(—)-NICOTINE AMELIORATES CORTICOSTERONE’S POTENTIATION OF N-METHYL-D-ASPARTATE RECEPTOR-MEDIATED CORNU AMMONIS 1 TOXICITY
Abstract—Hypercortisolemia, long-term exposure of the brain to high concentrations of stress hormones (i.e. corti- sol), may occur in patients suffering from depression, alco- holism, and other disorders. This has been suggested to produce neuropathological effects, in part, via increased function or sensitivity of N-methyl-D-aspartate (NMDA)-type glutamate receptors. Given that cigarette smoking is highly prevalent in some of these patient groups and nicotine has been shown to reduce toxic consequences of NMDA receptor function, it may be suggested that nicotine intake may atten- uate the neurotoxic effects of hypercortisolemia. To investi- gate this possibility, organotypic hippocampal slice cultures derived from rat were pre-treated with corticosterone (0.001– 1 µM) alone or in combination with selective glucocorticoid receptor antagonists for 72-h prior to a brief (1-h) NMDA exposure (5 µM). Pre-treatment with corticosterone (0.001– 1 µM) alone did not cause hippocampal damage, while NMDA exposure produced significant cellular damage in the cornu ammonis (CA)1 subregion. No significant damage was ob- served in the dentate gyrus or CA3 regions following NMDA exposure. Pre-treatment of cultures with corticosterone (0.1– 1 µM) markedly exacerbated NMDA-induced CA1 and dentate gyrus region damage. This effect in the CA1 region was prevented by co-administration of the glucocorticoid recep- tor antagonist RU486 (>1 µM), but not spironolactone (1– 10 µM), a mineralocorticoid receptor antagonist. In a second series of studies, both acute and pre-exposure of cultures to (—)-nicotine (1–10 µM) significantly reduced NMDA toxicity in the CA1 region. Co-administration of cultures to (—)-nicotine (1–10 µM) with 100 nM corticosterone prevented corticoste- rone’s exacerbation of subsequent CA1 insult. This protec- tive effect of (—)-nicotine was not altered by co-exposure of cultures to 10 µM dihydro-β-erythroidine but was blocked by co-exposure to 100 nM methyllycaconitine, suggesting the involvement of nicotinic acetylcholine receptors possessing the α7* subunit.
The present studies suggest a role for hypercortisolemia in sensitizing the hippocampal NMDA receptor system to pathological activation and indicate that prolonged nicotine exposure attenuates this sensitization. Thus, it is possible that one consequence of heavy smoking in those suffering from hypercortisolemia may be a reduction of neuronal injury and sparing of cellular function. © 2004 IBRO. Published by
Key words: glucocorticoid, excitotoxicity, hippocampus, alpha7, RU486, methyllycaconitine.
Several clinical conditions, including alcohol dependence, mood disorders, Cushing’s syndrome, and Alzheimer’s disease involve long-term dysregulation of the hypotha- lamic–pituitary–adrenal axis, characterized by chronic hy- percortisolemia (Sapolsky and Plotsky, 1990; Adinoff et al., 1991). Though short-term elevations in circulating glu- cocorticoid concentrations clearly impact upon glucose availability and reduce inflammation in response to stres- sors (Tsigos and Chrousos, 2002), long-term exposure of the brain to high concentrations of cortisol may well impact negatively on neuronal function and/or survival. Indeed, individuals with these neuropsychiatric disorders often present impairments of cognitive functioning (Starkman et al., 1992; Bremner et al., 1993; Oscar-Berman, 1994; She- line et al., 1999) and decreases in the volume of multiple brain regions, including the hippocampus (Starkman et al., 1992; Convit et al., 1997; Mann et al., 2001).
There exists a vast animal literature confirming the deleterious effects of chronic glucocorticoid exposure on neuronal function, particularly in the hippocampus. Re- straint stress and exogenous corticosterone exposure de- creased the number of branch points and the length of cornu ammonis (CA) 3 dendrites in rodent and non-human primate hippocampus (Woolley et al., 1990; Watanabe et al., 1992a,b; Magarinos and McEwen, 1995; Magarinos et al., 1996, 1997; Galea et al., 1997). Chronic elevations in plasma glucocorticoids (up to 1 µM) induced by water immersion, restraint stress, social stress, or exogenous exposure also produce neuronal loss in the hippocampus of both species (Uno et al., 1989; Sapolsky et al., 1990; Mizoguchi et al., 1992). Administration of glucocorticoids exacerbated toxicity associated with kainic acid adminis- tration, ischemia, oxidative stress, and the human immu- nodeficiency virus glycoprotein gp120 in rodent hippocam- pus (Sapolsky and Pulsinelli, 1985; Sapolsky, 1985, 1986; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a, b; Brooke et al., 1997). In rat magnocel- lular nucleus basalis, chronic administration of corticoste- rone (310 – 650 nM) potentiated the toxicity of the excito- toxin N-methyl-D-aspartate (NMDA; Abraham et al., 2000). Additionally, pre-treatment with the synthetic glucocorticoid receptor agonist dexamethasone potentiated the cell damage caused by intrastriatal injection of NMDA (Supko and Johnston, 1994). Corticosterone’s negative effects may be related, in part, to increased number or function of NMDA receptors (Weiland et al., 1997), as well as, in- creased levels of excitatory amino acids (Stein-Behrens et al., 1992, 1994), free radical formation (McIntosh and Sapolsky, 1996a, b), and elevated intracellular Ca2+ levels (Kerr et al., 1992; Elliott and Sapolsky, 1993; Karst et al., 1994). As a whole, these data suggest a role for long-term elevations in circulating glucocorticoid concentrations in sensitizing the brain to different insults, particularly those involving activation of excitatory amino acid receptor systems.
