing nicotine's effects on
neurotrophins.
INTRODUCTION
Recent research has shown that nicotine
enhances the concentration of several different
neurotrophins, including nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF),
and fibroblast growth factor-2 (FGF-2) in the
hippocampus, frontal cortex, and striatum,
respectively (French et al., 1999; Kenny et al.,
2000; Maggio et al., 1997, 1998). Neurotrophins
are known to play an important role in the
survival, differentiation, and maintenance of
developing and mature neurons and in the
formation of synaptic cir- cuitry in the brain
(Horch, 2004; Korsching, 1993). Both NGF and
BDNF are present in both the hippo- campus and
frontal cortex, two brain areas known to be of
primary importance for cognitive performance. It
has been hypothesized that NGF plays an
important role in neuronal survival and
differentiation (Hartikka and Hefti, 1988;
Hefti, 1986), whereas BDNF has been shown to be
important in the modification of synaptic
transmission (Binder and Scharfman, 2004).
Both of these neurotrophins have been shown
to be important in cognitive processes, as
infusion of either NGF or BDNF has been shown to
enhance cognitive perform- ance (Fernandez et
al., 1996; Scali et al., 1994; Walz et al.,
2000). Past data from our laboratory and a
collaborating laboratory have shown that
neonatal treatment of rats with quinpirole, a DA
D2/D3 agonist, results in super-sensitization of
the DA D2 receptor, a phenomenon known as
'priming' (Brown et al., 2004a; Kostrzewa et
al., 1991, 2004). Priming of the D2 receptor is
veri- fied through analyzing yawning behavior in
response to an acute injection of quinpirole in
adulthood. Yawn- ing has been shown to be a DA
D2 receptor-mediated behavioral event (Cooper et
al., 1989; Eguibar et al., 2003). Quinpirole has
been given across several differ- ent
developmental periods of drug treatment
(P1&endash;21, P1&endash;11, P11&endash;21, and
P21&endash;35) utilizing several differ- ent
doses ranging from 50 lg/kg/day to 2 mg/kg/day,
and in all cases, D2 receptors were primed into
adult- hood (Kostrzewa et al., 1991, 2004).
Behaviorally, we have found that neonatal
quinpirole treatment results in several
different behavioral abnormalities, includ- ing
prepulse inhibition deficits (Brown et al.,
manu- script submitted), an enhancement or
deficit in skilled reaching depending on the
neonatal period in which quinpirole was
administered (Brown et al., 2002, 2004a), and
cognitive deficits, when D2-primed rats were
tested as early postweanlings or as adults
tested on the Morris water maze (MWM) (Brown et
al., 2002, 2004b, 2005). We have recently shown
that subchronic adminis- tration of nicotine to
adult rats that were D2 receptor- primed as
neonates (P1&endash;21; 1 mg/kg quinpirole) com-
pletely alleviated cognitive deficits on the MWM
(Brown et al., 2004a). Importantly, these
animals were tested on the MWM for 3 consecutive
days after nico- tine administration had ceased,
demonstrating that nicotine's neurophysiological
effects were sufficient to mediate cognitive
improvement.
However, in this past study, brain tissue
was not harvested until 4 weeks after nicotine
administration was completed (P110) because
these animals did not finish behavioral testing
until that time. Brain tissue assays showed that
neo- natal quinpirole treatment produced a
significant decrease in NGF and a near
significant decrease of BDNF in the hippocampus,
but nicotine did not allevi- ate either
decrease, and there were no significant effects
in the frontal cortex. We have also recently
shown that neonatal quinpirole treatment results
in significant decreases of hippocampal NGF and
near significant decreases of hippocampal BDNF
when brain tissue was harvested in younger
animals at P30 (Brown et al., 2004b). These data
suggest that signifi- cant decreases in these
neurotrophins produced by neo- natal quinpirole
treatment may be present throughout early
postnatal and adolescent development, possibly
affecting synaptic maintenance and development
of hippocampal circuitry. The present study was
designed to test the hypothesis that nicotine
will alleviate the sig- nificant decreases in
neurotrophins in the hippocampus and frontal
cortex produced by neonatal quinpirole treatment
when brain tissue was harvested 48 h after
nicotine treatment had ceased. Additionally, the
D2- receptor mediated yawning behavior was
analyzed 24 h after nicotine treatment to
determine whether nicotine is alleviating
significant decreases of neurotro- phins through
a reduction in the priming of the D2 receptor in
rats neonatally treated with quinpirole.
