Inhibition of monoamine oxidase isoforms modulates nicotine withdrawal syndrome in the rat.

AIMS: There have been many reports of monoamine oxidase (MAO) inhibition by non-nicotine ingredients in tobacco smoke, persisting for days after smoking cessation. This study determined the effect of inhibiting MAO and its isoforms on nicotine withdrawal syndrome. MAIN METHODS: Rats were rendered nicotine-dependent by seven days of subcutaneous (s.c.) 9 mg/kg/day infusion of nicotine bitartrate. Twenty-two hours after termination of infusion, they were observed over 20 min for somatically expressed nicotine withdrawal signs. Three hours before observation, rats were injected intraperitoneally (i.p.) with 4 mg/kg each of the MAO A antagonist clorgyline and the MAO B antagonist deprenyl, or with saline alone. A similar experiment was performed with non-dependent, saline-infused rats. Another experiment compared nicotine-dependent rats that received injections of either saline or 4 mg/kg clorgyline alone. A further experiment compared rats receiving either saline or 4 mg/kg deprenyl alone. KEY FINDINGS: Combined treatment with both MAO inhibitors markedly and significantly exacerbated somatically expressed nicotine withdrawal signs in nicotine infused rats, while having no significant effects in saline-infused rats. Rats injected s.c. with 4 mg/kg clorgyline alone had significantly more withdrawal signs than saline-injected rats, while deprenyl-injected rats had significantly fewer signs than saline controls. Assays confirmed that clorgyline thoroughly reduced MAO A enzymatic activity and deprenyl thoroughly reduced MAO B activity. SIGNIFICANCE: The results suggest that inhibition of MAO A may contribute to the intensity of withdrawal syndrome in smoking cessation.

Hippocampal injections of amyloid beta-peptide 1-40 impair subsequent one-trial/day reward learning.

The injection of amyloid beta-peptide (Abeta) into rat CNS has been reported to induce cellular neuropathology. The present study investigated whether multiple intrahippocampal injections of Abeta 1-40 would impair one-trial/day reward learning 14 days later. Twenty-four male Sprague-Dawley rats, 3-4 months old, were injected with either Abeta 1-40 or distilled water into seven hippocampal sites bilaterally. Ten rats received 3 nmol Abeta 1-40 in 2 microl of distilled water per injection site, while 14 rats received distilled water alone. Following a 9-day recovery period, rats were gradually food deprived to 82% of their initial body weight. Fourteen days after the intrahippocampal injection, all rats received an initial training trial and three subsequent daily retention trials. Rats receiving Abeta 1-40 were significantly impaired on the second retention trial in terms of accuracy (number of unbaited alleys entered) and on the second and third retention trials in terms of speed (reciprocal of latency to reward). Histological analysis showed that Abeta 1-40 injections produced significant neuronal loss and gliosis. Abeta 1-40 immunoreactivity persisted locally at the injection site and in macrophages 2 weeks following the hippocampal injections. These effects appear to be sequence-specific; rats receiving Abeta 1-42 with a scrambled peptide sequence did not differ significantly from rats receiving distilled water alone in retention of the learning task or degree of histological damage.

Passive immunization against nicotine prevents nicotine alleviation of nicotine abstinence syndrome.

Passive immunization against nicotine interferes with its locomotor and pressor effects. The current study determined whether immunization could prevent another nicotine action: the reversal of nicotine abstinence syndrome. IgG containing 4.4-5.6% nicotine-specific antibody was isolated from rabbits immunized with 3'-amino-methyl-nicotine conjugated to a carrier protein. Twenty rats were rendered dependent by 7 days of subcutaneous infusion of 3.15 mg/kg/day nicotine (expressed as the base). Upon termination of nicotine infusion, each rat was injected intraperitoneally with 150 mg of IgG from normal serum (n=13) or from nicotine antiserum (n=7). Twenty-two and one-half hours later, all rats were observed over 15 min for baseline nicotine abstinence signs. Two and one-half hours after baseline observations, seven of the 13 rats pretreated with control IgG and all seven rats pretreated with nicotine-specific IgG were then challenged by 0.12 mg/kg (sc) nicotine. The remaining six rats pretreated with control IgG were challenged with saline alone. All rats were then observed again for abstinence signs. Nicotine injection caused significantly less reduction of abstinence signs in the immunized rats. The nicotine effect in immunized rats was comparable to the saline effect in nonimmunized rats. Immunization also significantly reduced free serum nicotine concentration and nicotine distribution to the brain. These results raise the possibility that immunization might prevent nicotine consumption from relieving the discomforts of smoking cessation.