Many of the clinical populations described, particularly those with mood disorders or who are alcohol-dependent, demonstrate nicotine dependence at a rate markedly higher than that observed in the general population. In- deed, estimates of the prevalence of alcohol and tobacco co-dependency in Western societies range from approxi- mately 50% (Marks et al., 1997) to nearly 95% (Sobell et al., 1990) while major depression is a significant risk factor for maintenance of cigarette smoking (Breslau et al., 1993; Covey et al., 1998). In contrast, approximately 20% of those in the general United States population smoke heavily (Marks et al., 1997). Clearly, many factors contrib- ute to high rates of cigarette smoking in these patient populations, including the putative antidepressant and an- xiolytic properties of nicotine delivered via tobacco smoke (Hughes et al., 2000). However, little work has examined the potential effects of chronic nicotine delivery via tobacco smoke on neuronal function in these groups as it relates to adverse effects of hypercortisolemia on neuronal function or survival. Previous evidence suggests that nicotine and synthetic nicotinic receptor agonists may reduce the toxic effects of NMDA receptor activation, such as that observed following long-term exposure to high concentrations of glucocorticoids. Indeed, both acute and chronic nicotine (1–10 µM) treatment has been reported to attenuate NMDA receptor-mediated, Ca2+-dependent neuronal death induced by withdrawal from long-term ethanol expo- sure (Prendergast et al., 2000b), NMDA exposure (Marin et al., 1994; Prendergast et al., 2001a,b), and glutamate exposure (Akaike et al., 1994; Shimohama et al., 1996; Kaneko et al., 1997) in several regions of rodent brain. Thus, one consequence of long-term nicotine delivery in those suffering from mood disorders and/or alcohol depen- dence may be the reduced likelihood of glucocorticoid- induced neuropathology. It must be noted, however, that nicotine delivery via cigarettes has been reported to in- crease plasma cortisol levels in human smokers (Pomer- leau and Pomerleau, 1990; Pomerleau et al., 1992), though the implications of this for neuronal viability are uncertain.
The present studies were designed to examine effects of prolonged corticosterone exposure on cell survival alone and in combination with subsequent NMDA-induced insult in rat hippocampal slice cultures. Further, these experiments ex- amined the role of mineralocorticoid and glucocorticoid receptors in mediating the potentially toxic effects of corticoste- rone. Finally, a separate series of experiments were com- pleted to assess the potential neuroprotective effects of acute and prolonged (—)-nicotine treatment on corticosterone- induced neurotoxicity.
EXPERIMENTAL PROCEDURES
Hippocampal slice culture preparation
Preparation of hippocampal explants followed procedures de- scribed by Stoppini and colleagues (1991). Whole brains from 8-day old male Sprague–Dawley rat pups (Harlan, Indianapolis, IN, USA) were aseptically removed and placed into dissection medium (4 °C). Dissecting medium is made of Minimum Essential Medium plus 2 mM L-glutamine, 25 mM HEPES, and 50 µM penicillin/streptomycin solutions. Bilateral hippocampi were dis- sected out and placed into culture medium (4 °C) made of dis- secting medium with the addition of 36 mM glucose, 25% (v/v) Hanks’ balanced salt solution and 25% heat-inactivated horse serum. Using a McIllwain tissue chopper (Mickle Laboratory En- gineering Co. Ltd., Gomshall, UK), each hippocampus was coro- nally sectioned at 200 µm and placed into fresh culture medium. Each unilateral hippocampus yielded approximately 12 slices. Three slices were transferred onto individual Millicell-CM 0.4 µm biopore membrane insert (Millipore Corporation, Bedford, MA, USA) and then placed in 35 mm six-well culture plates containing 1 ml of pre-incubated cell culture medium. Excess medium on top of slices was aspirated to ensure explants remained exposed to the atmosphere of 5% CO2/95% air. Explants were kept at 37 °C in an incubator and were allowed to become attached to mem- brane inserts in culture medium for 5 days prior to the start of experiments. Gibco BRL (Gaithersburg, MD, USA) supplied all culture medium solutions with the exception of heat-inactivated horse serum (Sigma, St. Louis, MO, USA). Care of all animals was carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publications no. 80-23) in an effort to minimize the number of animals used and their suffering. Also, the care and use of, as well as all procedures involving, animals have been approved by the University of Kentucky’s Institutional Animal Care and Use Committee.
