In Animals, Receptor Puts Brakes on Nicotine Consumption

Findings appear to pinpoint a source of individual differences in smoking rates.
March 2012
By Lori Whitten, NIDA Notes Staff Writer

Some people smoke a few cigarettes now and then, while others smoke 30, 40, or more per day. NIDA-supported research now suggests that smokers' differences in tobacco consumption reflect, in part, differences in the functional efficacy of one subtype of nicotinic acetylcholine receptor (nAChR).

The new animal research, by Dr. Paul Kenny and colleagues at Scripps Research Institute in Jupiter, Florida, indicates that the nAChR subtype that incorporates the α5 structural subunit (termed α5* nAChR) promotes an aversive state in response to nicotine that increases in strength as blood concentrations of the drug rise. Among the evidence: Eliminating the α5 subunit greatly increased the amount of nicotine that animals would self-administer.

The new findings may explain previous observations that people who have a gene variant that results in a less functional version of α5* nAChR are far more likely to become dependent on nicotine than those with the normal version. The results open up the possibility that medications to stimulate the activity of α5* nAChRs might help smokers smoke less, reducing their risk for addiction and other consequences of exposure to tobacco.

A Tale of Two Pathways

As anyone who has inhaled tobacco for the first time has experienced, nicotine's effects are not all rewarding; some can be extremely unpleasant. A smoker's motivation to light up depends on the balance between anticipation of the rewarding effects and aversion to the noxious ones. The balance tilts more toward aversion as nicotine concentrations in the blood rise.

Dr. Kenny's team theorized that each smoker's rate of tobacco consumption depends on his or her tipping point, and that the tipping point, in turn, depends on how strongly two opposing brain pathways respond to nicotine. One, the reward pathway, which links the ventral tegmental area (VTA) to the nucleus accumbens (NAc), responds to nicotine stimulation by generating pleasurable feelings and motivation to repeat the experience. The other, the medial habenula-interpeduncular pathway (MHb-IPN), encodes states of aversion, thereby opposing the effects of the VTA-NAc reward systems and limiting further nicotine intake.

The figure shows the brains of a normal rat and a knockdown rat that has reduced levels of the α5 subunit of the nicotine receptor in the aversive pathway from the habenula to the interpeduncular nucleus. In the knockdown rat brain, that pa Two Opposing Pathways Shape Rats’ Responses to Nicotine: Rats' motivation to self-administer nicotine reflects the balance of activity in two brain pathways. Nicotine stimulation of receptors in the reward pathway, from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) generates pleasurable feelings and motivation to take more of the drug. In contrast—particularly at higher doses—nicotine stimulation of the pathway from the medial habenula (MHb) to the interpeduncular nucleus (IPN) produces an aversive state. Reducing the functionality of the α5 subunit of the nicotine acetylcholine receptor weakened activity in the aversive MHb-IPN pathway of knockdown rats, increasing the influence of the reward system over the rats’ behavior.

In support of this hypothesized role for the MHb-IPN, the Scripps researchers showed that suppressing neuronal activity in each of the two pathways in mice altered the animals' nicotine consumption in opposite ways. Rats decreased nicotine self-administration when the researchers injected either the anesthetic lidocaine or a substance that prevents the neurotransmitter glutamate from activating neurons in the VTA. The animals increased nicotine self-administration when the same substances were injected to deactivate the IPN.

The results for deactivation of the MHb supported the proposed role but were more complex. The animals increased their nicotine intake, but only when high concentrations of the drug were available—suggesting that MHb activation of the IPN occurs preferentially when high nicotine doses are consumed.

A Receptor for Restraint

The Scripps team noted that the MHb-IPN pathway contains a dense concentration of α5* nAChRs, which have an excitatory impact on neurons. Accordingly, nicotine stimulation of these receptors might constitute a primary mechanism by which the drug activates the pathway underlying aversive responses to the drug.

Schematic drawing shows a cell in the medial habenula and its synapse with a cell in the interpeduncular nucleus. Blown-up images show that a medial habenula cell that has functioning α5 nicotinic acetylcholine receptors releases more gluta Hypothesized Mechanism: Deficient Receptors Reduce Signal to Inhibit Nicotine: High levels of nicotine stimulate nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit in the neural pathway from the medial habenula (MHb) to the interpeduncular nucleus (IPN). Such stimulation enhances release of the neurotransmitter glutamate that binds to NMDA receptors, triggering an inhibitory signal that reduces motivation for further consumption of the drug. A deficiency of α5* nAChRs diminishes the magnitude of the inhibitory signal and the curb on consumption.