Nicotine dependence: studies with a laboratory model.

Simple, rapid preclinical models of nicotine physical dependence and abstinence syndrome are needed to identify underlying neurobiological mechanisms and screen potential therapies. One such model induces dependence by 7 days of continuous subcutaneous nicotine infusion in the rat. Abstinence is initiated through termination of infusion or injection of nicotinic antagonist drugs. The result is an abstinence syndrome involving a pattern of behaviors somewhat resembling opiate abstinence in the rat as well as weight gain and depressed locomotor activity. The model has met a number of validity criteria and its essential features have been replicated in several laboratories. Several research groups have modified or extended the model by measuring emotional/motivational changes associated with nicotine abstinence such as conditioned aversion, intracranial self-stimulation (ICSS) thresholds and the startle response. Dependence models have been used to identify neurobiological systems that contribute to nicotine dependence, particularly endogenous opiate systems and the mesolimbic dopamine pathway. It is hypothesized that these different systems contribute to different behavioral aspects of nicotine abstinence syndrome. Increasingly used as a preclinical screening tool, the model has proved sensitive to various abstinence-alleviating therapeutic approaches, including some with already demonstrated clinical effectiveness.

Nociceptin/orphanin FQ (N/OFQ) induces a quasi-morphine abstinence syndrome in the rat.

Nociceptin/orphanin FQ (N/OFQ) is a neuropeptide that exerts antiopiate effects under some circumstances, and there is evidence that it contributes to opiate tolerance. This raises the question, might N/OFQ also contribute to opiate dependence and abstinence? Twenty-two male Sprague-Dawley rats were cannulated in the third ventricle and challenged 7 days later by third ventricle injection of 50, 200 or 1,000 ng N/OFQ or saline alone. Each rat was observed under "blind" conditions for 30 min beginning 15 min after onset of the third ventricle injection. There was a significant positive linear trend of signs as a function of N/OFQ dose. Subjects receiving saline had 18.0+/-2.0 (mean+/-SEM) overall abstinence-like signs, whereas subjects receiving 50, 200 or 1000 ng N/OFQ had 35.2+/-3.6, 49.8+/-2.6 and 63.5+/-9.7 signs, respectively. In 16 additional rats, abstinence-like signs induced by 1000 ng N/OFQ were significantly attenuated by low SC doses of morphine or clonidine. These results raise the possibility that N/OFQ might contribute to opiate dependence and subsequent abstinence syndrome. On the other hand, N/OFQ over a wide dose range induced abstinence signs with similar potency in morphine dependent and non-dependent rats.

A rodent model of cocaine abstinence syndrome.

This study introduces a rat model of cocaine abstinence syndrome based on quantitation of spontaneously emitted behaviors following termination of continuous drug exposure (analogous to established methods of assessing morphine and nicotine abstinence). Groups of eight male S-D rats were infused SC for 7 days via an osmotic minipump with saline alone or with 40 or 60 mg/kg/day cocaine HCl. Pumps were removed and rats were observed at 12, 24, 36, and 48 h postremoval. Each 15-min observation employed a checklist of abstinence signs including ptosis, chews, teeth chatters, gasps, writhes, seminal ejaculations, head shakes, and tremors. The high infusion rate group displayed significantly more signs than the low infusion rate group, which in turn, displayed significantly more signs than the saline group. Cocaine injection significantly reduced signs by 83.3%, while saline injection reduced them by only 4.9%. In another experiment, rats infused with 60 mg/kg/day showed significantly more signs 36 h postinfusion than before infusion, during infusion and 84 h postinfusion. Finally, 6.5 days of infusion resulted in significantly more abstinence signs than did 1.5 days of infusion. This rapid and simple model quantitated cocaine abstinence syndrome in a manner that was cocaine-reversible and related to the rate and duration of drug infusion.