NMDA-induced neurotoxicity
At 8 days in vitro, a portion of all cultures (n=15–18/group) was transferred to culture plates containing 1 ml of Ca2+ Locke’s buffer (150 mM NaCl, 5.4 mM KCl, 5 mM NaOH, 2.5 mM CaCl2, 10 mM HEPES, and 36 mM glucose, pH 7.4) to wash culture medium off of cultures. Cultures were then placed in new plates containing 1 ml of Ca2+ Locke’s buffer on the top and bottom alone or with the addition of 5 µM NMDA (Sigma) and 10 µM glycine (ICN Biomedicals Inc., Aurora, OH, USA) and returned to an incubator at 37 °C for 1-h to produce excitotoxicity. This concentration of NMDA was selected because of its ability to produce minimal, yet significant neurotoxicity (after Mulholland and Prendergast, 2003). Additional cultures were exposed to NMDA in buffer with 20 µM (+)-MK-801 (maleate; ICN Biomedicals Inc.), a non-competitive NMDA receptor antagonist. The NMDA challenge was stopped at 1-h by washing wells in 1 ml of fresh culture medium on the top and bottom. Cultures were then placed in 1 ml of culture medium with the addition of 2.5 µg/ml of the fluorescent dye propidium iodide (Molecular Probes, Eugene, OR, USA), which is a marker of non-vital cells and returned to the incubator for 24-h.
Corticosterone pre-treatment
At 5 days in vitro, additional cultures (n=15–18/group) were ran- domly transferred to culture plates containing 1 ml in each well of
either standard culture medium or physiologically relevant corti- costerone (1 nM-1 µM; Sigma) concentrations in standard culture medium. Additional cultures were exposed to 1 ml of culture medium with the glucocorticoid receptor antagonist RU486 (1– 10 µM; Sigma) or the mineralocorticoid receptor antagonist spi- ronolactone (1–10 µM; Sigma) alone or in combination with 100 nM corticosterone to assess the effects of glucocorticoid receptor activation. All six-well culture plates were then returned to the incubator for 72-h. For all experiments, corticosterone, RU486, and spironolactone stock solutions were prepared in 100% di- methyl sulfoxide (DMSO; Sigma) and diluted in normal culture medium to a final concentration of 1.0% DMSO. One-half of control cultures were also exposed to an equivalent concentration of DMSO in cell culture medium. Additional controls were not exposed to DMSO. After 72-h of continuous glucocorticoid expo- sure in an incubator, cultures were then either assessed for neu- ronal damage or subjected to 1-h of NMDA exposure, as de- scribed previously. To determine neuronal damage, cultures were placed in 1 ml of fresh culture medium with the addition of pro- pidium iodide and returned to the incubator for 24-h. Twenty-four hours later, delayed neuronal death was measured by determina- tion of propidium iodide uptake in the dentate gyrus, CA3, and CA1 regions of all cultures.
Nicotine treatment
A final series of studies was conducted to examine the possible neuroprotective effects of nicotine against corticosterone’s exac- erbation of NMDA-induced toxicity. (—)-Nicotine (bitartrate salt; 0.1–10 µM; Sigma) was acutely applied to cultures during the 1-h exposure to NMDA in cultures treated in the presence or absence of 100 nM corticosterone for 72-h. These concentrations of nico- tine were selected based on previous neuroprotection studies in hippocampal slice cultures (Prendergast et al., 2000b, 2001a,b). Additional cultures were exposed to (—)-nicotine or were co- exposed to (—)-nicotine and 100 nM corticosterone for 72-h prior to 1-h NMDA administration. Naturally expressed nicotinic acetyl- choline receptors (nAChRs) possessing α7* subunits may possi- bly contain additional variations of subunits; the ongoing nature of such research is indicated by an asterisk (Lukas et al., 1999). To assess the possible roles of specific nAChR subtypes in the neuroprotective actions of (—)-nicotine, additional studies were conducted to examine the ability of methyllycaconitine citrate (MLA; 100 nM; Tocris, Ballwin, MO, USA) or dihydro-β- erythroidine hydrobromide (DHβE; 10 µM; Sigma) to antagonize nicotine’s protective effect. MLA is a potent reversible, competitive inhibitor of α7* nAChR while DHβE is a competitive antagonist of function of nAChRs containing the β2 subunit. It must be noted, however, that recent work suggests that MLA possesses signifi- cant affinity for nAChRs that possess α3 or α6 subunits (Mogg et al., 2002). In that these subunits of nAChRs are not expressed in abundance in the hippocampus, use of MLA use is still postulated to reflect a predominantly α7*-mediated effect.
