Abstract
To identify the brain nicotinic acetylcholine receptor (nAChR) subtypes that may be involved in nicotine addiction, we investigated the actions of bupropion, a drug used in cigarette smoking cessation programs, and nicotine on three pharmacologically identified nAChRs in rat hippocampal slices, namely, type IA, type II, and type III nAChRs, likely representing α7, α4β2, and α3β4 subunits, respectively. Using whole-cell patch-clamp recordings from interneurons of acute hippocampal slices prepared from male rat pups, we studied the effect of nicotine on in vivo up-regulation and in vitro desensitization of nAChRs. Two subcutaneous injections of nicotine (0.586 mg/kg free base, in less than a day) to rats at postnatal days 14 to 15 significantly enhanced the magnitude of functional responses arising from type III and type II, but not type IA nAChRs. This treatment did not increase the functional affinity for acetylcholine at type II nAChRs. A single injection of nicotine also produced a significant increase in type III nAChR response. In addition, type III and type II, but not type IA nAChRs, are desensitized by in vitro exposure to nicotine at concentrations found in the venous blood of cigarette smokers. Bupropion at 1 μM produced 56, 15, and 0% inhibition of type III, type II, and type IA nAChR responses, respectively, in the slices. Our results suggest that in vivo-nicotine-induced nAChR up-regulation observed in neurons of intact brain tissue is a physiologically relevant phenomenon and that early up-regulation of type III and type II nAChRs could be an important biological signal in nicotine addiction.
The biological basis of nicotine addiction is attributed primarily to the actions of this tobacco alkaloid on various nicotinic acetylcholine receptor (nAChR) subtypes in the brain (Dani and Heinemann, 1996). Although the nAChR subunits β2 (Picciotto et al., 1998) and α4 (Tapper et al., 2004) have been implicated in the reinforcing properties of nicotine and β4 in the withdrawal effects of nicotine (Salas et al., 2004), the relative importance of each native brain nAChR subtype in nicotine addiction is still unknown. Among the various known actions of nicotine, nAChR up-regulation is a well documented observation, believed to play a central role in nicotine addiction (Buisson and Bertrand, 2002; Gentry and Lukas, 2002). Up-regulation of nAChRs has been observed in human brain tissue of smokers (Breese et al., 1997), animal brain after a prolonged exposure to nicotine (Flores et al., 1992; Marks et al., 1992; Zhang et al., 1994; Rowell and Li, 1997; Nguyen et al., 2004), and to a greater extent in cell lines expressing different combinations of nAChR subunits (Peng et al., 1994; Fenster et al., 1999; Buisson and Bertrand, 2001; Gentry and Lukas, 2002). However, it is unclear whether the up-regulation of nAChRs as determined by an increase in the number of binding sites is always functional and physiologically relevant because it represents mainly intracellular pools (Gentry and Lukas, 2002; Sallette et al., 2004). Considering that nAChRs present in different brain regions are the natural targets for the actions of nicotine and may also have different compositions than the receptors studied in artificial expression systems, a clear understanding of nicotine addiction requires an in depth knowledge of the functional status of nAChRs present in brain tissue after in vivo nicotine exposure. However, with the exception of a handful of studies in which functional nAChR up-regulation was directly evaluated in intact neurons using primary cultures (Almeida et al., 2000; Kawai and Berg, 2001; Nashmi et al., 2003), we are not aware of any studies that investigated functional nAChR up-regulation using identified neuron types present in intact brain tissue.
