Alzheimer's pathology is associated with altered cognition, brain volume, and plasma biomarker patterns in traumatic encephalopathy syndrome.

BACKGROUND: Traumatic encephalopathy syndrome (TES) is a clinical phenotype sensitive but non-specific to underlying chronic traumatic encephalopathy (CTE) neuropathology. However, cognitive symptoms of TES overlap with Alzheimer's disease (AD), and features of AD pathology like beta-amyloid (Aß) plaques often co-occur with CTE, making clinical-to-pathological conclusions of TES diagnoses challenging. We investigated how Alzheimer's neuropathological changes associated with cognition, brain volume, and plasma biomarkers in patients with repetitive head impacts (RHI)/TES, clinical AD, or typically aging controls. METHODS: We studied 154 participants including 33 with RHI/TES (age 61.5 ± 11.5, 100% male, 11/33 Aß[ +]), 62 with AD and no known prior RHI (age 67.1 ± 10.2, 48% male, 62/62 Aß[ +]), and 59 healthy controls without RHI (HC; age 73.0 ± 6.2, 40% male, 0/59 Aß[ +]). Patients completed neuropsychological testing (memory, executive functioning, language, visuospatial) and structural MRI (voxel-based morphometry analysis), and provided plasma samples analyzed for GFAP, NfL, IL-6, IFN-γ, and YKL-40. For cognition and plasma biomarkers, patients with RHI/TES were stratified as Aß[ +] or Aß[ -] and compared to each other plus the AD and HC groups (ANCOVA adjusting for age and sex). Differences with at least a medium effect size (Cohen's d > 0.50) were interpreted as potentially meaningful. RESULTS: Cognitively, within the TES group, Aß[ +] RHI/TES performed worse than Aß[-] RHI/TES on visuospatial (p = .04, d = 0.86) and memory testing (p = .07, d = 0.74). Comparing voxel-wise brain volume, both Aß[ +] and Aß[ -] RHI/TES had lower medial and anterior temporal lobe volume than HC and did not significantly differ from AD. Comparing plasma biomarkers, Aß[ +] RHI/TES had higher plasma GFAP than HC (p = .01, d = 0.88) and did not significantly differ from AD. Conversely, Aß[ -] RHI/TES had higher NfL than HC (p = .004, d = 0.93) and higher IL-6 than all other groups (p's ≤ .004, d's > 1.0). CONCLUSIONS: Presence of Alzheimer's pathology in patients with RHI/TES is associated with altered cognitive and biomarker profiles. Patients with RHI/TES and positive Aß-PET have cognitive and plasma biomarker changes that are more like patients with AD than patients with Aß[ -] RHI/TES. Measuring well-validated Alzheimer's biomarkers in patients with RHI/TES could improve interpretation of research findings and heighten precision in clinical management.

Plasma Tau and Neurofilament Light in Frontotemporal Lobar Degeneration and Alzheimer Disease.

OBJECTIVE: To test the hypothesis that plasma total tau (t-tau) and neurofilament light chain (NfL) concentrations may have a differential role in the study of frontotemporal lobar degeneration syndromes (FTLD-S) and clinically diagnosed Alzheimer disease syndromes (AD-S), we determined their diagnostic and prognostic value in FTLD-S and AD-S and their sensitivity to pathologic diagnoses. METHODS: We measured plasma t-tau and NfL with the Simoa platform in 265 participants: 167 FTLD-S, 43 AD-S, and 55 healthy controls (HC), including 82 pathology-proven cases (50 FTLD-tau, 18 FTLD-TDP, 2 FTLD-FUS, and 12 AD) and 98 participants with amyloid PET. We compared cross-sectional and longitudinal biomarker concentrations between groups, their correlation with clinical measures of disease severity, progression, and survival, and cortical thickness. RESULTS: Plasma NfL, but not plasma t-tau, discriminated FTLD-S from HC and AD-S from HC. Both plasma NfL and t-tau were poor discriminators between FLTD-S and AD-S. In pathology-confirmed cases, plasma NfL was higher in FTLD than AD and in FTLD-TDP compared to FTLD-tau, after accounting for age and disease severity. Plasma NfL, but not plasma t-tau, predicted clinical decline and survival and correlated with regional cortical thickness in both FTLD-S and AD-S. The combination of plasma NfL with plasma t-tau did not outperform plasma NfL alone. CONCLUSION: Plasma NfL is superior to plasma t-tau for the diagnosis and prediction of clinical progression of FTLD-S and AD-S. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that plasma NfL has superior diagnostic and prognostic performance vs plasma t-tau in FTLD and AD.

Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Regulation of α4ß2α5 nicotinic acetylcholinergic receptors in rat cerebral cortex in early and late adolescence: Sex differences in response to chronic nicotine.

Chronic nicotine administration in animals, and smoking in humans, causes up-regulation of α4ß2* neuronal nicotinic receptors (nAChRs), which has been hypothesized to contribute to the addictive actions of nicotine. We used a rat model to test whether such up-regulatory effects differ in adolescents versus adults, and in males versus females. Following chronic treatment with nicotine or saline via subcutaneous osmotic minipumps, we measured α4ß2 and α4ß2α5 nAChRs in cerebral cortex using [3H]epibatidine to label assembled nAChRs, and selective antibodies to measure the individual subunits via immunoprecipitation. For the first time, we provide a detailed characterization of the response of both α4ß2 and α4ß2α5 nAChRs in female adolescent rat cerebral cortex. We found differences in nicotine-induced up-regulation between males and females in early adolescence that are absent in both late adolescence and adulthood. Males showed significant up-regulation at PN28 which was absent in age-matched females. These results demonstrate sex differences in the susceptibility of α4ß2* nAChRs to the effects of chronic nicotine exposure in the cerebral cortex based on age.

Frontotemporal dementia.

Frontotemporal dementia (FTD) encompasses several clinical syndromes that involve a progressive change in behavior and/or language; it is more common than Alzheimer's disease in early-onset dementia under the age of 60 years. In the behavioral variant of FTD (bvFTD) patients have social and emotional changes with prominent disinhibition, apathy, lack of empathy, changes in diet, and repetitive behaviors. Motor neuron disease or parkinsonism are seen in association with bvFTD. Frontal and/or temporal atrophy are often seen on structural brain imaging. Several pathological entities can cause bvFTD, and they are defined by the presence of specific abnormal protein accumulations. Most cases are characterized by accumulation of the proteins tau, TAR-DNA-binding protein-43 (TDP-43), and fused in sarcoma (FUS). Though most cases are sporadic, a variety of genes have been identified that cause autosomal dominant forms of FTD. The most common mutations occur in C9ORF72, MAPT, and GRN. No disease-modifying treatments have been currently identified, but limited evidence supports the use of antidepressants or neuroleptics in symptomatic management, and education regarding nonpharmacologic methods may be helpful to caregivers.

A randomized clinical trial of trans-dermal nicotine replacement in pregnant African-American smokers.

We compared acceptability, adherence and efficacy of trans-dermal nicotine patches and cognitive behavioral therapy (Group 1) to cognitive behavioral therapy alone (Group 2) in minority pregnant smokers. This is a randomized controlled trial. 52 women were recruited during pregnancy with a mean gestational age 18.5 ± 5.0 weeks and followed through delivery. Randomization was by site and initial cotinine levels. Interventionists and interviewers were blinded to group assignment. Two different nicotine replacement therapy dosing regiments were administered according to the baseline salivary cotinine level. A process evaluation model summarized patient adherence. The main outcome measure was self-report of cessation since last visit, confirmed by exhaled carbon monoxide. Analyses of categorical and continuous measures were conducted as well as linear trend tests of salivary cotinine levels. Women lost to follow-up were considered treatment failures. Participants were on average 27.5 ± 5.4 years old, 81 % were single, 69 % unemployed and 96 % were Medicaid eligible. A process evaluation indicated patients in both groups were adherent to scheduled program procedures through Visit 4, but not for Visits 5 and 6. Confirmed quit rates were: at visit 3, 23 (Group 1) and 0 % (Group 2) (p = 0.02); at visits 4 and 5, no difference; at visit 6, 19 (Group 1) and 0 % (Group 2) (p = 0.05). Group 1 delivered infants with a mean gestational age of 39.4 weeks versus 38.4 weeks in Group 2 (p = 0.02). 73 % (52/71) of the eligible smokers agreed to participate and 65 % (17/26) of Group 1 completed the protocol (i.e. attended 6 visits). A comparison of Group 1 and 2 quit rates confirmed a non-significant difference.

Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain.

Chronic nicotine administration increases α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4ß2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ∼150 times its affinity for α4ß2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4ß2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons.

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [¹²5I]epibatidine ([¹²5I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, ß2 and α5 subunits, but α2, α3, α6 and ß4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the ß2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.