The pharmacology and distribution of [3H]methyllycaconi- tine binding corresponds with [125I]-α-bungarotoxin binding in ro- dent brain suggesting activity at α7-containing nAChRs at low nanomolar concentrations (Davies et al., 1999; Whiteaker et al., 1999). This concentration of DHβE was selected for this study based on its ability to block nicotine-induced elevated free intra- cellular Ca2+ levels in HEK293 cells expressing the β2 subunit (Chavez-Noriega et al., 2000). Further, 10 µM DHβE was selected because it reduced the open probability of nAChRs expressing β subunits in rat hippocampal CA1 stratum radiatum interneurons (Shao and Yakel, 2000). Determination of neuronal death as indicated by uptake of propidium iodide was measured 24-h later.
Assessment of excitotoxicity
Cell death (propidium iodide staining of dead and dying cells) was detected by fluorescent microscopy 24-h after NMDA insult. Up- take of propidium iodide was visualized using a 4× objective on a Nikon TE200 microscope (Nikon Inc., Melville, NY, USA) fitted for fluorescence detection (Mercury-arc lamp) connected to a per- sonal computer via a CCD camera (Gel Expert; Nucleotech Cor- poration, San Mateo, CA, USA). Propidium iodide has a peak excitation wavelength of 536 nm and was excited using a band- pass filter exciting the wavelengths between 510 and 560 nm. The emission of propidium iodide in the visual range is 620 nm. Inten- sity of propidium iodide fluorescence was analyzed by optical intensity using NIH Image with the experimenter blind to treatment condition. Each culture was coded prior to quantitative analysis. Optical intensity, in arbitrary optical units, was determined in the dentate gyrus, CA3, and CA1 regions of each individual explant using NIH Image. A background optical intensity was also mea- sured for each explant from the visual field surrounding the ex- plant. This background measurement was subtracted individually from those obtained for the explant subregions before statistical analysis to account for potential daily variation in camera perfor- mance using the following formula: (S—B), where S was the intensity of fluorescence for a given region in a slice and B was background intensity for that slice. Statistical analysis was then performed on the data after subtraction of the background inten- sity. For illustrative purposes, all data points were normalized to percentage of control using the following formula: (S—B)/C, where S was the intensity of fluorescence for a given region in a slice, B was background intensity for that slice, and C was the mean fluorescence for a particular region in control slices.
Statistical analysis
Data were first analyzed using two-way analyses of variance (ANOVA) to compare propidium iodide uptake in different treat- ment groups on sex (treatment×sex) in each hippocampal sub- region. No sex differences were observed in any of these studies; thus, data were collapsed across sex and re-analyzed using a two-way ANOVA comparing different treatment groups in different hippocampal subregions (dentate gyrus, CA1, and CA3 regions). When appropriate, post hoc analyses were conducted using the Tukey honestly significant difference test. Each experiment was replicated in triplicate representing three culture batches from approximately 18 different animals yielding n=45–54/group. The level of significance was set at P<0.05. RESULTS NMDA-induced neurotoxicity Initial studies were designed to examine the effects of NMDA exposure on cell death in cultures at 8 days in vitro. Relative to control cultures, NMDA exposure resulted in significant cell damage (i.e. uptake of propidium iodide) in pyramidal layer of the CA1, but not the CA3 or dentate gyrus regions [two-way interaction with brain region and treatment; F(6, 158)=5.290, P<0.001, post hoc P<0.05]. MK-801 exposure alone did not produce significant alter- ations in neurotoxicity in any hippocampal region in com- parison with control cultures. However, co-exposure to MK-801 (20 µM) during administration of NMDA signifi- cantly reduced excitotoxicity observed in the CA1 region, to near control levels. Fig. 1. Potentiation of neurotoxicity in corticosterone pre-treated hippocampal cultures for 72-h prior to 1-h of 5 µM NMDA exposure. In the CA1 pyramidal neuronal layer, NMDA exposure produced a significant increase in cell loss, which was potentiated by prior exposure to 100 nM and 1 µM corticosterone. Prior exposure to 1.0 µM corticosterone resulted in dentate gyrus neurotoxicity following insult, a finding that was not demonstrated with NMDA alone. No significant propidium iodide uptake was observed in the CA3 region. Data expressed as percentage of untreated control (mean±S.E.M.). Dashed line represents control value. * P<0.05 vs. untreated control. ** P<0.05 vs. NMDA. *** P<0.05 vs. 100 nM corticosterone. Corticosterone