In view of such paucity of information and the fact that hippocampus contains multiple subtypes of nAChRs (Alkondon and Albuquerque, 2004) that are also present in other brain regions (Aramakis and Metherate, 1998; Zoli et al., 1998; Wooltorton et al., 2003) and are likely to be involved in nicotine addiction, we sought to evaluate the effects of exposure to in vivo and in vitro nicotine on three pharmacologically identified nAChR subtypes in the CA1 interneurons of rat hippocampal slices. The three subtypes in the hippocampal slices (Alkondon et al., 2003; Alkondon and Albuquerque, 2004) are referred here as type IA, type II, and type III nAChRs because they exhibit a pharmacological profile similar to that of the nAChRs in cultured hippocampal neurons (Albuquerque et al., 1997) and also in mouse brain slice neurons (Zoli et al., 1998). Evidence from several studies suggests that type IA nAChRs consist of α7 subunits, type II nAChRs consist of α4β2 subunits, and type III nAChRs contain of at least α3β4 subunits (Albuquerque et al., 1997; Zoli et al., 1998; Alkondon et al., 2003). Here, we investigated the changes in the magnitude of nAChR responses after either two injections or a single injection of nicotine because an early effect of this alkaloid could signify the development of lasting cues associated with nicotine addiction as it is the case for other drugs of abuse in general (Saal et al., 2003). Furthermore, we compared the desensitizing effects on various hippocampal nAChRs of acute in vitro nicotine exposure, because desensitization seems to be essential and often precedes receptor up-regulation in other systems (Peng et al., 1994; Fenster et al., 1999). Finally, we tested the effects of bupropion on native nAChR subtypes in hippocampal slices because this drug, currently used in smoking cessation programs, has shown some degree of selectivity toward α3 subunit-containing nAChRs (Fryer and Lukas, 1999; Slemmer et al., 2000). In this study, we demonstrate that type III and type II nAChRs are sensitive to functional up-regulation after in vivo nicotine, are desensitized by nicotine at concentrations similar to those found in cigarette smokers, and are blocked by bupropion at clinically relevant concentrations. Thus, our results support the notion that both type III and type II nAChRs play important roles in nicotine addiction.
Materials and Methods
Hippocampal Slices. Slices of 250-μm thickness were obtained from the hippocampus of 15- to 19-day-old Sprague-Dawley rats according to the procedure described previously (Alkondon et al., 2003). Animal care and handling were done strictly in accordance with the guidelines set forth by the Animal Care Committee of the University of Maryland (Baltimore, MD). Slices were stored at room temperature in artificial cerebrospinal fluid (ACSF), which was bubbled with 95% O2 and 5% CO2 and composed of 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 25 mM glucose. Stratum radiatum (SR) and stratum lacunosum moleculare (SLM) interneurons in the CA1 field of the slices were visualized by means of infrared-assisted videomicroscopy. Additionally, biocytin labeling was used to identify the neurons morphologically (Alkondon et al., 2003).
In Vivo Nicotine Injection. Male rat pups at postnatal days 14 or 15 were injected subcutaneously twice in less than 24 h with either saline or nicotine. The first injection was done at 4:00 to 5:00 PM, and the second at 8:00 to 9:00 AM the next morning. Brains were collected for electrophysiological experiments 1 to 2 h after the second saline or nicotine injections. In another set of experiments, a single injection of saline or nicotine was given on postnatal day 15, and 17 h later, experiments were conducted. Typically, two male rat pups from the same litter were used on the same day, one for saline and the other for nicotine. Nicotine hydrogen tartrate dissolved in sterile saline was injected at a dose of 3.6 μmol/kg (equivalent to 0.586 mg/kg free base) in a 10 ml/kg volume. A 10-min visual observation of the animal behavior after the injections revealed that there were occasional tremors and a slight increase in locomotor activity in the nicotine-treated group.
Electrophysiological Recordings. Excitatory postsynaptic currents (EPSCs) and agonist-evoked whole-cell currents were recorded from the soma of CA1 interneurons according to the standard patch-clamp technique using an LM-EPC7 amplifier (List Electronics, Darmstadt, Germany). Agonists were applied to the slices via a U-tube, and antagonists were applied both via the U-tube and bath perfusion (Alkondon et al., 2003). Signals were filtered at 2 kHz and either recorded on a video tape recorder for later analysis or directly sampled by a microcomputer using the pCLAMP 9 program (Axon Instruments Inc., Union City, CA). Neurons were superfused with ACSF at 2 ml/min. Atropine (1 μM) was added to the ACSF to block the muscarinic receptors. Bicuculline (10 μM) was added to ACSF to block GABAA receptor activity. Methyllycaconitine (MLA, 10 nM) was included in the ACSF while studying nontype IA nAChR responses. Patch pipettes were pulled from borosilicate glass capillary (1.2-mm outer diameter), and when filled with internal solution had resistance between 2 and 5 MΩ. The series resistance ranged from 8 to 20 MΩ.At ∼68 mV, the leak current was generally between 50 and 150 pA, and when it exceeded 200 pA, the data were not included in the analysis. The internal pipette solution contained 0.5% biocytin in addition to 10 mM ethylene-glycol bis(β-amino-ethyl ether)-N-N′-tetraacetic acid, 10 mM HEPES, 130 mM Cs-methane sulfonate, 10 mM CsCl, 2 mM MgCl2, and 5 mM lidocaine N-ethyl bromide (QX-314) (pH adjusted to 7.3 with CsOH; 340 mOsM). Membrane potentials were corrected for liquid junction potentials. All experiments were carried out at room temperature (≈20–22°C).