A nicotine conjugate vaccine reduces nicotine distribution to brain and attenuates its behavioral and cardiovascular effects in rats.

Vaccination of animals to elicit drug-specific antibodies, or the passive transfer of such antibodies from other animals, can reduce the behavioral effects of drugs such as cocaine and heroin. To study the potential application of this approach to treating nicotine dependence, IgG was isolated from rabbits immunized with a nicotine-protein conjugate vaccine. Anesthetized rats received immune IgG containing nicotine-specific antibodies (Nic-IgG) or control-IgG i.v.. Thirty minutes later, rats received nicotine at 0.03 mg/kg i.v., equivalent on an mg/kg basis to the nicotine intake from two cigarettes by a smoker. Compared to control-IgG, Nic-IgG reduced the brain nicotine concentration in a dose-related manner (65% reduction at the highest IgG dose). Pretreatment with Nic-IgG also reduced the distribution to brain of five repeated doses of nicotine (equivalent to the nicotine intake from 10 cigarettes) administered over 80 min. To study blood pressure effects, rats received control-IgG or Nic-IgG 1 day prior to administering nicotine. Nicotine-induced systolic blood pressure increases were attenuated by Nic-IgG in a dose-related manner, and were almost completely blocked by the highest Nic-IgG dose. Pretreatment with Nic-IgG also completely prevented the nicotine-induced stimulation of locomotor activity observed in rats receiving control-IgG. Nic-IgG did not prevent locomotor activation from cocaine, demonstrating its specificity for nicotine. These data demonstrate that the administration of nicotine-specific antibodies can reduce or prevent some of the pharmacokinetic, cardiovascular, and behavioral consequences of nicotine in rats. Effects were observed at nicotine doses and nicotine serum concentrations equal to or exceeding those typically associated with nicotine exposure in cigarette smokers. A potential role for immunization in the treatment of nicotine dependence is suggested.

Nicotine abstinence syndrome precipitated by the competitive nicotinic antagonist dihydro-beta-erythroidine.

Rats infused subcutaneously with 9 mg/kg/day nicotine tartrate for 7 days exhibit behavioral abstinence signs following either termination of nicotine infusion or injection of the noncompetitive nicotinic antagonists mecamylamine (s.c.) or hexamethonium (ic.c.v.). This study examined the abstinence precipitating effects of dihydro-beta-erythroidine (DHbetaE), a competitive nicotinic antagonist. Twenty-four nicotine-dependent rats were injected in the third ventricle with 10, 18, or 25 microg DHbetaE in 20 microl saline or with saline alone and observed for abstinence signs over a 20-min period. There was a significant positive linear trend of overall abstinence signs as a function of dose, p < 0.01. In 12 nondependent rats, the high dose of DHbetaE did not induce more abstinence-like signs than saline alone. In a second experiment, 18 nicotine-dependent rats were injected s.c. with 1 or 6 mg/kg of the muscarinic antagonist scopolamine or with saline alone. Few abstinence signs were observed in any group: there was no significant drug effect. The results suggest that nicotine abstinence signs observed in the rat are specific to reduced stimulation of previously overstimulated nicotinic receptors.

The nitric oxide synthesis inhibitor nitro-L-arginine (L-NNA) attenuates nicotine abstinence syndrome in the rat.