pre-treatment Hippocampal cultures were exposed to glucocorticoid ago- nists and/or antagonists for 72-h, starting at 5 days in vitro, to assess potential neurotoxic effects in the absence of NMDA insult. Corticosterone (1 nM–1 µM), RU486 (1–10 µM), or spironolactone (1–10 µM) exposure for 72-h did not significantly alter neuronal survival (i.e. uptake of propidium iodide) in any hippocampal subregion, in com- parison with control cultures (data not shown). Further, DMSO treatment of control cultures did not alter the uptake of propidium iodide. Hippocampal cultures pre-treated with corticosterone (100 nM–1 µM) for 72-h prior to NMDA exposure displayed a concentration-dependent potentiation of subsequent NMDA-mediated CA1 neurotoxicity [two-way interaction with brain region and treatment; F(10, 260)=12.661,P<0.001, post hoc P<0.05]. Whereas exposure of cul- tures to NMDA produced an approximate 35% increase in cell death relative to controls, NMDA-induced toxicity in cultures pre-treated with corticosterone (100 nM–1 µM) demonstrated an approximate 83–200% increase in pro- pidium iodide uptake in CA1 region (Fig. 1). There were no differences in propidium iodide uptake between controls exposed to DMSO and those not exposed to DMSO. Fur- ther, pre-treatment with 1 µM corticosterone prior to NMDA exposure resulted in significant cell death in the dentate gyrus, an effect not seen in cultures exposed to NMDA alone. Cultures exposed to 1–10 nM corticosterone for 72-h prior to NMDA exposure did not display potentiated NMDA-induced toxicity in the CA1 region. Subsequent experiments were conducted to examine the effects of glucocorticoid receptor antagonists on potentiation of hippocampal neurotoxicity resulting from 72-h corticosterone pre-exposure. Pre-treatment of cultures with 1 or 10 µM RU486, a glucocorticoid receptor antag- onist, and 100 nM corticosterone resulted in a significant reduction of corticosterone’s potentiation of NMDA-in- duced CA1 neuronal damage, to near-NMDA levels [F(8, 176)=8.619, P<0.001, post hoc P<0.05]. In contrast, pre- treatment of cultures with spironolactone (1 or 10 µM), a mineralocorticoid antagonist, and 100 nM corticosterone did not significantly alter the exacerbation of CA1 cell death [F(12, 278)=15.199, P<0.001, post hoc P<0.05;Fig. 2]. Co-exposure with 20 µM MK-801 during NMDA insult resulted in a marked attenuation of CA1 neurotoxicity in cultures pre-treated with 100 nM corticosterone, to near control values [F(10, 260)=12.661, P<0.001, post hoc P<0.05]. Pre-treatment with RU486 or spironolactone in the absence of corticosterone had no effect on NMDA- mediated CA1 toxicity. Fig. 2. Blockade of glucocorticoid and mineralocorticoid receptors during corticosterone (100 nM) treatment differentially affects potentiation of NMDA-induced neurotoxicity. Co-exposure to cultures with RU486 (10.0 µM) during corticosterone treatment attenuated subsequent potentiation of NMDA-induced CA1 damage, whereas concurrent exposure with spironolactone (10.0 µM) failed to reduce the exacerbation. Co-exposure to MK-801 and NMDA for 1-h significantly reduced corticosterone’s potentiation of insult to levels of control cultures. Data expressed as percentage of untreated control (mean±S.E.M.). Dashed line represents control value. * P<0.05 vs. untreated control. ** P<0.05 vs. NMDA and corticosterone+RU486. (—)-Nicotine neuroprotection In additional studies, cultures were pre-treated with (—)-nicotine (0.1–10 µM) for 72-h prior to NMDA applica- tion or were exposed to (—)-nicotine during the NMDA challenge. Extended exposure to nicotine for 72-h prior to NMDA insult produced significant reductions in subse- quent NMDA toxicity [F(10, 186)=7.761, P<0.001, post hoc P<0.05; Table 1]. Cellular uptake of propidium iodide was reduced by prior nicotine exposure by approximately 50% compared with NMDA-treated cultures at each con- centration in the CA1 region. Acute (—)-nicotine exposure with NMDA also abolished NMDA-induced CA1 damage, to near-control levels at all concentrations [F(12, 252)=7.696, P<0.001, post hoc P<0.05; Table 2]. A second series of studies was conducted to examine effects of neuroprotective nicotine concentrations on cortico- sterone’s potentiation of NMDA insult. Cultures which were previously exposed to 100 nM corticosterone for 72-h, were treated with varying concentrations of (—)-nicotine (0.1– 10 µM) during the 1-h NMDA exposure. (—)-nicotine expo- sure did not significantly reduce the exacerbated toxicity in any hippocampal region. However, there was a non-signifi- cant trend for a reduction of corticosterone’s potentiation by co-exposure to cultures with 10 µM (—)-nicotine and NMDA. In contrast, pre-exposure to explants with (—)-nicotine (1– 10 µM) and corticosterone abolished corticosterone’s poten- tiation of NMDA-induced CA1 neurotoxicity [F(14, 413)=7.258, P<0.001, post hoc P<0.05; Fig. 3]. Pre-treat- ment with 0.1 µM (—)-nicotine during corticosterone expo- sure did not attenuate corticosterone’s exacerbation of insult. Co-exposure of explants to 100 nM methyllycaconitine, 10 µM (—)-nicotine, and corticosterone resulted in a signi- ficant attenuation of (—)-nicotine’s neuroprotective ef- fect. This protective effect of nicotine, however, was not re- duced by co-exposure to 10 µM DHβE administration during (—)-nicotine and corticosterone exposure. When co-adminis- tered with corticosterone in the absence of (—)-nicotine, nei- ther methyllycaconitine nor DHβE significantly altered corti- costerone’s exacerbation of NMDA-induced CA1 excitotoxic- ity. Representative gray-scale images of fluorescent propidium iodide uptake in hippocampal cultures exposed to corticosterone and nicotine for 72-h are presented in Fig. 4. DISCUSSION Several different forms of neuronal injury are thought to be mediated by increases in extracellular levels of the excita- tory amino acid glutamate and subsequent NMDA recep- tor-mediated rises in intracellular Ca2+ concentrations (Arundine and Tymianski, 2003). Physiological events, such as prolonged hypercortisolemia, that are thought to increase the number and/or sensitivity of these receptors may be associated with overt neuropathology or with in- creased susceptibility to excitotoxic insults. Indeed, this series of events may be involved in the development of neuropathology observed in those with long-term depres- sion (Holsboer, 1999) and alcohol dependence (Menzano and Carlen, 1994), two of many clinical conditions associ- ated with prolonged hypercortisolemia. In the present re- port, we demonstrated that 72-h pre-treatment with corti- costerone exacerbated NMDA-mediated neurotoxicity in organotypic hippocampal slice cultures, an effect that was attenuate by additional pre-treatment with nicotine. Chronic corticosterone exposure leading to plasma concentrations up to 1 µM in the absence of subsequent insult has been reported to cause hippocampal damage in non-human primates (Sapolsky et al., 1990), while chronic elevations of corticosterone induced by stress have been correlated with dendritic atrophy of rat hippocampal neu- rons (Watanabe et al., 1992a,b). Typically, the above stud- ies characterized stress-induced hippocampal alteration and degeneration using paradigms where exposure to glu- cocorticoids lasted 3 or more weeks. Our data did not demonstrate cell loss in any subregion following 72-h glu- cocorticoid treatment. However, these findings are consis- tent with in vivo and in vitro reports, which utilized similar concentrations and exposure time (Sapolsky and Pulsinelli, 1985; Sapolsky, 1985, 1986; Sapolsky et al., 1988; Bodnoff et al., 1995; Vollmann-Honsdorf et al., 1997; Sousa et al., 1998, 1999). It is possible that non-lethal alterations in dendritic arborization may have gone unde- tected in the present study. Nonetheless, 72-h of pre-exposure to corticosterone, at concentrations that pro- duced no overt toxicity, concentration-dependently poten- tiated NMDA-induced CA1 degeneration in hippocampal explants. Several other reports using both in vivo and in vitro rodent models report related findings in demonstrat- ing that acute or subchronic pre-exposure of the brain to glucocorticoids potentiates the toxic effects of insults such as β-amyloid, gp120, and NMDA itself (Sapolsky and Pulsinelli, 1985; Sapolsky, 1985, 1986; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a; Supko and Johnston, 1994; Abraham et al., 2000). As a whole, these data are consistent with the suggestion that even short-lasting hypercortisolemia may render the CNS susceptible to excitotoxic insult. Fig. 3. Neuroprotection against corticosterone’s exacerbation of NMDA-mediated CA1 neurotoxicity induced by 72-h prior exposure to (—)-nicotine. Both 1.0 and 10.0 µM (—)-nicotine significantly ameliorated potentiation of CA1 insult when co-exposed with 100 nM corticosterone. This effect was blocked by concurrent administration with 100 nM methyllycaconitine, but not with 10.0 µM DHβE. Data expressed as percentage of untreated control (mean±S.E.M.). Dashed line represents control value. * P<0.05 vs. untreated control and corticosterone. ** P<0.05 vs. NMDA. It is interesting to note that both NMDA-induced toxicity and corticosterone potentiated toxicity were most readily observed in the CA1 region of hippocampal slice cultures. This greater CA1 sensitivity, relative to other regions of the hippocampal formation, may be related to the greater den- sity of NMDA receptors in this region (Martens et al., 1998) or to their resistance to block by Mg+ ex vivo (Sakaguchi et al., 1997). Additionally, there is recent evidence, suggest- ing that the sensitivity of the CA1 region to excitotoxic insult may be related, in