Data Analysis. The frequency, peak amplitude, 10 to 90% rise time, and decay-time constant of AMPA EPSCs were analyzed using WinEDR version 2.3 (Strathclyde Electrophysiology Software, Glasgow, Scotland). The peak amplitude of nicotinic currents and the net charge of NMDA receptor-mediated EPSCs and nicotinic currents were analyzed using the pCLAMP9 software (Axon Instruments Inc.). Typically, the net charge of agonist-evoked responses was calculated for the duration of the agonist pulse starting from the valve opening. Results are presented as mean ± S.E.M. and compared for their statistical significance by paired Student's t test, Wilcoxon signed rank test, or Mann-Whitney U test. Agonist concentration-response curves were fitted to a Hill equation: I = (Imax*An)/(An + EC50n), where I is the measured current amplitude or net charge, Imax is the maximum current amplitude or net charge, n is the Hill coefficient, and EC50 is the agonist concentration producing half-maximal response.
Drugs Used. ACh chloride, (–)-bicuculline methiodide, bupropion hydrochloride, choline chloride, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), (–)-nicotine hydrogen tartrate, dl-2-amino-5-phosphonovaleric acid (APV), QX-314, NMDA, glycine, and atropine sulfate were obtained from Sigma-Aldrich (St. Louis, MO). (±)-Mecamylamine HCl was a gift from Merck (Rahway, NJ). MLA was a gift from Professor M. H. Benn (Department of Chemistry, University of Calgary, Alberta, Canada). Stock solutions of all drugs were made in distilled water.
Results
Two Subcutaneous Nicotine Injections Do Not Cause Functional Up-Regulation of Type IA nAChRs. Application of choline at 10 mM, a concentration that is nearly saturating, induced type IA inward currents in CA1 SR interneurons at –68 mV (Fig. 1A). These currents decayed to the baseline before the end of agonist pulses. Such nicotinic currents, being sensitive to blockade by low concentrations of MLA and α-bungarotoxin (Albuquerque et al., 1997), represent the sole activation of α7 nAChRs in the SR interneurons. We compared the peak amplitude of functional type IA nAChRs in these interneurons after two injections of rats with either saline or nicotine (3.6 μmol/kg s.c. in less than a day). The peak amplitude of choline-evoked type IA currents, a measure of the activity of functional α7 nAChRs present in the somatodendritic regions of SR interneurons, ranged from 6 to 155 pA (n = 21 neurons) in saline-treated rats and from 6 to 432 pA (n = 24 neurons) in nicotine-treated rats (Fig. 1B). A statistical analysis of the data indicated that the mean values of the peak amplitude of choline-elicited currents are not significantly different (p > 0.05 by Mann-Whitney test) between saline-injected and nicotine-injected rats (Fig. 1C). We also compared the differences between the two groups based on net charge of choline-evoked currents as the net charge of the currents is considered a better measure of receptor activity than the peak amplitude of the currents under conditions where solution exchange around the neuron is slow (Papke and Papke, 2002) as it would occur in the brain slices. Similarly to those observed in the peak amplitude analysis, the net charge of choline-evoked currents also showed an identical pattern between the two groups of rats (Fig. 1, B and C). Furthermore, no significant differences between the two groups of animals were noted on the mean decay time constant of choline-induced currents (Fig. 1C). We conclude from the above results that two nicotine injections, at the dose used, are unable to functionally up-regulate to any significant extent the activity of type IA nAChRs and modify the properties of these receptors.
