Nicotineinduced Desensitization Of Da Release

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As discussed above, the most tenable explanation to account for the ability of chronic nicotine treatment to produce an upregulation of nAChRs in the brain is that nicotine acts as a functional antagonist by producing long-term nAChR desensitization (Marks et al., 1983; Schwartz and Kellar, 1985; Wonnacott, 1990; Ochoa and

FIGURE 3.2 Nicotine-induced desensitization of [3H]DA release from rat striatal synaptosomes. Striatal synaptosomes, prepared as described in Figure 3.1 were exposed to 0, 10 or 100-nM nicotine for 20 min followed by a 2 min challenge with 5 jiM nicotine. Samples of the superfusate were collected every 2 min; the resulting profile of [3H]DA release during this time is presented.

FIGURE 3.2 Nicotine-induced desensitization of [3H]DA release from rat striatal synaptosomes. Striatal synaptosomes, prepared as described in Figure 3.1 were exposed to 0, 10 or 100-nM nicotine for 20 min followed by a 2 min challenge with 5 jiM nicotine. Samples of the superfusate were collected every 2 min; the resulting profile of [3H]DA release during this time is presented.

McNamee, 1990) or inactivation (Aoshima, 1984; Simasko et al., 1986; Egan and North, 1986; Lukas, 1991; Rowell and Duggan, 1998). The ability of nicotine to produce desensitization of the nAChRs responsible for nicotine-evoked release of DA can readily be demonstrated to occur at very low concentrations of the drug. The use of in vitro superfusion of synaptosomes offers the advantage that drug access to and removal from the tissue can occur rapidly so that drug concentration, time-course, temperature, and other variables can be carefully controlled while still assessing the effects of treatment on endogenous receptors. Studies on striatal synaptosomes have shown that nicotine rapidly (within minutes of exposure) produces almost complete nAChR desensitization at very low concentration, with an ED50 of about 10 nM (Grady et al., 1994; Rowell and Hillebrand, 1994; Marks et al., 1994; Lippiello et al., 1995). This can be seen in Figure 3.2, which shows the effect of pretreatment of rat striatal synaptosomes with nanomolar concentrations of nicotine. It can be seen that a challenge with 5 ^M nicotine (arrow) produces a robust release of DA in nontreated tissue (panel A), but has virtually no effect after a 20 min exposure to 100 nM nicotine (panel C), although this concentration of nicotine produces some initial activation.

Since the studies cited indicate that nAChRs desensitize at nanomolar concentrations of nicotine, and desensitization is responsible for nAChR upregulation, it is important to consider the brain levels of nicotine achieved during administration of the drug. In human cigarette smokers, as might be expected, there is considerable individual variability in the nicotine blood levels achieved. Blood levels can range from 30 to 500 nM (5 to 90 ng/ml), depending upon the "smoking profile" of the individual (Russell, 1990); however, for most smokers, the blood levels of nicotine vary throughout the day from about 125 to 275 nM (Haines et al., 1974; Russell and Feyerabend, 1978; Bridges et al., 1990; Russell, 1990; Benowitz et al., 1997). The plasma half-life of nicotine in humans is 1 to 2 hours (Rosenberg et al., 1980; Kyerematen et al., 1982, 1990; Shiffman et al., 1992). Fortuitously, the plasma

FIGURE 3.3 Effect of chronic treatment on brain nicotine and nAChR levels. Male Sprague Dawley rats were administered nicotine, 0.6-4.8 mg/kg/day at a rate of 20 ^1/hr, subcutane-ously for 10 days via indwelling catheters connected through a Harvard swivel to an infusion pump. Plasma and whole brain (minus cerebellum and the areas dissected for nAChR analysis) nicotine levels were determined by GC with NP detection and nAChR density was determined by specific saturated binding with [3H]cytisine.