part, to overactivation of intrinsic hippocampal excitatory pathways and resulting “network excitation” terminating in the CA1 region of isolated ex- plants (Lahtinen et al., 2001; Mulholland and Prendergast, 2003). While we and others have previously reported the greater sensitivity of the CA1 region to excitotoxic insult, as compared with the CA3 and dentate regions, others have reported that glucocorticoid exposure did not potentiate adverse effects of kainic acid exposure on metabolism in CA1 region explants (Yusim et al., 2000a). However, this same group did report that corticosterone potentiated the toxic effects of the HIV-1 glycoprotein gp120 in the CA1 region of explants (Yusim et al., 2000b). Clearly, the re- gion-specific effects of glucocorticoids in the hippocampus vary markedly with the nature of a concurrent insult. Fig. 4. Representative grayscale hippocampal images of a: (A) control culture, (B) culture exposed to 5 µM NMDA for 1-h, (C) culture exposed to 100 nM corticosterone for 72-h subsequent to NMDA exposure, (D) culture co-exposed to corticosterone and 10.0 µM (—)-nicotine prior to NMDA, (E) culture co-exposed to corticosterone, (—)-nicotine, and 100 nM methyllycaconitine prior to NMDA, and (F) culture co-exposed to corticosterone, (—)-nicotine, and 10.0 µM DHβE prior to NMDA. There is evidence to suggest that chronic elevations in corticosterone exposure sensitize the brain to injury by means of increasing genomic expression of functional NMDA receptors subunits (Weiland et al., 1997). For ex- ample, chronic corticosterone treatment has been shown to increase [3H]MK-801 binding and expression of mRNA encoding for NR2A and NR2B subunits of the NMDA re- ceptor in the CA1 and dentate regions of the rodent hip- pocampal formation (Weiland et al., 1997). Moreover, cor- ticosteroids modulate the release of excitatory amino acids resulting in an increase in extracellular glutamate (Moghaddam et al., 1994) and treatment with supra- physiologic concentrations of corticosteroids produce in- creases in intracellular Ca2+ concentrations in rodent brain (Kerr et al., 1992; Elliott and Sapolsky, 1993; Karst et al., 1994). As a whole, these finding suggest that exposure to high concentrations of corticosteroids may increase the function and/or sensitivity of NMDA receptor systems. However, it must be noted that long-term corticosterone exposure has been reported to alter expression of the calcium buffering protein calbindin-D28k in rodent brain. Though chronic stress and hypercortisolemia have been show to both increase (Iacopino and Christakos, 1990; Krugers et al., 1996) and decrease expression of calbin- din-D28k in different regions of the rat brain (Stuart et al., 2001), this latter evidence in particular may suggest that the potentiation of NMDA toxicity observed reflects a de- creased ability to buffer accumulated Ca2+. Further, it is possible, if not likely, that this potentiation of NMDA toxicity produced by corticosterone exposure involves perturbation of other transduction pathways or expression of other neu- ronal proteins such as microtubule-associated protein and calcium– calmodulin dependent kinase (Gartside et al., 2003). A recent report has examined the effects of mineralocorticoid and glucocorticoid receptor activation on neuro- degenerative effects of corticosterone (Sousa et al., 1999). It was reported that exclusive glucocorticoid receptor acti- vation resulted in subfield-specific hippocampal loss. The present report attempted to extend the above findings by examining the differential effects of mineralocorticoid and glucocorticoid receptor blockade during corticosterone-in- duced potentiation of insult. Co-exposure to cultures with corticosterone and RU486, a glucocorticoid receptor an- tagonist, resulted in attenuation of corticosterone’s en- hanced neurotoxicity in the CA1 region. In contrast, when co-exposed with corticosterone, the mineralocorticoid re- ceptor antagonist spironolactone did not reverse the exac- erbation of CA1 damage. It may be assumed that concen- trations which significantly potentiated excitotoxicity re- sulted in substantial glucocorticoid receptor function. Thus, exposure to corticosterone concentrations which did not potentiate insult (i.e. 1–10 nM) may not have resulted in significant occupation and function of glucocorticoid recep- tors. In fact, at least one recent report supports this con- tention in that only high concentration of corticosterone were observed to potentiate toxic effects of intra-hip- pocampal NMDA injection in rodents (Abraham et al., 2000). Thus, these findings are consistent with reports that activation of glucocorticoid receptors, and not mineralocor- ticoid receptors, may lead to a potentiation of NMDA receptor-mediated neuronal degeneration in the hippocam- pus (Gould et al., 1990; Woolley