To test this idea, the researchers bred mice with a genetic mutation that halted production of α5* nAChRs. They compared these α5* nAChR-lacking (or knockout) animals with normal animals in experiments that showed:

  • Eliminating α5* nAChRs reduced activity in the IPN at high nicotine blood concentrations. The researchers injected mice with various doses of nicotine and then assayed levels of c-fos, a marker for neuronal activity, in the IPN. At low doses both normal and knockout mice exhibited similar levels of c-fos. At high nicotine doses that typically induce aversive effects, c-fos levels markedly increased in the normal mice, but not in the knockout mice.
  • Eliminating α5* nAChRs increased animals' consumption of the drug at higher doses. Mice self-administered nicotine infusions in a number of sessions, each with a higher dose per infusion. Normal animals reduced the number of infusions they took as the dose per infusion increased, keeping their average intake per session at about 1.5 mg/kg at all doses. Knockout mice, which did not restrain their intake as the dose per infusion rose, eventually consumed more than 5 mg/kg in the session with the highest dose.
Two graphs compare nicotine consumption between mice lacking the α5 subtype of nicotinic acetylcholine receptor, called knockout mice, and normal mice. The left graph shows that as the nicotine dose per infusion rose, control mice reduced t Receptor Subtype Influences Nicotine Intake: Knockout mice lacking α5 nicotinic acetylcholine receptors self-administered more nicotine than normal, or control, mice. As the nicotine dose per infusion rose, control mice took fewer infusions, maintaining roughly stable levels of drug consumption; whereas knockout mice took roughly the same number of infusions and consequently consumed much more drug.
  • Eliminating α5* nAChRs increased animals' motivation for the drug. Employing a progressive-ratio protocol—a type of self-administration trial in which animals must press a lever an increasing number of times for each successive dose—the researchers found that knockout mice worked harder for nicotine, showing more motivation than normal mice. Again, the difference between the two groups was more marked at high nicotine doses.
  • Restoring α5* nAChRs reinstated limits to consumption. When the researchers reversed the a5 genetic mutation in the MHb-IPN of the knockout mice, the animals showed the same limited nicotine self-administration as the normal mice.

To further explore the role of the α5* nAChR in the drive for, and consumption of, nicotine, Dr. Kenny and colleagues used molecular tools that selectively reduced the α5 subunit in a particular brain region to produce what they call α5 knockdown (KD) rats. These animals have about half the normal levels of α5* nAChRs in the MHb-IPN pathway and normal levels elsewhere in the brain. The KD rats demonstrated nicotine intake patterns similar to those of KO mice.

Titrating Reward

The researchers found that the reduction in the number of α5* nAChRs altered nicotine's aversive effects, but not its rewarding properties. When normal and knockdown rats could directly activate their reward pathway with electrical stimulation, both groups self-administered similar intensities when nicotine-free. When given low doses of nicotine known to be rewarding but not aversive, both groups self-administered far lower electrical intensities than normal, which reflects the stimulatory effects of nicotine on reward pathways.

When rats received high nicotine doses, however, normal and KD rats' responses diverged. Normal rats self-administered higher electrical intensities than they had with low nicotine doses, presumably requiring more reward stimulation to counter the mounting nicotine-induced aversive state underpinned by the MHb-IPN. In contrast, knockdown rats self-administered the same low levels of electrical intensity as they had when they received low nicotine doses, suggesting that eliminating the α5* nAChRs suppressed those aversive effects. Overall, at high nicotine doses, the knockdown rats' motivational balance was tipped toward reward seeking and nicotine consumption (see figure).

Together, these results confirm that the α5* nAChRs activate the MHb-IPN pathway and play a key role in the aversive effects induced by the drug. By doing so, the receptors dissuade further nicotine consumption as blood concentrations of the drug increase.

Endowed With Risk

Dr. Kenny and colleagues' results provide a physiological explanation for population genetic studies that have linked variation in the gene (CHRNA5) that encodes the α5 subunit of the nAChR to the risk for nicotine dependence. Compared with individuals having no copies of a high-risk variant, those with one copy had a 30 percent higher likelihood of nicotine addiction; two copies of the high-risk variant more than doubled the probability of addiction. This risk variant of the CHRNA5 gene results in less-functional α5* nAChRs than those typically found in individuals without the risk variant, according to Dr. Kenny.

"Our findings in animals suggest that individuals carrying CHRNA5 risk alleles can tolerate unusually high levels of nicotine without experiencing inhibitory effects of nicotine on reward pathways, increasing their risk for dependence and exposure to carcinogens," says Dr. Kenny. According to this reasoning, individuals with more copies of the high-risk alleles produce less-functional α5* nAChRs, and hence, experience weaker aversive effects of nicotine. Their resulting increased tolerance for higher doses of the drug allows them to consume more cigarettes, increasing their exposure to the nicotine-induced brain changes that underlie addiction and to toxic effects of the drug that cause cancer and other diseases.

The Scripps findings may lead to a new antismoking medication strategy. "My colleagues and I have launched a drug discovery campaign to identify compounds that stimulate nAChRs containing the α5 subunit, and we are preparing to test candidate compounds in animals very soon," says Dr. Kenny. Increased stimulation of the α5* nAChRs in the IPN could strengthen nicotine's aversive effects and put a lid on the desire to smoke. "In addition, the dramatic effect of this signaling protein on nicotine-related behavior has spurred my team's interest in its natural function in the brain, its neural circuitry, and its possible connection with the high rates of smoking among people with psychiatric illnesses," says Dr. Kenny.

"Dr. Kenny's cutting-edge research demonstrates how researchers can employ genetic techniques in animals to examine the cellular mechanisms underlying observations from human population genetics and epidemiology," says Dr. Minda Lynch of NIDA's Division of Basic Neuroscience and Behavioral Research. "These findings suggest that individual variation in the gene encoding the α5 subunit may render some individuals less sensitive to negative motivational signals—tipping the balance in favor of reward and consumption."


Fowler, C.D., et al. Habenular α5 nicotinic receptor subunit signaling controls nicotine intake. Nature 471(7340):597–601, 2011.

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