FIGURE 3.3 Effect of chronic treatment on brain nicotine and nAChR levels. Male Sprague Dawley rats were administered nicotine, 0.6-4.8 mg/kg/day at a rate of 20 ^1/hr, subcutane-ously for 10 days via indwelling catheters connected through a Harvard swivel to an infusion pump. Plasma and whole brain (minus cerebellum and the areas dissected for nAChR analysis) nicotine levels were determined by GC with NP detection and nAChR density was determined by specific saturated binding with [3H]cytisine.

half-life of nicotine in rats is almost the same as in humans: about one hour (Adir et al., 1976; Miller et al., 1977; Kyerematen et al., 1988; Benowitz et al., 1990). Therefore, nicotine blood levels comparable to those found in human smokers can be achieved by frequent administration or, more commonly, the constant infusion of 1.5 to 4 mg/kg/day nicotine via osmotic minipumps.

The relationship between nicotine brain levels and nAChR upregulation in animals is readily assessed by the chronic administration of nicotine and subsequent measurement of brain nAChR density and brain nicotine levels. Animals can be administered nicotine at any dose or frequency desired by means of an indwelling catheter connected to a nicotine-filled syringe controlled by a programmable pump. With this procedure nicotine has been administered at intervals from hourly to twice per day as well as constantly infused. Figure 3.3 shows brain and blood levels of nicotine achieved by the administration of 0.6 to 4.8 mg/kg/day via constant infusion along with the resulting changes in nAChR density in three brain areas.

Note that when rats are chronically treated with various doses of nicotine, a dose-dependent increase in nAChR takes place in the three brain areas examined. An assay of the blood and brain nicotine levels indicates that concentrations of nicotine much higher than the ED50 for nicotine-induced desensitization of DA release are achieved. At the highest doses of nicotine administered, brain levels of nicotine which will significantly stimulate DA release from NAc, striatal, and cortical tissue (> 500 nM) are achieved.

When one compares the concentrations of nicotine sufficient to achieve full desensitization of central nAChR in vitro (50 to 100 nM) with the blood levels of nicotine (125 to 275 nM) found in studies on experimental animals as well as human smokers that give rise to nAChR upregulation, one might assume that central nAChRs should be fully desensitized at all times in vivo. This is even more likely considering findings that the levels of nicotine in the brain following nicotine administration are 1.5 to 3 times higher than in the blood (Benowitz, 1990; Plowchalk et al., 1992; Henningfield et al., 1993; Sastry et al., 1995; Rowell and Li, 1997). This begs the question of why animals (and human smokers) self-administer nicotine if their receptors are in a continuous state of desensitization.

Behavioral or microdialysis experiments in living animals should be able to shed light on the dopaminergic responses to a nicotine exposure in chronically treated animals. Unfortunately, most studies of chronic nicotine treatment that study such things as nicotine self-administration, locomotion, place preference, or DA levels via in vivo microdialysis have administered nicotine to rats at infrequent intervals (once or twice per day) so there would be no continuous exposure to the drug between sessions. With a half-life of only about 1 hour, the concentration of nicotine in the brain between experiments could fall to very low levels so any nAChR desensitization might have recovered before the next session. For example, it can be calculated that the nicotine blood level from a 1 mg/kg injection would fall below that required to maintain significant nAChR desensitization (« 5 nM) after about 8 hours. Thus, it is not surprising that daily injections of nicotine could lead to an increase in nicotine-evoked DA release from NAc (Benwell and Balfour, 1992; Marshall et al., 1997; Balfour et al., 1998).

This typical nicotine delivery regimen in animal studies is clearly different from that with most cigarette smokers where frequent smoking, with consequent high nicotine levels, should result in continuous nAChR desensitization. Only before the first cigarette of the day might nicotine levels fall below that producing desensiti-zation. Even then, nicotine blood levels do not typically fall below 30 nM (« 5 ng/ml) due to the somewhat longer half-life of nicotine in humans, as well as more recent nicotine exposure just before retiring (Benowitz et al., 1990). Therefore, unlike most studies in animals, the brain nAChRs in humans should still be desensitized before each exposure to nicotine.