Procedure Yawning behavior
One day after nicotine treatment had ceased,
all animals were given an acute injection of
quinpirole (100 lg/kg), and the number of yawns
was recorded for 1 h. A collaborating laboratory
has previously shown, utilizing a dose-response
curve with three dif- ferent doses of
quinpirole, that the 100-lg/kg dose of
quinpirole was sufficient to induce a maximal
yawn- ing response in comparison with that of
rats neona- tally treated with saline (Nowak et
al., 2001). For this test, animals were
individually placed into plastic polycarbonate
cages that were without bedding. The cage was
inverted during behavioral testing and slightly
elevated above a table, so that the plastic
poly- carbonate cage served as the top and sides
of the cage, and animal droppings would go
through the cage top.
The rationale for this method is that
animals tend to chew on the bedding or droppings
when injected with quinpirole, and we have
observed that chewing can supersede yawning.
Estrous cycle stage verification One day after
the yawning test and immediately before
harvesting brain tissue, the estrous cycle stage
was verified utilizing a damp cotton swab that
was inserted into the vagina and smeared onto a
clean microscope slide. The slides were analyzed
utilizing an Olympus light microscope with 203
magnification. The stage of the estrous cycle
was determined by com- paring the vaginal smear
with reference photographs of cell appearance
during different stages of the estrous cycle,
from a study by Marcondes et al. (2002).
Neurotrophic factor assays One day after the
yawning test, brains were harvested following
rapid decapitation. Whole hippo- campus and
medial frontal cortex were dissected away from
the rest of the brain, and brain tissues were
rapidly frozen by submergence in cold isopentane
(assayed. For the nerve growth factor (NGF) and
brain-derived neurotrophic factor (BDNF) assay,
tis- sues were homogenized in 1 ml of cold lysis
buffer, and an aliquot was used for assessment
of BDNF, NGF, or protein.
The BDNF or NGF EMAX immuno- assay protocol
was followed as defined by the supplier
(Promega, Madison, WI). For the BDNF assay, in
brief, 10 ll of the antiBDNF monoclonal antibody
(mAb) was added to 9.99 ml of carbonate coating
buffer (pH 9.7). There was 100 ll of this
mixture added to each well of a polystyrene
ELISA plate (Nunc, MaxiSorb) and incubated
overnight at 48C. All wells were washed using a
TBST wash buffer, incu- bated at room
temperature for 1 h, and nonspecific binding was
blocked through adding a block and sample 13
buffer and deionized water mixture to each well
and incubated at room temperature for 1 h. The
BDNF standard curve was prepared using the BDNF
standard supplied from the manufacturer (1
lg/ml). The standard was diluted 1:2000 in Block
3 Sample 13 buffer to achieve a concentration of
500 pg/ml.
Both the hippocampal and medial frontal
cortex were further diluted 1:2, prior to being
assayed. The standards and samples were
incubated with shak- ing at room temperature for
2 h. Antihuman BDNF pAB was then added to each
well plate and incubated at room temperature (2
h), which was followed by incubation (1 h) with
antiIgY horseradish peroxidase (HRP) conjugate.
Finally, 100 ll of TMB one solution was added to
each well followed by a 10 min incuba- tion at
room temperature. The entire reaction was
stopped in each well with the addition of 100 ll
of 1 N hydrochloric acid and read within 30 min.