Dipstick tests for secondhand smoke exposure.

INTRODUCTION: We tested TobacAlert and NicAlert immunochromatographic strips for use as indicators of secondhand smoke (SHS) exposure. METHODS: Urine samples collected from SHS-unexposed volunteers were spiked with cotinine to concentrations of 5, 8, 13, and 23 ng cotinine/ml urine. One sample was not spiked and used as a control. According to manufacturer's instructions, 45 NicAlert and 45 TobacAlert dipsticks were prepared. The exercise was repeated once. Cotinine levels in urine samples were measured using liquid chromatography/mass spectrometry (LC/MS). STATA was used for statistical analyses. RESULTS: Ninety NicAlert and 90 TobacAlert dipsticks were tested. Each strip was read by 3 different readers, for 270 NicAlert and 270 TobacAlert readings; 98/270 (36%) NicAlert and 104/270 (39%) TobacAlert readings agreed with the readings predicted by LC/MS-determined cotinine levels. Spearman's rho for the NicAlert strips was .13 and for the TobacAlert strips .23. Both were statistically significant. Using a dichotomous scheme to interpret any strip reading >or=1 as "positive," indicating SHS exposure, NicAlert strips were 94% sensitive and 31% specific, while TobacAlert strips were 89% sensitive and 60% specific. DISCUSSION: NicAlert and TobacAlert strips performed poorly at low cotinine levels. While the strips could be used to prescreen samples prior to more accurate testing, their use in the clinical or research setting to indicate SHS exposure should be restricted to carefully selected scenarios.

Evaluation of the immediate impact of the Washington, D.C., smoke-free indoor air policy on bar employee environmental tobacco smoke exposure.

OBJECTIVE: On January 2, 2007, thw Washington, D. C., City Council banned smoking in restaurants and bars. We sought to determine the immediate impact of the ban on cotinine-confirmed environmental tobacco smoke (ETS) levels and respiratory symptom reports of a random sample of bar employees. METHODS: We conducted an assessment of 66 employees from 41 randomly selected bars in December 2006, a month before the ban went into effect. After analyses of baseline data, 52 employees were eligible and 49 of them (94%) had a post-ban assessment in February 2007. Three participants were excluded due to high cotinine levels at the post-ban assessment, yielding a final sample size of 46 bar employees. ETS exposure levels were documented using saliva cotinine analyses by tandem liquid chromatography and mass spectrometry. Employee respiratory and sensory symptoms reports were assessed by a standardized, validated form: the International Union Against Tuberculosis and Lung Disease Bronchial Symptoms Questionnaire. Employee ETS exposure reports at work were eliminated after the ban. RESULTS: Sensory symptoms reports (at < or = 4 weeks) declined significantly by 70% to 100% (p = 0.0016); respiratory symptoms results were inconclusive due to a lack of data. Saliva cotinine medians declined significantly by 70% (p < 0.0001), from a pre-ban mean of 2.11 nanograms per millileter (ng/mL) to a post-ban mean of 0.29 ng/mL, confirming reports of no ETS exposure at work. CONCLUSION: We concluded that the indoor air law was effective, eliminating employee ETS exposure reports, dramatically reducing their cotinine levels, and almost eliminating reports of sensory symptoms.

Prenatal exposure of rats to nicotine causes persistent alterations of nicotinic cholinergic receptors.

We examined for immediate and persistent changes in nAChRs in cerebral cortex, thalamus and striatum of male rats caused by prenatal exposure to nicotine from gestational day 3 to postnatal day 10 (PN10), and how such exposure affected the responses of adolescents to subsequent nicotine challenge. Receptor numbers were assessed by [(3)H]epibatidine binding and receptor function was measured by acetylcholine-stimulated (86)Rb efflux (cerebral cortex and thalamus) and nicotine-stimulated dopamine release (striatum). Immediate effects of prenatal nicotine, assessed in PN10 animals, were not detected for any parameter. A subsequent 14 day nicotine exposure in adolescence revealed persistent changes caused by prenatal nicotine exposure. Nicotine exposure in adolescents caused up-regulation of binding in all three regions; however, this up-regulation was lost in thalamus from animals prenatally exposed to nicotine. Nicotine exposure in adolescents caused decreased nicotine-stimulated dopamine release in striatum; this effect was lost in animals prenatally exposed to nicotine. Comparison of parameters in PN10 and PN42 rats revealed developmental changes in the CNS cholinergic system. In thalamus, binding increased with age, as did the proportion of (86)Rb efflux with high sensitivity to acetylcholine. In cortex, binding also increased with age, but there was no change in total (86)Rb efflux, and the proportion of high to low sensitivity efflux declined with age. Nicotine-stimulated striatal dopamine release (both total and alpha-conotoxin MII-resistant release) increased with age in naïve animals, but not in those prenatally exposed to nicotine. These findings demonstrate that prenatal exposure to nicotine causes alterations in nAChRs and in their regulation by nicotine that persist into adolescence. These changes may play a role in the increased risk for nicotine addiction observed in adolescent offspring of smoking mothers.