A few studies have investigated nicotine's behavioral and neurochemical effects in vivo during delivery regimens that would presumably result in nicotine levels sufficient to maintain receptor desensitization. Hakan and Ksir (1991) studied locomotor activity of rats injected with nicotine every 20 min. They observed a progressive decline in activity with subsequent injections of nicotine up to a maximal effect at 60 min. Since locomotor activity, like positive reinforcement, is mediated in large part through DA release from the NAc (Clarke et al., 1988), this would be an indirect indication of nicotine-stimulated DA release in the brain. Sharp and coworkers have measured nicotine-stimulated norepinephrine release via microdialysis in rats injected with nicotine at various time intervals (Sharp and Matta, 1993; Fu et al., 1998). They found that a nicotine injection rapidly desensitized the response to subsequent injections. Benwell et al. (1995) found that constant infusion of 4 mg/kg/day nicotine for 14 days abolished the sensitizing effect of systemic nicotine injections to increase DA release in the VTA, indicating that the nAChRs are desensitized in vivo by this treatment.

The animal studies cited indicate that frequent or constant administration of nicotine does indeed produce an in vivo desensitization of nicotine-evoked responses in the brain. Why, then, would animals continue to self-administer nicotine for long periods of time and cigarette smokers smoke throughout the day if nAChRs were in a state of desensitization? One suggestion is that nicotine is self-administered to keep a population of receptors in a continual state of desensitization (Balfour, 1994; Pidopli-chko et al., 1997), implying that a return of these receptors to the active state leads to dysphoria and/or withdrawal. While this is a possibility, it seems unlikely since functional MRI studies of nicotine administration in smokers indicate that it, like many other drugs of dependence with positive reinforcing properties, increases rather than decreases neuronal activity in the NAc (Stein et al., 1998). Unless nicotine is modulating the activity of an inhibitory neurotransmitter or pathway, it appears that nAChR activation, rather than desensitization, results from nicotine administration.

A more likely possibility is that, even during desensitization, nAChRs may be able to produce an increased release of DA above the basal (non-nicotine) condition. Collins and coworkers have shown that continued exposure of brain tissue to nicotine results in a prolonged elevation of DA release (about 20% of the initial stimulation response) as long as the drug is present (Grady et al., 1997). They determined that this persistent release is mediated by the same nAChRs responsible for initial stimulation. In a similar fashion, experiments in our lab have shown that, although very low concentrations of nicotine do not produce an initial transient neurotrans-mitter spike (see Figure 3.2), prolonged exposure does lead to a gradual elevation in release above baseline (Rowell, 1995). These results suggest that, although the blood levels of nicotine in cigarette smokers and nicotine-treated animals may be above that sufficient to achieve nAChR desensitization, the desensitized receptors are, nonetheless, able to produce an elevation in DA release above that produced in the absence of the drug. Of course, these experiments were performed with isolated nerve endings of the striatum. It is quite probable that nAChRs in the VTA provide an even greater response because of increased glutamate release resulting in enhanced burst firing of the neuron with sustained elevated DA levels in the NAc.

Experiments directly comparing the effects of nicotine on nAChRs of the VTA with those on the terminals of the NAc have, in fact, suggested that there are differences in the activation and/or desensitization properties of nAChRs in these two tissues. Differences in the extent of desensitization between terminal and den-drosomatic receptors are indicated by the experiments of Nisell et al. (1994a) in which the continued application of nicotine in the NAc increased DA levels for only

20 min while the response from nicotine application to the VTA was maintained throughout an 80 min application. While electrophysiological studies of Dani and coworkers have shown that nAChRs in the VTA are desensitized by nanomolar concentrations of nicotine (Pidoplichko et al., 1997; Fisher et al., 1998), the desen-sitization characteristics are variable, and receptors, even within the same brain area, can behave quite differently based on the influence of neighboring neurons (Dani et al., 2000). In fact, Yin and French (2000) have demonstrated that, while nicotine stimulates both dopaminergic and nondopaminergic neurons in the VTA, the nAChRs on dopaminergic neurons are comparatively resistant to desensitization.