Nitric oxide synthesis contributes to opiate tolerance and dependence. Nicotine dependence and abstinence syndrome in the rat appear to involve opiate mechanisms. Therefore, it was postulated that nitric oxide synthase (NOS) activity might be essential for the expression of nicotine abstinence syndrome. Twenty-one rats were rendered dependent by SC infusion of 9 mg/kg per day nicotine tartrate via Alzet osmotic minipump. Rats were pretreated SC with vehicle alone, or with 18 or 30 mg/kg of the NOS inhibitor L-NNA (nitro-L-arginine). Thirty minutes later, rats were challenged by 1 mg/kg of the nicotinic antagonist mecamylamine SC and observed for 30 additional minutes. Rats pretreated with vehicle displayed a total of 68.7+/-8.0 mecamylamine-precipitated abstinence signs (mean+/-SEM), while those receiving 18 or 30 mg/kg L-NNA had 12.7+/-2.0 and 5.1+/-1.7 signs, respectively. All three groups differed significantly from one another according to Dunn's post-hoc procedure. Rats pretreated with L-NNA combined with an excess of the NOS substrate L-arginine had significantly more mecamylamine-precipitated abstinence signs than rats receiving L-NNA combined with D-arginine. Also, D-NNA, which does not selectively bind to NOS, was significantly less effective than L-NNA in preventing mecamylamine-precipitated abstinence syndrome. Additional studies determined the effect of L-NNA on spontaneous nicotine abstinence syndrome. Rats were assessed for abstinence signs at 17 and 20 h after termination of nicotine infusion. They received injections of 9, 18, or 30 mg/kg L-NNA SC or vehicle alone immediately before the 20-h observation; all rats were observed for 30 min. Signs at 20 h (post-injection) as a percentage of signs at 17 h (pre-injection) declined significantly as a function of L-NNA dose. Once again, this effect was attenuated significantly more by co-administration of L-arginine than by D-arginine. The overall pattern of results suggests that nitric oxide synthesis is critical to the expression of nicotine abstinence syndrome.

Nicotine abstinence syndrome precipitated by central but not peripheral hexamethonium.

A rodent model of nicotine dependence has been developed based on continuous subcutaneous (s.c.) infusion of nicotine tartrate. Nicotine abstinence syndrome was precipitated by s.c. injection of the nicotinic antagonist mecamylamine, which freely crosses the blood-brain barrier. In contrast, the nicotinic antagonist hexamethonium crosses the blood-brain barrier very poorly. This study determined whether central or peripheral administration of hexamethonium could precipitate nicotine abstinence. In the first experiment, 26 nicotine-dependent rats were injected s.c. with 0.5, 5 or 10 mg/kg hexamethonium dichloride or saline alone and observed for 20 min. Few abstinence signs were observed in any group; there was no significant drug effect. In the second experiment, 18 rats were cannulated in the third ventricle and rendered nicotine dependent. One week later, rats were injected through the cannula with 12 or 18 ng hexamethonium or saline alone and observed for 20 min. Both dose groups differed significantly from the saline-injected group, and there was a significant positive linear trend of signs as a function of dose. The high dose had no significant effect in 14 nondependent rats. We conclude that hexamethonium is much more potent by the central route, and there is a major central nervous system component in nicotine dependence.

Nicotine abstinence syndrome precipitated by an analog of neuropeptide FF.

In a recently introduced rodent model of nicotine abstinence syndrome the observed behavioral signs closely resembled those typical of rat opiate abstinence syndrome. Nicotine-induced release of endogenous opioids may contribute to nicotine dependence; morphine potently reverses nicotine abstinence signs, while naloxone precipitates abstinence signs and prevents nicotine from alleviating them. Considerable evidence suggests that neuropeptide FF, an endogenous antiopiate peptide, contributes to opiate dependence. Third ventricle injection of neuropeptide FF precipitates abstinence syndrome in morphine-dependent rats, as does SC injection of its lipophilic analogs, dansyl-PQRFamide and dansyl-RFamide. Might NPFF also play a role in nicotine dependence? In the present study, SC injection of 15 or 25 mg/kg dansyl-RFamide or vehicle alone dose dependently precipitated an abstinence syndrome in nicotine-dependent rats. There was a significant, p < 0.01, positive linear trend of abstinence signs as a function of dose. Categories of abstinence signs had the same rank ordering by frequency as observed in spontaneous nicotine abstinence. Injection of 25 mg/kg dansyl-RFamide SC had no significant effect in nondependent rats.

Enhanced antiopiate activity and enzyme resistance in a peptidomimetic of FMRFamide containing E-2,3-methanomethionine and E-2,3-methanophenylalanine.