A very similar protocol for NGF analysis was
fol- lowed, and the Promega kit (Madison, WI)
was utilized. For the NGF assay, the hippocampal
samples were diluted 1:15 and the medial frontal
cortex samples 1:25, prior to being added to the
plates. For NGF analysis, flat bottom 96-well
plates were coated with antiNGF polyclonal
antibody (pAb), which binds solu- ble NGF. The
captured NGF is bound by a second spe- cific
mAb. After washing, the amount of specifically
bound mAb was detected using a species-specific
anti- body conjugated to HRP as a tertiary
reactant. The unbound conjugate was removed by
washing and following an incubation with a
chromogenic substrate, the color change was
measured. For both BDNF and NGF, the content was
expressed as the pg of neuro- trophic factor per
mg protein. Protein concentrations were
quantified with the BCA protein assay kit
(Pierce, Rockford, IL).
DISCUSSION
There are two primary findings of the
current study. First, nicotine alleviated
significant decreases of hippocampal NGF and
BDNF produced by neonatal quinpirole treatment
in female rats that were not influenced by the
estrous cycle. Although a mecha- nism is yet to
be identified, it does not appear that nicotine
produces its effects on neurotrophins in D2-
primed rats through alleviating priming of the
D2 receptor, as nicotine did not affect
significant in- creases in yawning in D2-primed
rats. Second, nicotine produced a significant
decrease of NGF and BDNF in the hippocampus and
frontal cortex in rats neonatally treated with
saline. We hypothesize that this signifi- cant
decrease of NGF and BDNF in controls may be due
to increases of stress due to nicotine
withdrawal, which may interact with increased
sensitivity to nicotine in females (Faraday et
al., 2005; Rhodes et al., 2001; Yilmaz et al.,
1997). In agreement with this hypothesis there
are data demonstrating that in adult animals
increased levels of corticosterone can produce
decreases in neurotrophins (Scaccianoce, 2000;
Schaaf, 1998).
Finally, the significant decrease in hip-
pocampal NGF produced by neonatal quinpirole
treat- ment replicates findings from two other
recent studies from this laboratory that have
shown that neonatal quinpirole treatment
produces significant decreases of NGF in the
hippocampus at P29 and P110 days of age (Brown
et al., 2004a,b). The significant decrease in
hippocampal BDNF produced by neonatal quinpirole
treatment is similar to past findings that have
shown that neonatal quinpirole treatment
produced a near significant decrease in BDNF in
the hippocampus at these same two ages. One
possible explanation for nicotine to alleviate
significant decreases in NGF and BDNF in
D2-primed rats may be through its actions on
hyperactive dopa- minergic pathways. Nicotine's
actions on the DA system have been
well-characterized, and are known to be mediated
through presynaptic nicotine receptors found on
dopaminergic terminals (Clarke and Reuben, 1996;
Nisell et al., 2004; Risso et al., 2004; Vizi
and Lendvai, 1999; Wonnacott, 1997; Zoli et al.,
2002).
Several studies have shown that nicotine
increases the release of DA through action on
these presynaptic nicotinic receptors.
Hypothetically, when nicotine increases the
release of DA in D2-primed rats, DA subsequently
binds to primed postsynaptic D2 recep- tors,
quite likely resulting in an overall increased
dopaminergic response. This increased
dopaminergic response would result in a
significant increase in synaptic activity, and
several studies have reported that both the
neurotrophic factors NGF and BDNF are
activity-dependent (Lessmann et al., 2003; Zafra
et al., 1990). Therefore, nicotine may be
increasing synaptic activity through its action
on the dopaminer- gic system, resulting in an
alleviation of significant decreases of NGF and
BDNF produced by neonatal quinpirole
treatment.
In support of this hypothesis, past studies
have shown that chronic administration of the
psychostimulants amphetamine or cocaine
correlates with increases of the neurotrophins
BDNF, basic fibroblast growth factor (bFGF), and
neurotro- phin-3 (NT-3) in the ventral tegmental
area, nucleus accumbens, hypothalamus, and
basolateral amygdala (Grimm et al., 2003;
Meredith et al., 2002; Yamada and Nabeshima,
2004). It has been hypothesized that this
increase in neurotrophic factor content is due
to drug-induced increases of dopaminergic
synaptic activ- ity in these brain areas
(Meredith, 1999). Although it is possible that
nicotine's effects on neurotrophic factor
content in D2-primed rats are through
hyperactive dopaminergic pathways, one of the
more accepted hypotheses of nicotine's action on
neurotrophic factors has been through its
agonist action in the acetylcholinergic system.
