Lynn Wecker

Chronic Nicotine Administration Differentially Affects Neurotransmitter Release from Rat Striatal Slices

Abstract: The objective of these experiments was to determine whether the chronic administration of nicotine, at a dose regimen that increases the density of nicotine binding sites, alters the nicotine‐induced release of [3H]dopamine ([3H]DA), [3H]norepinephrine ([3H]NE), [3H]serotonin ([3H]5‐HT), or [3H]acetylcholine ([3H]ACh) from rat striatal slices. For these experiments, rats received subcutaneous injections of either saline or nicotine bitartrate [1.76 mg (3.6 µmol)/kg, dissolved in saline] twice daily for 10 days, and neurotransmitter release was measured following preloading of the tissues with [3H]DA, [3H]NE, [3H]5‐HT, or [3H]choline. Chronic nicotine administration did not affect the accumulation of tritium by striatal slices, the basal release of radioactivity, or the 25 mM KCl‐evoked release of neurotransmitter. Superfusion of striatal slices with 1, 10, and 100 µM nicotine increased [3H]DA release in a concentration‐dependent manner, and release from slices from nicotine‐injected animals was significantly (p < 0.05) greater than release from saline‐injected controls; release from the former increased to 132, 191, and 172% of release from the controls following superfusion with 1, 10, and 100 µM nicotine, respectively. Similarly, [3H]5‐HT release increased in a concentration‐related manner following superfusion with nicotine, and release from slices from nicotine‐injected rats was significantly (p < 0.05) greater than that from controls. [3H]5‐HT release from slices from nicotine‐injected rats evoked by superfusion with 1 and 10 µM nicotine increased to 453 and 217%, respectively, of release from slices from saline‐injected animals. The nicotine‐induced release of [3H]NE from striatal slices was also concentration dependent but was unaffected by chronic nicotine administration. [3H]ACh release from striatal slices could not be detected when samples were superfused with nicotine but was measurable when tissues were incubated with nicotine. The release of [3H]ACh from slices from nicotine‐injected rats was significantly (p < 0.05) less than release from controls and decreased to 36, 83, and 77% of control values following incubation with 1, 10, or 100 µM nicotine, respectively. This decreased [3H]ACh release could not be attributed to methodological differences because slices from nicotine‐injected rats incubated with nicotine exhibited an increased [3H]DA release, similar to results from superfusion studies. In addition, it is unlikely that the decreased release of [3H]ACh from striatal slices from nicotine‐injected rats was secondary to increased DA release because [3H]ACh release from slices from hippocampus, which is not tonically inhibited by DA, also decreased significantly (p < 0.05) in response to nicotine; hippocampal slices from nicotine‐injected rats incubated with 1 and 10 µM nicotine decreased to 42 and 70%, respectively, of release from slices from saline‐injected animals. Results indicate that the chronic administration of nicotine increases the ability of nicotine to induce the release of [3H]DA and [3H]5‐HT and decreases the ability of nicotine to evoke the release of [3H]ACh but does not alter the nicotine‐induced release of [3H]NE from brain slices.

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µM nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration‐dependent manner with IC50 values in the 10 nM range following incubation for 24 h and in the 1 nM range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 nM nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first‐order process for both subtypes; half‐lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first‐order for both subtypes; half‐lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor‐mediated neurotransmitter release.

Choline administration: modification of the central actions of atropine.

The anticholinergic agent atropine decreases acetylcholine concentrations and increases high-affinity choline uptake in cortical and hippocampal regions of rat brain. Administration of choline 1 hour before atropine prevents both of these atropine-induced alterations. These findings suggest that alterations in acetylcholine precursor availability may modify the effects of centrally active anticholinergic agents.