Clarke and coworkers have recently conducted in vitro superfusion studies in which they have characterized and compared the efficacy of nicotinic agonists to stimulated the release of [3H]DA from striatal terminals with their activity on den-drosomes prepared from the SN (Reuben et al., 2000). These investigators took advantage of the finding that the dopamine transporter (DAT) is located in relatively high density on the dendrites and dendritic spines of neurons of the SN (Nirenberg et al., 1996) and that dendrosomes prepared from the SN incorporate and release [3H]DA in response to K+ depolarization (Hefti and Lichtensteiger, 1978; Silbergeld and Walters, 1979; Marchi et al., 1991). It was found that, at the synaptosomes of the terminals, nicotine is somewhat less efficacious than epibatidine in evoking [3H]DA release, whereas at the dendrosomes of the SN, nicotine has equal or greater activity than epibatidine (Reuben et al., 2000). Moreover, whereas the epibatidine evoked [3H]DA release from striatal synaptosomes was almost completely blocked by mecamylamine, at large portion of the release from dendrosomes was mecamy-lamine insensitive. These experiments indicate differences in the nAChR activation properties and/or processes for nicotine-evoked release of DA between nerve terminals compared to the dendrites of nigrostriatal dopaminergic neurons.

Similar in vitro superfusion experiments are underway comparing nicotine's effects at the terminals and dendrites of mesolimbic neurons. In these experiments, the nicotine-evoked efflux of 86Rb+ is compared in tissue slices of the NAc and VTA. The brain is cooled and cross-sectional cuts are made just anterior to the NAc (at 3.3 mm rostral to bregma), just posterior to the VTA (at 6.6 mm caudal to bregma) and then midway between these two sections to yield two approximately 5-mm-thick coronal brain sections. These are placed on a cold block with the area of interest facing upwards and 800 mm punches are removed from each side at the appropriate regions. These cylinders are placed on a McIlwine tissue slicer and 4 to 5 400 ^m slices of the NAc or VTA are taken and placed in artificial CSF buffer. The entire dissection and slicing procedure is performed in a cold room at approximately 5°C. The slices are warmed to 32°C and "loaded" with 86Rb+ for 30 min (Marks et al., 1993a) at which time individual slices are placed on 22-mm-diameter GF/C filters in an open chamber two-pump superfusion system (Grady et al., 1992) and perfused with artificial CSF buffer at 1 ml/min. The results of a 1 min challenge with nicotine in these two brain areas is shown in Figure 3.4.

These results suggest that nicotine is slightly more potent as an agonist in the VTA compared to the NAc. The efficacy of nicotine in the VTA plateaus at about a 35% increase in basal release in the VTA, while the activity in the NAc continues to increase with increasing concentrations of nicotine. The slopes of the concentration-response

FIGURE 3.4 Nicotine-stimulated 86Rb+ efflux from NAc and VTA slices. Tissue slices, prepared as described earlier, were superfused for 30 min in oxygentated aCSF, at which point they were exposed to a 1-min pulse of nicotine at the indicated concentrations. Results are means ± S.E.M. (n = 10-16).

curves between 0.2 and 1 ^M nicotine are similar in both tissues. Since these experiments are conducted in relatively intact tissue slices, it is possible that part of the 86Rb+ efflux measured is coming from the terminals of neighboring nondopaminergic neurons which also possess nAChRs, as evidenced by the finding that part of the nicotine-stimulated 86Rb+ efflux is calcium dependent.


The experiments considered thus far have centered on the ability of nicotine to interact with nAChRs on the membranes of dopaminergic neurons and directly or indirectly stimulate the release of DA. In addition to this activity, there is some evidence to indicate that nicotine also interacts with other proteins of dopaminergic neurons to produce an enhancement of DA levels in the synapse. In the case of DA synthesis, nicotine does not appear to have any direct effect on the synthetic enzymes. In particular, the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of DA, is not directly affected by nicotine treatment in vivo or in vitro (Naquira et al., 1978; Carr et al. 1989), although nicotine can increase tyrosine hydroxylase mRNA indirectly (Gueorguiev et al., 1999). The increased turnover of DA that can be measured as a result of nicotine treatment is apparently an indirect response which accompanies the stimulation of neurotransmitter release. No experimental evidence exists that nicotine directly affects the vesicular uptake pump or storage proteins. Interestingly, however, the nicotinic alkaloid, lobeline, has been shown to inhibit both the uptake of DA into synaptic vesicles and the DA transporter