The acetylcho- linergic system and NGF have been
shown to be posi- tively correlated in a number
of studies (Fernandez et al., 1996; Hernandez
and Terry, 2004; Walz et al., 2000).
Therefore, nicotine's ability to alleviate
the sig- nificant decrease of neurotrophins in
rats neonatally treated with quinpirole may not
be through the dopa- minergic system but through
its agonistic action on acetylcholinergic
pathways. This acetylcholinergic mechanism
cannot be ruled out at this time; however,
studies have shown that the D2 receptor is
important in regulation of acetylcholine release
in the hippocam- pus although its precise role
is yet to be identified (Day and Fibiger, 1993,
1994; Imperato et al., 1993; Umegaki et al.,
2001). Ultimately, nicotine may be increasing
dopaminergic activity in D2-primed rats
resulting in increases of acetylcholine, and
subse- quently increase neurotrophin factor
concentrations in the hippocampus and frontal
cortex. Another intriguing finding in the
current study was that chronic nicotine
administration to control ani- mals (Group
S&endash;N) produced a significant decrease of
hippocampal NGF and an overall significant
decrease of BDNF in the frontal cortex as
compared with that of rats given saline in
adulthood.
This is contrary to findings of several
other studies that have reported that nicotine
induces increases of bFGF, fibroblast growth
factor-2, NGF and BDNF activity or mRNA
expression in several different brain areas
analyzed up to 72 h after nicotine
administration (French et al., 1999; Kenny et
al., 2000; Maggio et al., 1997, 1998). One
critical difference between the present study
and these past findings was that female rats
were utilized as subjects in the present study,
and all of the previous studies analyzing
nicotine's effects on neurotrophic factors
utilized male rats, male mice, or were performed
in vitro. A second critical difference is that
these past studies analyzed genetic expression
of neurotrophic factors and not the actual
protein, whereas in the present study the
protein was analyzed utilizing the ELISA
technique. We hypothesize that the
nicotine-induced decrease in neurotrophic factor
content in control females may be due to the
stress response during nicotine withdrawal,
which may interact with increased sensitivity to
nico- tine in females. Rasmussen (1998) has
shown that plasma serum corticosterone levels
are increased dur- ing the initial withdrawal
period for up to 3 days after systemic nicotine
administration (0.4 mg/kg free base once daily
for 28 days) had ceased, and increases of
corticosterone levels have been shown to be cor-
related with significant decreases in
neurotrophin expression in several brain
regions, including the hippocampus (Gubba et
al., 2004; Schaaf et al., 2000).
However, Rasmussen (1998) also utilized only
male rats, so it is not known whether this would
also occur in females. On the other hand,
nicotinic receptor stimulation has been shown to
produce a significantly higher dose-related
corticosterone response in female as compared
with that of male rats (Rhodes et al., 2001),
supporting the notion that increases in HPA axis
activity due to nicotine withdrawal in female
rats may be greater than males, and thus helping
to explain the significant decrease in
neurotrophins pro- duced by chronic nicotine
administration. Regardless, identifying the
precise mechanism is quite likely out- side of
the scope of one experiment, and the present
results demonstrate that females demonstrate a
sig- nificant difference in neurotrophic factor
protein response to nicotine as compared with
past studies utilizing male subjects (French et
al., 1999; Kenny et al., 2000).
Underlying mechanisms of this gender
difference will have to be tested in future
studies. In our previous study, we showed that a
0.3 mg/kg free base of nicotine given twice
daily to D2-primed rats in adulthood was
sufficient to completely al- leviate cognitive
impairment in these animals when tested on the
Morris water maze. The present results show that
this identical nicotine treatment regimen was
sufficient to alleviate significant decreases of
NGF and BDNF that may be important in explaining
nicotine's effects on plasticity and behavior.
Although this nicotine treatment regimen
resulted in signifi- cant decreases of NGF and
BDNF in control animals, we hypothesize that
this may be due to gender differ- ences in
response to the drug, which may be related to
increased stress response. Future studies will
aim at identifying mediating mechanisms of the
effects reported here.
