Role for α6 nicotinic receptors in l-dopa-induced dyskinesias in parkinsonian mice.

L-Dopa-induced dyskinesias are a serious side effect that develops in most Parkinson's disease patients on dopamine replacement therapy. Few treatment options are available to manage dyskinesias; however,recent studies show that nicotine reduces these abnormal involuntary movements (AIMs) in parkinsonian animals by acting at nicotinic acetylcholine receptors (nAChRs). Identification of the nAChR subtypes that mediate this reduction in AIMs is important as it will help in the development of nAChR subtype selective drugs for their treatment. Here we investigate the role of α6ß2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using a6 nAChR subunit null mutant (α6⁻/⁻) mice.Wildtype and α6⁻/⁻ mice were lesioned by unilateral injection of 6-hydroxydopamine (3 mg/ml) into the medial forebrain bundle. They were then given L-dopa (3 mg/kg) plus benserazide (15 mg/kg) 2e3 wk later. L-dopa-induced AIMs developed to a similar extent in α6⁻/⁻ and wildtype mice.However, AIMs in α6⁻/⁻ mice declined to ~50% of that in wildtype mice with continued L-dopa treatment. Nicotine treatment also decreased AIMs by ~50% in wildtype mice, although not in α6⁻/⁻ mice. There were no effects on parkinsonism under any experimental condition. To conclude, the similar declines in L-dopa-induced AIMs in nicotine-treated wildtype mice and in α6⁻/⁻ mice treated with and without nicotine indicate an essential role for α6ß2* nAChRs in the maintenance of L-dopa-induced AIMs.These findings suggest that α6ß2* nAChR drugs have potential for reducing L-dopa-induced dyskinesias in Parkinson's disease.

Nicotine reduces L-DOPA-induced dyskinesias by acting at beta2* nicotinic receptors.

L-DOPA-induced dyskinesias or abnormal involuntary movements (AIMs) are a debilitating adverse complication associated with prolonged L-DOPA administration for Parkinson's disease. Few treatments are currently available for dyskinesias. Our recent data showed that nicotine reduced L-DOPA-induced AIMs in parkinsonian animal models. An important question is the nicotinic acetylcholine receptor (nAChR) subtypes through which nicotine exerts this beneficial effect, because such knowledge would allow for the development of drugs that target the relevant receptor population(s). To address this, we used ß2 nAChR subunit knockout [ß2(-/-)] mice because ß2-containing nAChRs are key regulators of nigrostriatal dopaminergic function. All of the mice were lesioned by intracranial injection of 6-hydroxydopamine into the right medial forebrain bundle. Lesioning resulted in a similar degree of nigrostriatal damage and parkinsonism in ß2(-/-) and wild-type mice. All of the mice then were injected with L-DOPA (3 mg/kg) plus benserazide (15 mg/kg) once daily for 4 weeks until AIMs were fully developed. L-DOPA-induced AIMs were approximately 40% less in the ß2(-/-) mice compared with the wild-type mice. It is interesting to note that nicotine (300 µg/ml in drinking water) reduced L-DOPA-induced AIMs by 40% in wild-type mice but had no effect in ß2(-/-) mice with partial nigrostriatal damage. The nicotine-mediated decline in AIMs was much less pronounced in wild-type mice with near-complete degeneration, suggesting that presynaptic nAChRs on dopaminergic terminals have a major influence. These data demonstrate an essential role for ß2* nAChRs in the antidyskinetic effect of nicotine and suggest that drugs targeting these subtypes may be useful for the management of L-DOPA-induced dyskinesias in Parkinson's disease.

Nicotinic receptor agonists decrease L-dopa-induced dyskinesias most effectively in partially lesioned parkinsonian rats.

L-dopa therapy for Parkinson's disease leads to dyskinesias or abnormal involuntary movement (AIMs) for which there are few treatment options. Our previous data showed that nicotine administration reduced L-dopa-induced AIMs in parkinsonian monkeys and rats. To further understand how nicotine mediates its antidyskinetic action, we investigated the effect of nicotinic receptor (nAChR) agonists in unilateral 6-OHDA-lesioned rats with varying striatal damage. We first tested the drugs in L-dopa-treated rats with a near-complete striatal dopamine lesion (>99%), the standard rodent dyskinesia model. Varenicline, an agonist that interacts with multiple nAChRs, did not significantly reduce L-dopa-induced AIMs, while 5-iodo-A-85380 (A-85380), which acts selectively at α4ß2* and α6ß2* subtypes, reduced AIMs by 20%. By contrast, both varenicline and A-85380 reduced L-dopa-induced AIMs by 40-50% in rats with a partial striatal dopamine lesion. Neither drug worsened the antiparkinsonian action of L-dopa. The results show that selective nicotinic agonists reduce dyskinesias, and that they are optimally effective in animals with partial striatal dopamine damage. These findings suggest that presynaptic dopamine terminal α4ß2* and α6ß2* nAChRs are critical for nicotine's antidyskinetic action. The current data have important implications for the use of nicotinic receptor-directed drugs for L-dopa-induced dyskinesias, a debilitating motor complication of dopamine replacement therapy for Parkinson's disease.