Two Subcutaneous Nicotine Injections Functionally Up-Regulate Type II nAChRs. Application of ACh (1 mM) in the presence of MLA induced slowly decaying type II inward currents at –68 mV in all the SLM interneurons (n = 42) and 25% of SR interneurons (n = 40) tested. Such slowly decaying nicotinic currents, being sensitive to blockade by dihydro-β-erythroidine, poorly activated by cytisine, represent the activation of α4β2 nAChRs in the CA1 interneurons (Alkondon et al., 2000, 2003). In saline- and nicotine-treated rats, ACh (1 μM to 3 mM) evoked concentration-dependent increase in the current amplitude with higher values observed in the nicotine group (Fig. 2A). In saline-treated rats, the plot of the peak amplitude of the inward currents against ACh concentration yielded a single sigmoidal curve with an EC50 of 112 μM (Fig. 2B; Table 1). The plot of net charge of these currents against agonist concentration gave an EC50 value of 117 μM (Fig. 2C; Table 1). In nicotine-treated rats, both peak amplitude and net charge of nicotinic currents were higher than in saline-treated rats at ACh concentration >1 μM, the differences were statistically significant (p < 0.05 by Mann-Whitney test) at several ACh concentrations between 10 μM and 3 mM (Fig. 2, B and C; Table 1). On average, nicotine treatment caused about 2-fold increase in the net charge of ACh (1 mM)-induced currents (Fig. 2C). In the nicotine group, the plot of the peak amplitude against concentration yielded an EC50 of 123 μM and that of net charge against concentration gave an EC50 of 104 μM. The EC50 values between saline and nicotine groups were not significantly different (Table 1), suggesting that the affinity for ACh was unchanged by nicotine treatment. We conclude from the above-mentioned results that two nicotine injections cause significant functional up-regulation of type II nAChRs.
Two Subcutaneous Nicotine Injections Functionally Up-Regulate Type III nAChRs. Our previous studies and that of Zoli et al. (1998) have suggested that type III nAChRs are represented by α3β4-like subunits, and activation of type III nAChRs in the hippocampal slices induces vesicular glutamate release that was detected in CA1 SR interneurons by the appearance of EPSCs mediated via AMPA and NMDA receptors (Alkondon et al., 2003). Here, we determined the effect of two subcutaneous injections of nicotine on type III nAChR responses by recording ACh-elicited AMPA EPSCs or NMDA EPSCs in SR interneurons.
At –68 mV, in the presence of GABAA receptor antagonist bicuculline, occasional spontaneous inward AMPA EPSCs were observed in the whole-cell recordings from SR interneurons at postnatal day 15 in saline-injected rats. Two in vivo nicotine injections caused a slight increase (but not significantly different from control; Table 2) in the frequency of spontaneous AMPA EPSCs. In the saline-treated rats, U-tube application of ACh induced a marginal increase at 10 μM and a significant increase at 100 μM in the frequency of AMPA EPSCs (Fig. 3; Table 2). In the nicotine-injected rats, ACh at both 10 and 100 μM induced a larger increase in the frequency of AMPA EPSCs (Fig. 3; Table 2). The effect of both concentrations of ACh was significantly higher in the nicotine group compared with saline group (n = 8–9 neurons in each group; Table 2). Nicotine injections did not affect the rise time or the decay time constant of ACh-evoked AMPA EPSCs (Table 2). However, the peak amplitude of AMPA EPSCs evoked by 100 μM ACh was significantly higher in the nicotine group compared with saline control (Table 2). Furthermore, these high-amplitude events were also associated with a slow NMDA component, particularly in the nicotine-treated group (Fig. 3, inset).
To evaluate ACh-evoked NMDA EPSCs in greater detail, recordings were carried out at positive membrane potentials (+40 mV) to remove Mg2+ block of NMDA channels. In saline-injected rats, 10 μM ACh evoked NMDA EPSCs in five of seven neurons. Increasing the concentrations of ACh to 30, 100, and 1000 μM produced proportionately larger magnitude NMDA EPSCs (Fig. 4, A and B), suggesting a concentration-dependent activation of type III nAChRs. On the other hand, in nicotine-injected rats, 10 μM ACh induced NMDA EPSCs in all nine interneurons tested. Higher concentrations of ACh induced much larger responses in nicotine-treated rats than in saline-treated rats (Fig. 4, A and B). At each of the four ACh concentrations, the values of net charge of NMDA EPSCs obtained in the nicotine group were significantly higher than those in the saline group (p < 0.02; Mann-Whitney test). On average, nicotine treatment caused about 3-fold increase in the net charge of NMDA EPSCs induced by 1 mM ACh (Fig. 4B).