et al., 1991; Hassan et al., 1996; McIntosh and Sapolsky, 1996; Sousa et al., 1999). A large body of evidence suggests that exposure to nicotine and nAChR agonists reduce the neurotoxic con- sequences of NMDA receptor overactivity, such as that observed during or following exposure to high concentra- tions of corticosteroids. Further, evidence of an interaction between nicotine and corticosteroids in regards to neuro- nal survival may suggest that cigarette smoking impacts the neuropathological effects of hypercortisolemia in some patient populations. The present studies found that both pre-exposure (72-h) and acute exposure to nicotine during NMDA insult produced a significant reduction in the neu- rotoxicity observed in tissue not exposed to corticosterone. In contrast, in corticosterone pre-exposed tissue, acute exposure to nicotine with NMDA for 1-h did not prove to be neuroprotective. We have also reported that with at least one other form of excitotoxic insult, ethanol withdrawal, only chronic nicotine (10 µM) pre-exposure proved to be neuroprotective (Prendergast et al., 2000a). Thus, acute nicotine exposure appears to be neuroprotective against excitotoxic insult only under highly specific conditions whereas chronic nicotine pre-treatment produces a marked protection against many forms of Ca2+-mediated toxicity. We believe the ability of chronic nicotine treatment to up-regulate expression of the Ca2+ buffering protein calbindin-D28k is a key mediator of nicotine’s protective effects (Prendergast et al., 2001a; Mulholland et al., 2003). This suggestion is supported by the finding that 72-h pre- treatment with corticosterone and nicotine, at concentra- tions comparable to those found in the serum of chronic smokers (Henningfield et al., 1993), completely prevented the potentiation of NMDA-mediated CA1 toxicity. Addition- ally, this difference in protective effects of acute and chronic nicotine exposure may be related to the ability of chronic corticosterone exposure to interact with nicotinic cholinergic systems in a manner such that prolonged re- ceptor occupancy is required to produce nicotine’s protec- tive effect. Indeed, chronic exposure to corticosterone re- duced [125I]-α bungarotoxin (α-BTX) binding in the mouse hippocampus (Pauly and Collins, 1993; Grun et al., 1995; Robinson et al., 1996), likely reflecting a decrease in the number of α7*-bearing nAChRs. Additionally, nicotine co- administration with corticosterone reversed corticoste- rone’s decrease in α-BTX and [3H]-nicotine binding in mouse hippocampus (Robinson et al., 1996). The neuro- protective effect of nicotine exposure observed in these studies was reversed by co-administration with methylly- caconitine, an antagonist of α7*-bearing nAChRs, but not by DHβE, an antagonist of nAChRs possessing the β2 subunit. It must be noted that 50 nM methyllycaconitine has also been found to potently antagonize function of α3- and α6-containing nAChRs (Mogg et al., 2002); while these subtypes of nAChRs are not found in abundance in the hippocampus, their role in observed effects of nicotine cannot be discounted. However, we cannot conclude from these studies that nicotine exposure directly reverses the deleterious effects of corticosterone on the hippocampus,especially how it relates to expression and/or function of nAChRs. In conclusion, these data suggest that elevated glu- cocorticoids levels, at concentrations not themselves dam- aging, may sensitize glutamatergic receptor systems to pathological activation caused by other toxins or neuro- traumas, in a glucocorticoid receptor-dependent manner. Further, prolonged nicotine exposure may attenuate the ability of corticosterone to sensitize the hippocampus to a neurotoxic insult, in a manner suggesting the involvement of α7*-bearing nAChRs. The exact neurobiological sub- strate for the protective effect of nicotine against cortico- sterone’s potentiation of insult remains unclear. However, this suggests that while heavy smoking in patients suffer- ing from hypercortisolemia clearly increases the risk for development of smoking-related illness, one additional consequence may be the sparing of neuronal survival and function. For example, some work has demonstrated a negative correlation with cigarette smoking and Parkin- son’s disease (Tanner et al., 2002) and a positive correla- tion with smoking and the delayed onset of Alzheimer’s disease (van Duijn and Hofman, 1991). In female alcohol- ics, one recent study did report a modest positive correla- tion between smoking history and corpus callosum volume (Pfefferbaum et al., 2002). Although nicotine delivery via cigarette smoking in humans has been reported to in- crease plasma cortisol levels (Pomerleau and Pomerleau, 1990; Pomerleau et al., 1992), it is unclear how this may affect any neuroprotective effect of nicotine.