Multiple roles for nicotine in Parkinson's disease.

There exists a remarkable diversity of neurotransmitter compounds in the striatum, a pivotal brain region in the pathology of Parkinson's disease, a movement disorder characterized by rigidity, tremor and bradykinesia. The striatal dopaminergic system, which is particularly vulnerable to neurodegeneration in this disorder, appears to be the major contributor to these motor problems. However, numerous other neurotransmitter systems in the striatum most likely also play a significant role, including the nicotinic cholinergic system. Indeed, there is an extensive anatomical overlap between dopaminergic and cholinergic neurons, and acetylcholine is well known to modulate striatal dopamine release both in vitro and in vivo. Nicotine, a drug that stimulates nicotinic acetylcholine receptors (nAChRs), influences several functions relevant to Parkinson's disease. Extensive studies in parkinsonian animals show that nicotine protects against nigrostriatal damage, findings that may explain the well-established decline in Parkinson's disease incidence with tobacco use. In addition, recent work shows that nicotine reduces l-dopa-induced abnormal involuntary movements, a debilitating complication of l-dopa therapy for Parkinson's disease. These combined observations suggest that nAChR stimulation may represent a useful treatment strategy for Parkinson's disease for neuroprotection and symptomatic treatment. Importantly, only selective nAChR subtypes are present in the striatum including the alpha4beta2*, alpha6beta2* and alpha7 nAChR populations. Treatment with nAChR ligands directed to these subtypes may thus yield optimal therapeutic benefit for Parkinson's disease, with a minimum of adverse side effects.

Nicotine is neuroprotective when administered before but not after nigrostriatal damage in rats and monkeys.

Nicotine reduces dopaminergic deficits in parkinsonian animals when administered before nigrostriatal damage. Here we tested whether nicotine is also beneficial when given to rats and monkeys with pre-existing nigrostriatal damage. Rats were administered nicotine before and after a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle, and the results compared with those in which rats received nicotine only after lesioning. Nicotine pre-treatment attenuated behavioral deficits and lessened lesion-induced losses of the striatal dopamine transporter, and alpha6beta2* and alpha4beta2* nicotinic receptors (nAChRs). By contrast, nicotine administered 2 weeks after lesioning, when 6-hydroxydopamine-induced neurodegenerative effects are essentially complete, did not improve these same measures. Similar results were observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. Nicotine did not enhance striatal markers when administered to monkeys with pre-existing nigrostriatal damage, in contrast to previous data that showed improvements when nicotine was given to monkeys before lesioning. These combined findings in two animal models suggest that nicotine is neuroprotective rather than neurorestorative against nigrostriatal damage. Receptor studies with (125)I-alpha-conotoxinMII and the alpha-conotoxinMII analog E11A were next performed to determine whether nicotine treatment pre- or post-lesioning differentially affected expression of alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR subtypes in striatum. The observations suggest that protection against nigrostriatal damage may be linked to striatal alpha6alpha4beta2* nAChRs.

Cigarette smoke, nicotine and cotinine protect against 6-hydroxydopamine-induced toxicity in SH-SY5Y cells.

Epidemiological studies consistently demonstrate a reduced incidence of Parkinson's disease in smokers. As an approach to evaluate whether nicotine in tobacco may be involved in this apparent protective effect, we compared the effect of mainstream 1R4F cigarette smoke solutions, which contain chemicals inhaled by active smokers, and nicotine against 6-hydroxydopamine (6-OHDA)-induced toxicity in an in vitro cell culture system. For this purpose we used terminally differentiated SH-SY5Y neuroblastoma cells that exhibit a catecholaminergic phenotype and express nicotinic receptors. Cells were pre-incubated for 24 h in mainstream-cigarette smoke solutions (0.06, 0.2, or 0.6 cigarette puffs/ml) made from University of Kentucky 1R4F research brand cigarettes, followed by the addition of 6-OHDA for another 24-48 h. The 0.2, but not 0.06, puffs/ml dose, significantly protected against 6-OHDA-induced toxicity in SH-SY5Y cells. This dose yielded final nicotine concentrations of approximately 5 x 10(-7) M, which is similar to plasma smoking levels. Although the 0.6 puffs/ml dose caused significant toxicity on its own, it also appeared to protect against 6-OHDA-induced damage. We next tested the effect of nicotine, as well as its metabolite cotinine. These agents protected against the toxic effects of 6-OHDA in SH-SY5Y cells at concentrations ranging from 10(-7) to 10(-5) M. These combined results support the idea that nicotine is one of the components in cigarette smoke that has a protective effect against neurotoxic insults. These data suggest that nicotine may be of potential therapeutic value for Parkinson's disease.

Pre-synaptic dopaminergic compensation after moderate nigrostriatal damage in non-human primates.