To rule out the possibility that nicotine-induced enhancement of net charge of ACh-evoked NMDA EPSCs resulted from an increase in the postsynaptic NMDA receptor responsiveness, we compared currents (Fig. 5A) evoked by U-tube application of NMDA to SR interneurons in both groups of animals. As illustrated in Fig. 5C, the net charge of NMDA-evoked currents in the CA1 SR interneurons did not reveal differences between saline- and nicotine-treated rats. Furthermore, if nicotine enhanced the net charge of ACh-evoked NMDA EPSCs by increasing the number of glutamate synapses on the dendrites, then one should expect that activation of glutamate synapses via a non-nicotinic mechanism would also produce enhanced responses after nicotine treatment. To verify this possibility, we activated glutamate synapses by applying 500 mM sucrose, a hyperosmotic solution known to induce transmitter release from the terminals in a Ca2+-independent manner (Rosenmund and Stevens, 1996). U-tube applied sucrose induced an outward current at +40 mV that resulted from the summation of several NMDA EPSCs because bath-applied APV (50 μM) attenuated the response dramatically (Fig. 5B). However, the magnitude of sucrose-evoked currents remained the same in both groups of animals (Fig. 5, B and C), indicating that nicotine's effect on NMDA EPSCs did not arise from a change in the number of glutamate synapses. We conclude from the above-mentioned results that two nicotine injections functionally up-regulate type III nAChRs to a significant extent.
Single Subcutaneous Nicotine Injection Functionally Up-Regulates Type III nAChRs. Next, we tested the effect of single injection of nicotine on the functional up-regulation. In this protocol, a single injection of saline or nicotine (3.6 μmol/kg s.c.) was given to rats on postnatal day 15, and experiments were conducted 17 h later on day 16. DMPP, another nicotinic agonist, was used to activate the NMDA EPSCs in the presence of 10 nM MLA. As illustrated in Fig. 6A, DMPP induced NMDA EPSCs in SR interneurons from both groups of rats; however, the magnitude of the response was larger in the nicotine-treated rats. Analysis of the net charge of DMPP-evoked NMDA EPSCs revealed a significant increase in the values in the nicotine group; about 2.7-fold that of saline-treated rats (Fig. 6B). These results indicated that single injection of nicotine is as effective as two injections with respect to up-regulation of type III nAChRs. Furthermore, because the agonist DMPP is not hydrolyzed by acetylcholinesterase, these experiments ruled out any changes in acetylcholinesterase activity as an explanation for increased ACh response after in vivo nicotine injections in the above-mentioned experiments.
In another set of experiments, we tested the pharmacological sensitivity of type III nAChR in control and nicotine-injected rats to find out whether in vivo nicotine exposure altered the properties of these receptors. We have shown previously that 1 μM mecamylamine inhibits about 80% of type III nAChR response in control rats (Alkondon et al., 2003). Here, in nicotine-injected rats (one injection, 17 h), 1 μM mecamylamine induced a mean inhibition of about 83% (n = 6 neurons) of the type III nAChR response, a value not significantly different between control and nicotine-injected rats (Fig. 7). Our previous study also revealed that MLA at 10 nM, a concentration known to inhibit α7 nAChR-mediated currents, failed to affect the type III nAChR response (Alkondon et al., 2003). Here, we tested the effect of MLA at 50 nM, a concentration found to inhibit α6-containing nAChRs in the rat striatum (Mogg et al., 2002). We observed that 50 nM MLA produced about 30% (n = 6) and 26% (n = 6) inhibition of type III nAChR in control and nicotine-injected rats, respectively. There were no significant differences between control and nicotine-injected rats in the magnitude of inhibition by MLA (Fig. 7). These results suggest that there is no major alteration in the pharmacological sensitivity of type III nAChR after in vivo nicotine injections.