Effects of nicotine on sensorimotor gating impairment induced by long-term treatment with neurotoxic NMDA antagonism.

In order to develop a model of persistent sensorimotor gating that did not require acute NMDA (N-methyl-D-aspartate) receptor blockade, adult female Sprague-Dawley rats were pre-treated with N-methyl-scopolamine (1 mg/kg s.c.), then administered MK-801 (dizocilpine, 5 mg/kg i.p.) along with two separate doses (5 mg/kg) of pilocarpine. The drug regimen was repeated four and eight days later. Controls received saline in lieu of any drug. Ten days after the last neurotoxic treatment, rats had a significant impairment (reduction) in pre-pulse inhibition (PPI). Each treatment group (neurotoxic treated and control) was then divided into two groups for treatment with saline or 0.5 mg/kg nicotine, administered s.c. twice daily from days 10 to 23. The rats were tested for sensorimotor gating on days 17 and 22 shortly after the morning nicotine administration. Nicotine did not affect the PPI in control animals. On day 17, PPI impairment was sustained in neurotoxically treated rats, regardless of saline or nicotine treatment. On day 22, however, the effect of neurotoxic treatment on PPI was totally absent in saline treated rats, whereas in nicotine treated rats, PPI impairment was still evident. Combination of nicotine and neurotoxic treatment also caused an up-regulation of high affinity nicotinic receptors in the cortex and the thalamus and apparent normalization of low affinity nicotinic receptors in the hippocampus. The findings indicate that muscarinic activation, in conjunction with neurotoxic NMDA receptor antagonism, produces relatively long-term impairment in auditory gating, a result relevant to modeling clinical observations of schizophrenia-associated symptoms. Contrary to expectation, nicotine administration in this model resulted in further impairment rather than amelioration of PPI. The results suggest a sustainable model of PPI impairment and possible role of nicotinic receptors in selective brain regions in this behavior.

Adult and periadolescent rats differ in expression of nicotinic cholinergic receptor subtypes and in the response of these subtypes to chronic nicotine exposure.

Adolescence is a time of significant brain development, and exposure to nicotine during this period is associated with higher subsequent rates of dependence. Chronic nicotine exposure alters expression of nicotinic acetylcholine receptors (nAChRs), changing the pattern of nicotine responsiveness. We used quantitative autoradiography to measure three major subtypes of nAChRs after chronic nicotine exposure by osmotic minipump in adult and periadolescent rats. Comparison of control animals at the two different ages revealed that periadolescents express consistently greater numbers of alpha4beta2* nAChRs compared to the same brain regions of adults. Similar but less pronounced increases in alpha7 nAChRs were found in control periadolescent rats compared to adults. Binding of [(125)I]alpha-conotoxin MII (largely to alpha6* nAChRs) did not systematically differ between adults and periadolescents. The response to chronic nicotine exposure also differed by age. Up-regulation of alpha4beta2* nAChRs was prominent and widespread in adult animals; in periadolescents, alpha4beta2* up-regulation also occurred, but in fewer regions and to a lesser extent. A similar pattern of response was seen with alpha7 receptors: adults were more responsive than periadolescents to nicotine-induced up-regulation. In adult animals, chronic nicotine exposure did not cause up-regulation of alpha6* nAChRs; binding was down-regulated in three regions. Unlike the other subtypes, the response of alpha6* nAChRs to chronic nicotine was greater in periadolescents, with more regions showing greater down-regulation compared to adults. These differences in receptor expression and regulation between age groups are likely to be important given the unique vulnerability of adolescents to nicotine-induced behavioral changes and susceptibility to drug abuse.

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo.