Central cholinergic mechanisms underlying adaptation to reduced cholinesterase activity

Abstract Paraoxon (Px), an irreversible cholinesterase (ChE) inhibitor, was administered acutely and chronically to rats in order to elucidate the mechanisms involved in the process of adaptation. Brain levels of acetylcholine (ACh) as well as ChE and cholineacetyltransferase (CAT) activities were investigated to further understand the relationship between chronically reduced ChE levels and neurotransmitter mobilization. In acutely treated animals, cholinergic symptoms were evident 15 min after injection and were accompanied by a 100 per cent increase in total brain ACh and an 83 per cent decrease in ChE activity. In low-dose chronically treated animals, symptoms did not ensue until 3 days of treatment, when total ACh levels reached a maximum of 50 per cent above control and ChE activity was 55 per cent inhibited. Free ACh levels increased in both acutely and chronically treated animals, whereas bound ACh levels increased significantly only in the acutely treated group. These results suggest that (1) cholinergic central nervous system symptoms can be correlated to an increase (50 per cent) in brain ACh, and (2) chronic ChE inhibition may lead to alterations in the pre-synaptic mobilization and storage of ACh.

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

Abstract: The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague‐Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c, twice daily for 10 days), and total cytochrome P450 content, the activity of N ADPH‐cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine‐injected rats: the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH‐cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O‐dealkylase (BROD) and pentoxyresorufin O‐dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213‐227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH‐cytochrome c reductase, BROD, and PROD, and levels of P4502B1 /2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.

A randomized trial of varenicline (chantix) for the treatment of spinocerebellar ataxia type 3.

Objective: The objective of this double-blind, placebo-controlled, randomized study was to evaluate the efficacy of varenicline (Chantix), a partial agonist at α4β2 neuronal nicotinic acetylcholine receptors used for smoking cessation, in patients with spinocerebellar ataxia (SCA) 3. Methods: Patients with genetically confirmed SCA3 were randomly assigned to receive either varenicline (4 weeks for titration and 4 weeks at a dose of 1 mg twice daily) or placebo. Outcome measures included changes in the Scale for the Rating and Assessment of Ataxia (SARA) scores at endpoint (8 weeks) compared with baseline, a timed 25-foot walk and 9-hole peg test, measurements of mood and anxiety, and adverse events. Results: Twenty patients with SCA3 (mean age = 51 ± 10.98 years; mean disease duration = 14 ± 9.82 years; mean SARA score = 16.13 ± 4.67) were enrolled in the study, and data on 18 patients were analyzed in period I. The most common side effect associated with varenicline was nausea. Improvements were noted in the SARA subsections for gait (p = 0.04), stance (p = 0.03), rapid alternating movements (p = 0.003), and timed 25-foot walk (p = 0.05) and Beck Depression Inventory scores (p = 0.03) in patients taking varenicline compared with those taking placebo at endpoint, with a trend toward improvement in the SARA total score (p = 0.06) in the varenicline group. Conclusions: In this controlled study, varenicline significantly improved axial symptoms and rapid alternating movements in patients with SCA3 as measured by SARA subscores and was fairly well tolerated. Classification of evidence: This study provides Class II evidence that varenicline improved the axial functions of gait, stance, and timed 25-foot walk but did not improve appendicular function, except for rapid alternating movements, in adult patients with genetically confirmed SCA3.