In Vitro Nicotine Exposure Differentially Desensitizes Three nAChRs in the Hippocampal Slices. Receptor desensitization has been linked to receptor up-regulation in several in vitro model systems (Peng et al., 1994; Fenster et al., 1999; Buisson and Bertrand, 2001). This raises the possibility that variable degree of desensitization could account for the observed differences in the up-regulation of hippocampal nAChRs by nicotine injections. To test this hypothesis, we studied the concentration dependence for nicotine to induce in vitro desensitization of three nAChR subtypes in the hippocampal slices. Nicotine-induced desensitization of type III nAChRs was assessed from inhibition of ACh (100 μM)-evoked NMDA EPSCs recorded from CA1 SR interneurons. A 10-min exposure of the slices to nicotine (1–100 nM) caused a concentration-dependent inhibition of ACh (100 μM)-evoked NMDA EPSCs (Fig. 8B). At 100 nM nicotine, ACh-evoked NMDA EPSCs were abolished (Fig. 8A). The plot of the net charge of ACh-evoked NMDA EPSCs versus concentration of nicotine yielded an IC50 of 12.8 ± 0.1 nM and a Hill coefficient of 1.97 ± 0.02 for inhibiting type III nAChR responses (Fig. 8B). Nicotine-induced desensitization of type II nAChRs was assessed from inhibition of ACh (100 μM)-evoked slow inward currents recorded from SLM interneurons in the continuous presence of MLA (10 nM). A 10-min exposure of the slices to nicotine (10–500 nM) inhibited in a concentration-dependent manner ACh-evoked slow inward currents (Fig. 8B). At 100 nM nicotine, there was only partial inhibition of ACh-evoked currents (Fig. 8A). Although nicotine at 500 nM produced substantial inhibition, nearly 15% of the currents still remained (Fig. 8B). The plot of the net charge of ACh-evoked slow inward currents versus concentration of nicotine yielded an IC50 of 124 ± 17 nM and a Hill coefficient of 1.33 ± 0.19 for inhibiting type II nAChR-mediated currents (Fig. 8B). Nicotine-induced desensitization of type IA nAChRs was assessed from inhibition of choline (10 mM)-evoked inward currents recorded from CA1 SR interneurons. Data from a previous study on type IA nAChRs (Alkondon et al., 2000) was used here for comparison with other subtypes. A 10-min exposure of the slices to nicotine (100–2500 nM) inhibited in a concentration-dependent manner choline-evoked inward currents (Fig. 8B). At 100 nM nicotine, there was practically no inhibition observed (Fig. 8A). The plot of the peak amplitude of choline-evoked inward currents versus concentration of nicotine yielded an IC50 of 653 ± 26 nM and a Hill coefficient of 2.78 ± 0.26 for inhibiting type IA nAChRs (Fig. 8B).
In Vitro Exposure to Bupropion Potently Inhibits Type III nAChR-Mediated NMDA EPSCs. From the above-mentioned results, it is evident that type III nAChRs are a sensitive target for the in vivo and in vitro actions of nicotine. Therefore, one can predict that blockade of this native subtype could be beneficial to some extent in nicotine dependence and addiction. To verify this, we tested the effects of a well known antismoking agent, bupropion, on ACh-elicited NMDA EPSCs. A 10-min bath application of bupropion (1 μM) attenuated ACh-induced NMDA EPSCs (Fig. 9A). The inhibitory effect was completely reversible within 20-min wash with drug-free ACSF. On average, there was about 56% inhibition with 1 μM(n = 6) bupropion. The effect at 1 μM was statistically significant (p < 0.01 by Student's paired t test). At 1 μM, bupropion produced marginal but significant (p < 0.05 by paired “t” test) inhibition of type II nAChR-mediated currents (Fig. 9, B and E), but it did not alter currents originating from either type IA nAChRs (Fig. 9, C and E) or NMDA receptors (Fig. 9, D and E). The degree of blockade induced by 1 μM bupropion at type III, type II, and type IA nAChRs (Fig. 9) is comparable with that observed for α3β4, α4β2, and α7 nAChRs studied in artificial expression systems (Fryer and Lukas, 1999; Slemmer et al., 2000).
Discussion
The present results disclosed major differences in the susceptibility of three distinct brain nAChR subtypes to the in vivo and in vitro actions of nicotine and to the blocking effects of bupropion. By evaluating the activity of nAChRs, this study, for the first time, demonstrated the propensity of in vivo nicotine to functionally up-regulate nAChRs in identified neuron types present in intact brain tissue.
Functional Up-Regulation by in Vivo Nicotine. The main finding of this study is that nicotine injections in less than a day is able to functionally up-regulate type III and type II, but not type IA nAChRs in the hippocampal slices. Both type IA and type II nAChRs were evaluated directly by studying the receptor-mediated currents, whereas type III nAChR activity was assessed indirectly via ACh-evoked glutamate EPSCs. Although the indirect measurement of type III nAChRs is a limitation in this study, multiple lines of evidence prove that up-regulation by nicotine takes place at the type III nAChRs only. First, the net charge of ACh-induced NMDA EPSCs increased as a function of ACh concentration as would be predicted for responses due to agonist-receptor interaction. Second, enhancement by in vivo nicotine could be seen at both AMPA EPSCs (Fig. 3) and NMDA EPSCs (Fig. 4), reflecting enhanced release of glutamate from the terminals and not enhanced sensitivity of postsynaptic NMDA receptors (Fig. 5). These results support that nicotine-induced enhancement of glutamate EPSCs is a direct consequence of up-regulation in the number and/or the activity of type III nAChRs present at the glutamate neurons or terminals.