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the alpha5 subunit in the adult rat brain. Among the regions of brain we surveyed, the alpha5 subunit is associated in approximately 37% of the nAChRs in the hippocampus, approximately 24% of the nAChRs in striatum, and 11-16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the alpha5 subunit is associated with alpha4beta2* nAChRs exclusively. Importantly, in contrast to alpha4beta2 nAChRs, which are increased by 37-85% after chronic administration of nicotine, the alpha4beta2alpha5 receptors are not increased by nicotine treatment. These data thus indicate that the alpha4beta2alpha5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo, which suggests an important regulatory role for the alpha5 subunit. To the extent that nicotine-induced up-regulation of alpha4beta2 nAChRs is involved in nicotine addiction, the resistance of the alpha4beta2alpha5 subtype to up-regulation may have important implications for nicotine addiction.

Chronic nicotine differentially regulates alpha6- and beta3-containing nicotinic cholinergic receptors in rat brain.

We investigated the effects of chronic nicotine on alpha6- and beta3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [(125)I]alpha-conotoxin MII to selectively label alpha6(*) nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [(3)H]epibatidine in these two regions showed that chronic nicotine increased alpha4- and beta2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in alpha6-containing nAChRs in striatum but no change in superior colliculus. No changes in beta3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of alpha6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to alpha-conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by alpha-conotoxin MII was decreased by 56%. These findings indicate that the alpha6(*) nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a beta3 subunit with alpha6 may serve to inhibit nicotine-induced down-regulation of these receptors.

An autoradiographic analysis of [125I]alpha-bungarotoxin binding in rat brain after chronic nicotine exposure.

Chronic exposure to nicotine has been shown to increase binding to high affinity nicotinic cholinergic receptors in rat brain, but the effect of this treatment on the low affinity alpha7 nicotinic receptors has been less well characterized. Male Sprague-Dawley rats were treated with saline or nicotine (6 mg/kg/day, by osmotic minipump) for 14 days. Frozen brain sections were then prepared and processed for quantitative autoradiography using [(125)I]alpha-bungarotoxin to measure the effect of this treatment on low affinity nicotinic receptors. Nicotine exposure increased [(125)I]alpha-bungarotoxin binding in 26 of 52 brain regions analyzed; increases ranged from 10 to 70% over saline controls. Increases were seen in all areas of the brain, but were more prominent in forebrain areas, and especially in cerebral cortex. These data demonstrate that low affinity alpha7 nicotinic receptors are also up-regulated by chronic nicotine. This phenomenon may be relevant to the heavy use of tobacco products in diseases like schizophrenia, and needs to be considered in the design of pharmaceuticals directed at this receptor system.

Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment.

Chronic nicotine exposure up-regulates neuronal nicotinic receptors, but the functional consequences for these receptors is less well understood. Following 2 weeks of nicotine or saline treatment by osmotic minipump, the functional activity of nicotinic receptors was measured by concentration-response curves for epibatidine-stimulated (86)Rb efflux. Nicotine-treated animals had a significantly higher maximal efflux in cerebral cortex and superior colliculus, but not in thalamus or interpeduncular nucleus plus medial habenula. This increase was confirmed in a separate experiment with stimulation by single concentrations of epibatidine (cortex, superior colliculus) or nicotine (cortex only). Chronic nicotine did not alter (86)Rb efflux stimulated by cytisine, an alpha3beta4-selective agonist, or by potassium chloride, in any region. Short-term (16 h) nicotine exposure caused no changes in either (86)Rb efflux or receptor binding measured with [(3)H]epibatidine. Binding was significantly increased after 2 weeks nicotine exposure in cortex, superior colliculus and thalamus, but not in interpeduncular nucleus plus medial habenula. The increases in epibatidine-stimulated (86)Rb efflux in the four regions tested was linearly correlated with the increases in [(3)H]epibatidine binding in these regions (R(2) = 0.91), suggesting that rat brain receptors up-regulated by chronic nicotine are active. These results have important consequences for understanding nicotinic receptor neurobiology in smokers and users of nicotine replacement therapy.

Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography.

Subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) are differentially sensitive to up-regulation by chronic nicotine exposure in vitro. To determine whether this occurs in animals, rats were implanted with minipumps containing saline +/- nicotine (6.0 mg/kg/rat/day) for 14 days. Autoradiography with [125I]epibatidine using 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380) or cytisine as selective competitors allowed quantitative measurement in 33 regions of 3 families of nAChR binding, with properties of alpha4beta2, alpha3beta4, and alpha3/alpha6beta2. Chronic nicotine exposure caused increases of 20 to 100% for alpha4beta2-like binding in most regions surveyed. However, binding to this subtype was not increased in some regions, including habenulopeduncular structures, certain thalamic nuclei, and several brainstem regions. In 9 of 33 regions, including catecholaminergic areas and visual structures, alpha3/alpha6beta2-like binding represented >10% of total binding. Binding to this subtype was up-regulated by nicotine in only two of these nine regions: the nucleus accumbens and superior colliculus. alpha3beta4-Like binding represented >10% of total in 15 of the 33 regions surveyed. Binding to this subtype was increased by nicotine in only 1 of these 15 regions, and actually decreased in subiculum and cerebellum. These studies yielded two principal findings. First, chronic nicotine exposure selectively up-regulates alpha4beta2-like binding, with relatively little effect on alpha3/alpha6beta2-like and alpha3beta4-like binding in vivo. Second, up-regulation by chronic nicotine exposure shows considerable regional variation. Differential subtype sensitivity to chronic nicotine exposure may contribute to altered pharmacological response in individuals who smoke or use nicotine replacement therapy.

Role of intracellular calcium stores in cell death from oxygen-glucose deprivation in a neuronal cell line.

To determine the role of calcium homeostasis in ischemic neuronal death, the authors used an in vitro model of oxygen-glucose deprivation in neuronal cell lines. Exposure of human neuroblastoma SH-SY5Y cells to 10-to 16-hour oxygen-glucose deprivation decreased viability to 50% or less, and longer exposure times killed almost all cells. The death following 10-to 16-hour oxygen-glucose deprivation was not manifested until 24 to 72 hours after exposure. Deprivation of both glucose and oxygen together was required for expression of toxicity at these exposure times. Dantrolene, which blocks the release of endoplasmic reticulum Ca2+ stores, partially protected SH-SY5Y cells from oxygen-glucose deprivation toxicity. The addition of dantrolene during the deprivation phase alone produced the maximal drug effect; no further protection was obtained by continued drug exposure during the recovery phase. Prevention of Ca2+ influx by chelation or channel blockade or the chelation of cytosolic Ca2+ did not inhibit oxygen-glucose deprivation toxicity. In contrast, increasing extracellular Ca2+ or stimulating Ca2+ influx did inhibit toxicity. Calcium measurements with fura-2 acetoxymethylester revealed that oxygen-glucose deprivation caused a significant reduction in thapsigargin-releasable endoplasmic reticular stores of Ca2+. These studies suggest that an important component of the neuronal toxicity in cerebral ischemia is due to disruption of calcium homeostasis, particularly to the depletion of intracellular Ca2+ stores.

Depletion of intracellular calcium stores is toxic to SH-SY5Y neuronal cells.

Inhibiting Ca(2+) uptake by the sarcoendoplasmic reticular Ca(2+)-ATPase pump (SERCA) causes release of Ca(2+) from the endoplasmic reticulum (ER), increased cytosolic Ca(2+) ([Ca(2+)](cyt)) and depletion of ER Ca(2+) stores. These studies were designed to test the effects of SERCA inhibition on neuronal viability, using as a model the human neuroblastoma cell line, SH-SY5Y. Continuous exposure to the SERCA inhibitor thapsigargin (TG) decreased SH-SY5Y viability to <30% after 48 h exposure, and produced DNA laddering. Two other SERCA inhibitors, BHQ and cyclopiazonic acid CPA, were similarly toxic, although at 1000-fold higher concentrations. BHQ and CPA toxicity was prevented by removing drug within several hours, whereas TG toxicity was essentially irreversible. All three SERCA inhibitors caused an increase in [Ca(2+)](cyt) that was partially blocked by the ryanodine receptor inhibitors, dantrolene and DHBP. Pretreatment with 40 microM dantrolene gave substantial protection against TG- or BHQ-induced cell death but it did not inhibit death from staurosporine, which does not cause release of ER Ca(2+). DHBP (20-100 microM) also gave partial protection against TG toxicity, as did ruthenium red (2 microM), but not ryanodine (10 microM). Inhibition of capacitative Ca(2+) entry with EGTA or LaCl(3) or low extracellular Ca(2+), or chelation of [Ca(2+)](cyt) with BAPTA-AM, failed to inhibit TG toxicity, although they prevented increases in [Ca(2+)](cyt) caused by TG. Taken together, these data suggest that toxicity caused by SERCA inhibition in SH-SY5Y cells is caused by ER Ca(2+) depletion, which triggers an apparent apoptotic pathway.