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27 janvier 2008
The effects of adulthood nicotine treatment
on D2-mediated behavior and neurotrophins
of rats neonatally treated with quinpirole
Brown RW, Perna MK, Schaefer TL, Williams MT
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
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.
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).
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.