Our results on type II nAChR functional up-regulation by nicotine injection contrast with the results of another study (Nguyen et al., 2004) in which the authors find no significant increase in α4β2 nAChR function after a 16-h nicotine exposure via osmotic pumps. It is possible that either methodological differences (such as injection versus osmotic pump or young versus adult rats) or heterogeneity of native nAChR subtypes could account for the observed differences in the two studies.
In Vitro Desensitization Correlates with in Vivo nAChR Functional Up-Regulation. A striking difference in the concentrations of nicotine for in vitro desensitization of hippocampal nAChRs (Fig. 8B) validates our previous assumption that three pharmacologically classified nAChR subtypes are indeed separate structural entities. Comparison of nicotine's IC50 values (Fig. 8B) indicated that type III nAChRs are the most sensitive, type II nAChRs being moderately sensitive, and type IA nAChRs being the least sensitive for the desensitizing action of nicotine. Interestingly, comparison of the magnitude of nAChR functional up-regulation by nicotine indicated a 3-fold increase for type III nAChRs, 2-fold increase for type II nAChRs, and no significant increase for type IA nAChRs (Figs. 1, 2, and 4), suggesting an inverse relationship between desensitization IC50 and percentage of up-regulation. If this trend holds true for a wider in vivo nicotine doses, it can be predicted that a dose lower than 0.586 mg/kg free base of nicotine could functionally up-regulate only type III nAChRs. On the contrary, doses higher than 0.586 mg/kg and given for many days could functionally up-regulate even type IA nAChRs. In fact, our preliminary studies revealed that eight and 15 injections (4- and 8-day treatment), but not two injections of nicotine (0.586 mg/kg), induce significant functional up-regulation of type IA nAChRs when assessed from choline-induced GABAergic postsynaptic currents in SR interneurons, a measure of type IA nAChR activity in population of interneurons (Alkondon and Albuquerque, 2004).
Subunit Makeup of the Type III nAChRs That Modulates Glutamate Release onto the Interneuron. Previous pharmacological analyses based on cytisine's efficacy for activation and mecamylamine's potency for blockade suggested that the nAChRs that modulate glutamate release onto the SR interneurons may be composed of a minimum of α3 and β4 subunits (Alkondon et al., 2003). The partial block of type III nAChR response by 50 nM MLA (Fig. 7) rules out the involvement of α6 subunits because this concentration of MLA is known to block α6-containing nAChRs in the rat striatum (Mogg et al., 2002). The high-potency blockade by bupropion of ACh-induced NMDA EPSCs (Fig. 9) further validates the involvement of α3 subunit in this nAChR because previous studies have shown that α3-containing nAChRs are the most sensitive subtype for the blocking action of bupropion (Fryer and Lukas, 1999; Slemmer et al., 2000). A recent study demonstrating that bupropion inhibits potently nicotine-evoked norepinephrine release from hippocampal slices via its action on α3β4-like nAChRs also supports this conclusion (Miller et al., 2002). Other reports that some somatic signs of nicotine withdrawal in mice depend on β4-containing nAChRs (Salas et al., 2004) and that bupropion reverses the expression of somatic aspects of nicotine withdrawal syndrome in rats (Cryan et al., 2003) are in line with our results. However, in most studies in artificial cell systems, α3β4 nAChRs are not potently desensitized or up-regulated by nicotine (Fenster et al., 1997; Wang et al., 1998; Meyer et al., 2001; Sallette et al., 2004). Also, in the adult rats, chronic nicotine treatment for 2 weeks via osmotic pump fails to increase α3β4 nAChR function in the medial habenula and interpeduncular nucleus as assessed by cytisine-induced rubidium efflux (Nguyen et al., 2004). It is possible that type III nAChRs investigated in the hippocampal region of young postnatal rats (this study) are structurally and functionally distinct from the α3β4-like nAChR studied in the habenula region of adult rats (Nguyen et al., 2004). More detailed analysis may be required to assess the exact subunit composition of the type III nAChRs in neurons to understand its unique sensitivity to desensitization and up-regulation.