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. Peptidomimetics of FMRFamide substituted with conformationally constrained stereoisomers of Z-2,3-methanomethionine or E-2,3-methanomethionine precipitated abstinence syndrome far more potently than FMRFamide itself. The current study determined the effect on antiopiate potency of an additional rigid substitution. A peptidomimetic containing a stereoisomer of E-2,3-methanomethionine was compared with a peptidomimetic additionally substituted at the C-terminal with E-2,3-methanophenylalanine. Morphine abstinence signs were observed after varying doses (0.125-25.0 micrograms) of these two peptidomimetics were injected into the third ventricle of morphine-dependent rats. The peptidomimetic containing both rigid substitutions was far more potent than the peptidomimetic of FMRFamide containing methanomethionine alone. The increased potency appears to be related to enzyme resistance rather than receptor affinity.

Nitric oxide synthesis inhibition attenuates behavioral actions of neuropeptide FF.

Neuropeptide FF (NPFF) has certain antiopiate actions and may play a role in opiate tolerance and dependence. Third ventricle injection of 10 micrograms NPFF induces a quasimorphine abstinence syndrome in opiate-naive rats. Nitric oxide synthesis may also contribute to opiate tolerance and dependence. The present study tests the hypothesis that NPFF acts through stimulation of nitric oxide synthase (NOS). Third ventricular injection of 10 micrograms NPFF precipitated an average of 46 abstinence-like signs during a 20-min observation. Pretreatment (30 min earlier) with 7.5 or 15 mg/kg s.c. of the NOS inhibitor nitro-L-arginine (L-NNA) resulted in a significant and dose-dependent alleviation of NPFF-induced abstinence-like signs. The anti-NPFF activity of 15 mg/kg L-NNA was blocked by 750 mg/kg L-arginine, but not by the same amount of D-arginine, indicating that L-NNA attenuates NPFF activity through a stereospecific inhibition of NOS.

Nicotine alleviation of nicotine abstinence syndrome is naloxone-reversible.

In a recently introduced rodent model of nicotine abstinence syndrome, the observed signs closely resembled those typical of rat opiate abstinence syndrome. Signs were precipitated by naloxone and potently reversed by morphine as well as nicotine itself, suggesting that nicotine might relieve nicotine abstinence syndrome through releasing endogenous opioids. To test this hypothesis, rats were continuously infused subcutaneously (SC) for 7 days with 9 mg/kg per day nicotine tartrate. Each rat was observed for abstinence signs at 18 and 21 h after termination of infusion. Three minutes before the 21-h test, all rats received 0.35 mg/kg nicotine tartrate, SC; 5 min before the nicotine injection, subjects received 9 or 4.5 mg/kg naloxone or saline alone, SC. Abstinence reversal scores were calculated as signs at 21 h as a percentage of signs at 18 h. Naloxone prevented nicotine alleviation of nicotine abstinence in a dose-related manner. However, naloxone in the absence of a nicotine injection had no effect on abstinence severity in either highly dependent or moderately dependent rats (infused with 9 or 5 mg/kg per day nicotine tartrate, respectively). These results support the hypothesis that endogenous opioids play a role in nicotine dependence and abstinence.

Subcutaneous injection of an analog of neuropeptide FF prevents naloxone-precipitated morphine abstinence syndrome.

There is evidence that neuropeptide FF (NPFF) has antiopiate activity and may play a role in opiate dependence and subsequent abstinence syndrome. A fragment of NPFF was modified at the C-terminal in an effort to convert it to an NPFF antagonist. It was also dansylated at the N-terminal in an effort to render it more lipophilic and increase its penetration of the blood-brain barrier. Third ventricle administration of the resulting compound, dansyl-PQRamide (0.75 microgram and 1 microgram), dose-dependently antagonized the quasi-morphine abstinence activity of NPFF (10 micrograms) in opiate-naive rats. Subcutaneous injection of dansyl-PQRamide (13 mg/kg) in chronically morphine-infused rats attenuated opiate dependence as indicated by prevention of naloxone-precipitated abstinence syndrome. Dansyl-PQRamide displaced radiolabelled ligand from NPFF receptors in a concentration-dependent manner with a Ki of 13 microM, and had a half-life over 300 times longer than NPFF under aminopeptidase digestion.