(DAT) (Teng et al., 1997, 1998). Nicotine also does not appear to act directly on dopaminergic receptors, at least those of the D2 subtypes, as determined with in vitro experiments (Fung and Lau, 1996; Carr et al., 1989). Although chronic nicotine administration does not appear to produce a widespread alteration in dopaminergic receptor affinity or density (Kirch et al., 1992; Fung and Lau, 1992; Levin et al., 1997; Court et al., 1998), it may selectively alter DA autoreceptor function (Reilly et al., 1987; Janson et al., 1992; Harsing et al., 1992; Takaki, 1995).

Although nicotine does not directly affect the DAT, Izenwasser et al. (1991) found that very low concentrations of nicotine inhibit DA uptake in an intact striatal tissue preparation. The maximal inhibition was about 50%, and the IC50 was found to be 0.005 nM. The ability of nicotine to act at low picomolar concentrations would make this the most potent of nicotine's activities reported so far. Nicotine's inhibition of DA uptake was stereospecific, with the (-) isomer about 20 times more potent than the (+); other nicotinic agonists (carbachol and DMPP) were also effective, although at much higher concentrations. Because nicotine does not directly inhibit the DAT in synaptosomes (Carr et al., 1989; Izenwasser et al., 1991) it was suggested that nicotine acts indirectly to release other neurotransmitters or neuromodulators which in turn inhibit the DAT. Experiments have been unable to show an inhibition of DA uptake in intact striatal mince or slices (Rowell and Hill, 1993). Interestingly, it has been found that non-nicotine components of cigarette smoke or nicotine metabolites, which inhibit the activity of the DAT (Carr et al., 1991, 1992; Dwoskin et al., 1992) might exist and could act synergistically with nicotine, along with an as yet-unidentified monoamine oxidase (MAO) inhibitor present in cigarette smoke, to enhance the activity of nicotine in vivo.

With respect to DA metabolism, while nicotine does not inhibit MAO directly (Oreland et al., 1981; Carr and Basham, 1991), a component of tobacco appears to be a potent MAO inhibitor. Oreland et al. (1981) and Yong and Perry (1986) found that the platelet activity of MAO was lower in smokers than nonsmokers, and Yu and Bolton (1987) determined that aqueous solutions of cigarette smoke could irreversibly inhibit MAO in vitro. Studies in our lab were the first to find that some component of cigarette smoke produces a decrease in the activity of MAO in the brain (Carr and Rowell, 1990). It was found that, in animals which had been chronically exposed to cigarette smoker for two to three weeks, a significant decrease in the activity of cortical MAO-B took place. In humans, the studies of Fowler and coworkers (Fowler et al., 1996a, 1996b) found that the activities of both MAO-A and MAO-B are decreased in the brains of cigarette smokers. The component of tobacco responsible for this effect has not been definitively identified; it is possibly a quinoline or quinone derivative (Mendez-Alvarez et al., 1997; Khalil et al., 2000). A decreased metabolism of DA brought about by MAO inhibition from tobacco component(s) may possibly act synergistically with nicotine-stimulated DA release to potentiate the reinforcement and dependence of cigarette smoking (Fowler et al., 1998).

Finally, in addition to nicotine, alkaloids present in tobacco, pyrolysis products from cigarette smoke, and/or metabolic products of tobacco alkaloids are able to stimulate the release of DA from brain tissue. These include cotinine, nornicotine, anabaseine, and anabasine (Dwoskin et al., 1993, 1995, 1999; Teng et al., 1997).

Although somewhat less potent than nicotine, it is possible that these compounds could also potentiate the effects of nicotine to increase synaptic DA levels (Crooks and Dwoskin, 1997).

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