RELATIONSHIP BETWEEN CHOLINE AVAILABILITY AND ACETYLCHOLINE SYNTHESIS IN DISCRETE REGIONS OF RAT BRAIN

Abstract— The relationship between choline availability and the synthesis of acetylcholine in discrete brain regions was studied in animals treated with the organophosphorus cholinesterase inhibitor paraoxon. Administration of paraoxon (0.23 mg/kg) inhibited acetylcholinesterase activity by approx 90% in the striatum, hippocampus and cerebral cortex and increased acetylcholine levels to 149%, 124% and 152% of control values, respectively. Free choline levels were unaltered by paraoxon in the hippocampus and cerebral cortex, but were significantly decreased in the striatum to 74% of control. When animals were injected with choline chloride (60 mg/kg), 60 min prior to the administration of paraoxon, the paraoxon‐induced choline depletion in the striatum was prevented and the paraoxon‐induced acetylcholine increase was potentiated from 149% to 177% of control values. Choline pretreatment had no significant effect in either the hippocampus or cerebral cortex, brain regions that did not exhibit a decrease in free choline levels after paraoxon administration. Results indicate that choline administration, which had no significant effect on acetylcholine levels by itself, increased acetylcholine synthesis in the striatum in the presence of acetylcholinesterase inhibition. However, this effect was not apparent in either the hippocampus or the cerebral cortex at similar levels of enzyme inhibition. It appears that choline generated from the hydrolysis of acetylcholine may play a significant role in the regulation of neurotransmitter synthesis in the striatum, but not in the other brain areas studied. The evidence supports the concept that the regulatory mechanisms controlling the synthesis of acetylcholine in striatal interneurons may differ from those in other brain regions.

Repeated ethanol exposure during adolescence alters the developmental trajectory of dopaminergic output from the nucleus accumbens septi

Individuals who begin using alcohol prior to 14 years of age are 4 times more likely to progress to addiction than those who do not initiate use until 21 years of age. The nucleus accumbens septi undergoes dramatic developmental transitions during the adolescent period, and dopaminergic activity within this region has been identified as a central neurochemical mediator of drug reward, addiction and dependence. Thus, alcohol‐induced neurochemical alterations in dopaminergic activity within this brain region likely mediate the heightened vulnerability to addiction observed in adolescent alcohol users. To investigate this idea, Sprague–Dawley rats were exposed to intraperitoneal injections of either saline or ethanol (0.5, 1.0 or 2.0 g/kg) twice daily over four days beginning on postnatal day 21, 31, 41 or 56. Cannulas were implanted toward the nucleus accumbens septi, subsequent in vivo microdialysis was used to collect samples, and both basal and ethanol‐stimulated dopamine overflow was measured using high performance liquid chromatography with electrochemical detection. A developmental transition in basal levels of dopamine in the nucleus accumbens septi was apparent with peak levels at postnatal day 45. An ethanol challenge produced unique responses across ages, with greater peak effects relative to baseline in younger animals (postnatal day 25 and 35). Following repeated exposure to ethanol, a significant increase in basal dopamine was apparent for all ages, and when these animals were challenged with ethanol, peak effects relative to baseline were decreased in younger animals, but unchanged in older animals (postnatal day 45 and 60). Results indicate that there is a key developmental transition in the ability of rats to adapt to the effects of repeated ethanol exposure, which occurs between postnatal day 35 and 45. This alteration may explain the increased addiction vulnerability observed in individuals who initiate alcohol use during early adolescence.

Paraoxon-induced myopathy: Muscle specificity and acetylcholine involvement

Abstract Treatment of rats with Paraoxon, an organophosphorous cholinesterase (ChE) inhibitor, produces a progressive myopathy in skeletal muscle. The earliest stage of lesion development is characterized histologically by heavy trichrome staining of central nuclei with splitting fibers. This progresses to enlargement of central nuclei, more intense splitting, and breakdown of fiber architecture. Final stages are characterized by total fiber necrosis and phagocytosis. The myopathy is more severe in the diaphragm, with 60% tonic, slow fibers, than it is in the soleus, with a majority of intermediate fibers, or the gastrocnemius, a fast white muscle. Maximal inhibition of ChE activity in the muscles occurred during the first 30 min after the initial Paraoxon treatment. Within 24 hr, there was significant recovery of enzyme activity. Evidence suggests there may be a critical period of inhibition of ChE activity to initiate the myopathic process. Results are discussed in terms of a possible nerve-mediated muscle necrosis.

Concomitant increases in the levels of choline and free fatty acids in rat brain: evidence supporting the seizure-induced hydrolysis of phosphatidylcholine

Abstract: The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 μg/kg, s.c), at a dose that produced toxic effects, increased the levels of both free fatty acids (175–250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman‐induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman‐induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant‐mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.