The nicotinic antagonist mecamylamine precipitates nicotine abstinence syndrome in the rat.

Recently, a rodent model of nicotine abstinence syndrome has been developed based on observing the frequency of spontaneous behavioral signs following termination of continuous subcutaneous infusion of nicotine tartrate. In the present study, the nicotinic antagonist mecamylamine precipitated an abstinence syndrome in nicotine-dependent rats. Twelve rats were each infused for 7 days with 9 mg/kg per day nicotine tartrate in saline via Alzet osmotic minipumps; another 12 rats were sham-operated and remained nicotine-naive. Six rats from each group received 1 mg/kg mecamylamine in saline SC immediately before a 30-min observation, while the remaining six rats from each group received saline alone. Nicotine-infused rats receiving mecamylamine exhibited significantly more (P < 0.01), overall abstinence signs than all other groups. In terms of categories of signs, they displayed significantly more gasps/writhes, teeth chatter/chews, shakes/tremors and ptosis. In a second experiment utilizing only nicotine-naive rats, a far higher dose of mecamylamine (5 mg/kg sc) induced a quasi-nicotine abstinence syndrome. The results provide further validation for this rodent model of nicotine abstinence syndrome.

Enhanced antiopiate activity and enzyme resistance in peptidomimetics of FMRFamide containing (E)-2,3-methanomethionine.

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. The current study determined the antiopiate potency of FMRFamide and two conformationally constrained peptidomimetics of FMRFamide containing stereoisomers of (E)-2,3-methanomethionine. Morphine abstinence signs were observed after varying doses (0.25-25.0 microgram) of these substances were injected into the third ventricle of morphine-dependent rats. Both peptidomimetics were far more potent than FMRFamide itself. In addition, although both peptidomimetics bound with lower affinity than FMRFamide to rat spinal cord receptors for NPFF (the mammalian FMRFamide-like peptide), they were far more resistant than FMRFamide to enzymatic degradation by leucine aminopeptidase.

Chronic methanesulfonyl fluoride enhances one-trial per day reward learning in aged rats.

Aged (24-month-old) rats were treated chronically with methanesulfonyl fluoride (MSF), an acetylcholinesterase (AChE) inhibitor with selectivity for central nervous system AChE, or with injection vehicle alone. Twelve 0.22 mg/kg IP injections were given over 4 weeks. MSF rats showed significantly greater speed and accuracy on a 1 trial/day discriminative reward learning task. The chronic MSF treatment resulted in a 56% decrease in brain AChE activity but no discernable locomotor side effects and no liver damage as indicated by aspartate transferase activity.

Naloxone precipitates nicotine abstinence syndrome in the rat.

Recently, a rodent model of nicotine abstinence syndrome has been developed based on continuous subcutaneous infusion of nicotine tartrate and observing the frequency of spontaneous behavioral signs following termination of infusion. The observed signs closely resemble those commonly seen in rat opiate abstinence syndrome, raising the possibility that there is an endogenous opioid component in nicotine dependence. The present study demonstrates that the opiate antagonist naloxone can precipitate an abstinence syndrome in nicotine-dependent rats. Fourteen rats were infused for 7 days with 9 mg/kg/day nicotine tartrate in saline via an Alzet osmotic minipump. Fourteen rats were sham-operated and remained nicotine-naive. Half of each group received 4.5 mg/kg naloxone SC immediately before a "blind" 15-min observation, while the other half received saline alone. ANOVA revealed significant nicotine infusion, naloxone injection and interaction effects. Post-hoc analysis showed that the nicotine-infused rats injected with naloxone had significantly more signs than all other groups (P < 0.01). In a second experiment, 2 mg/kg morphine sulfate SC produced a significant (P < 0.01) 91.2% reduction of spontaneous abstinence signs observed 21 h after termination of nicotine infusion. These results are consistent with the hypothesized endogenous opioid component in nicotine dependence and abstinence syndrome.