Despite a dramatic loss of nigrostriatal dopaminergic neurons in Parkinson's disease, clinical symptoms only arise with 70-80% reduction of striatal dopamine. The mechanisms responsible for this functional compensation are currently under debate. Although initial studies showed an enhanced pre-synaptic dopaminergic function with nigrostriatal degeneration, more recent work suggests that functional compensation is not dopamine-mediated. To address this issue, we used cyclic voltammetry to directly measure endogenous dopamine release from striatal slices of control monkeys and animals with a moderate or severe MPTP-induced dopaminergic lesion. The moderately lesioned monkeys were asymptomatic, while the severely lesioned animals were parkinsonian. In monkeys with a moderate lesion, a 300% increase was obtained in endogenous striatal dopamine release. In contrast, in striatal slices from severely lesioned animals, a small % of evoked dopamine signals were similar in amplitude to control while the greater majority were undetectable. These findings suggest that pre-synaptic dopaminergic compensation develops in residual dopaminergic terminals with moderate lesioning, but that this response is lost with severe nigrostriatal damage. Such an interpretation is supported by the results of dopamine turnover studies. This enhanced pre-synaptic dopaminergic activity may be important in maintaining normal motor function during the initial stages of Parkinson's disease.

Chronic neonatal nicotine increases anxiety but does not impair cognition in adult rats.

Developmental nicotine exposure has been implicated in the association between maternal smoking and adverse outcomes in offspring. The 3rd trimester of human pregnancy is equivalent to the 1st postnatal week in rodents; both are periods of active brain growth during which nicotinic acetylcholine receptors are transiently upregulated. Chronic neonatal nicotine (CNN; 6 mg/kg/day) from postnatal Days 1 to 7 was given orally to rat pups to evaluate long-term behavioral effects. Males and females were tested as adolescents or as young adults. CNN significantly decreased center time, ambulatory behavior, and rearing in the open-field test and decreased the number of entrances and time spent in the open arm of the elevated plus-maze in both sexes and ages. CNN did not change performance in the T maze or in the water maze in adult males or females. Motor coordination was not altered. In summary, CNN had long-term effects on anxiety-like behavior but did not affect spatial learning and memory functions. This finding is particularly important when evaluating the benefits of nicotine-replacement therapies during human pregnancies, which may improve smoking cessation rates but could result in long-term behavioral consequences.

Effects of paraformaldehyde fixation on nicotinic acetylcholine receptor binding in adult and developing rat brain sections.

In vitro receptor autoradiography requires unfixed tissue sections, but incubation and washing procedures often result in substantial tissue damage in sections from developing brain, hindering quantitative and qualitative analysis. Formaldehyde fixation greatly preserves morphology. However, fixation can interfere with pharmacological properties of receptors, increase in non-specific background labeling, or even destroy ligand binding sites. Two mild fixation protocols, 0.2% paraformaldehyde (pFA) and pFA vapor fixation, were compared for their ability to improve tissue morphology in postnatal day 7 (P7) brain slices and maintain binding of [125I]-epibatidine and [125I]-alpha-bungarotoxin to heteromeric and homomeric nicotinic acetylcholine receptors, respectively. Fixation greatly improved the ability of P7 brain slices to withstand incubation and washing procedures during binding, resulting in minimal or no loss of tissue after prior 0.2% pFA or vapor fixation, respectively. In adults, distribution pattern of [125I]-epibatidine was identical in fixed and unfixed slices, with no difference in total and non-specific labeling. Distribution of [125I]-alphaBTX labeling was similarly unaffected by 0.2% pFA fixation, but vapor fixation increased total and non-specific binding signal. Thus, mild fixations greatly improve tissue quality during receptor binding procedures and can preserve pharmacological properties of nicotinic acetylcholine receptors. However, different receptors or ligands might exhibit differential sensitivity to fixation protocols.

Chronic nicotine induces growth retardation in neonatal rat pups.

In the United State, 20% of pregnant women smoke. One of the most consistent adverse outcomes is reduced birth weight in the off-spring. Animal studies using chronic nicotine, the major psychoactive tobacco ingredient, have shown conflicting results, questioning the role of nicotine in growth retardation. To evaluate the direct effects of nicotine during a period equivalent to the human third trimester, we developed an oral gastric intubation model using neonatal rat pups. Nicotine (6 mg/kg/day) was dissolve in milk-formula and delivered during three feedings daily from postnatal day (P)1 to P7. Nicotine immediately and significantly [P<0.05] decreased weight gain per day (WGD) by 13.5% (+/-) 1 day after onset of treatment in both genders and throughout the treatment period. This resulted in significantly lower body weight at P4 and P5 in male and female pups, respectively. After nicotine withdrawal, WGD returned to control level within 1 day, whereas total body weight recovered by P18. There were no long-term consequences on body weight or growth pattern in either gender. The nicotinic acetylcholine receptor (nAChR) antagonist dihydro-beta-erythroidine (DHbetaE) reversed nicotine's effects on WGD suggesting an involvement of heteromeric alpha4beta2, whereas methyllycaconitine (MLA) an antagonist for the homomeric alpha7-type receptor was ineffective. The immediate decrease of growth in neonatal pups suggests that nicotine's effect on birth weight results from direct anorexic rather then indirect effects due to placental dysfunction or increased fetal hypoxia. The postnatal oral gastric intubation model seems to accurately reflect the direct effects of nicotine in neonates.