Level of Up-Regulation in Cigarette Smoking. The concentration of nicotine in the blood of a smoker fluctuates depending on several factors. The mean arterial blood levels, which reflects the brain concentrations, range from 37 to 58 ng/ml (equivalent to 228–357 nM) when measured within 1 min after end of smoking a single cigarette, and individual values reaching up to 92.5 ng/ml (i.e., 570 nM) (Henningfield et al., 1993). The concentration of nicotine in the venous blood varies between 5 and 30 ng/ml after smoking a single cigarette, but rises to 10 and 50 ng/ml after smoking several cigarettes (Benowitz and Jacob, 1998). A plasma half-life of 2 h in humans (Benowitz and Jacob, 1998) allows the venous concentration to build gradually and to remain in the blood-stream for several hours, providing a condition in which the nAChRs in brain neurons can be desensitized and up-regulated. The results portrayed in Fig. 8 indicate that type III nAChRs will be desensitized completely, type II nAChRs will be desensitized significantly, and type IA nAChRs being spared from desensitization at the venous range of nicotine levels in cigarette smokers. Therefore, it is possible that in cigarette smokers, both type III and type II nAChRs are up-regulated at a time when the type IA nAChRs remain unaffected by the available nicotine concentrations, and such up-regulated nAChRs may contribute to altered neurotransmission in the brain of smokers.
Significance of Bupropion Blockade and Early Nicotine-Induced Up-Regulation of Type III nAChRs. The present results indicated that type III nAChRs are the first among the three identified subtypes in the hippocampus to be readily desensitized by smoker's level of nicotine, significantly up-regulated by in vivo nicotine in less than a day, and potently blocked by clinically relevant concentrations of bupropion (Fryer and Lukas, 1999; Slemmer et al., 2000). It is unlikely that a high-potency inhibition of type III response by bupropion is due to measurement of an indirect rather than a direct response because the magnitude of the indirect response was directly proportional to the agonist concentrations (Fig. 4). The type III nAChRs when activated by exogenously applied agonists triggers both AMPA receptor- and NMDA receptor-mediated EPSCs (Figs. 3 and 4) and thereby forms one of the means by which endogenous cholinergic impulse could regulate the excitability of interneurons (Alkondon et al., 2003). ACh-induced excitation of GABAergic interneurons is an important signal that improves attention and encoding by suppression of background activity in the cortical circuits (Hasselmo and McGaughy, 2004). It has been established that the activity of GABAergic interneurons is critical in setting up various hippocampal rhythms, indicative of the process of learning and memory (Cobb et al., 1999). Because a single injection of nicotine is able to induce rapid synaptic changes that underlies cue-related learning (Saal et al., 2003) and is also able to functionally up-regulate the nAChRs (Fig. 6), we propose that early up-regulation of type III nAChRs by nicotine may be a key mechanism involved in early cue-related learning associated with nicotine addiction. It is also likely that blockade of up-regulated type III and type II nAChRs remains one of the key mechanisms by which bupropion produces beneficial effects in antismoking therapies. This is also consistent with the reports that bupropion and other α3β4 nAChR antagonists are able to suppress nicotine self-administration in rats (Glick et al., 2002; Bruijnzeel and Markou, 2003; Rauhut et al., 2003).
Acknowledgments
The technical assistance of Barbara Marrow and Mabel Zelle is gratefully acknowledged. We are thankful to Bhagavathy Alkondon for excellent technical assistance in the preparation of hippocampal slices.
Footnotes
-
This work was supported by U.S. Public Health Service Grant NS41671 (to E.X.A.) and University of Maryland School of Medicine, Other Tobacco-Related Diseases (OTRD) research grant (to E.X.A.).
-
doi:10.1124/jpet.104.081232.
-
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ACSF, artificial cerebrospinal fluid; SR, stratum radiatum; SLM, stratum lacunosum moleculare; EPSC, excitatory postsynaptic current; MLA, methyllycaconitine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-d-aspartate; ACh, acetylcholine; DMPP, 1,1-dimethyl-4-phenylpiperazinium; APV, dl-2-amino-5-phosphonovaleric acid.
- Received November 22, 2004.
- Accepted January 11, 2005.
- The American Society for Pharmacology and Experimental Therapeutics