Choline exposure reduces potentiation of N-methyl-d -aspartate toxicity by corticosterone in the developing hippocampus

Developmental Brain Research 153 (2004) 203 – 211 www.elsevier.com/locate/devbrainres

Research report

Choline exposure reduces potentiation of N-methyl-d-aspartate toxicity by corticosterone in the developing hippocampus Patrick J. Mulhollanda, Rachel L. Self a, Barton R. Harrisb, John M. Littletonc, Mark A. Prendergasta,* a Department of Psychology, University of Kentucky, 115 Kastle Hall, Lexington, KY 40506-0044, USA Department of Behavioral Science, University of Kentucky College of Medicine, College of Medicine Office Building, Lexington, KY 40536-0086, USA c Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, A.B. Chandler Medical Center MS 305, Lexington, KY 40536-0298, USA b

Accepted 25 August 2004 Available online 25 September 2004

Abstract Exposure to high levels of glucocorticoids (GCs) may adversely affect neuronal viability, particularly in the developing hippocampus, via increased function or sensitivity of N-methyl-d-aspartate (NMDA)-type glutamate receptors. Conversely, choline supplementation in the developing brain may reduce the severity of subsequent insult. The present studies aimed to examine the extent to which short-term exposure to high concentrations of corticosterone would produce neuronal injury mediated by NMDA receptor activity. These studies also assessed the ability of choline to prevent this form of injury via interactions with nicotinic acetylcholine receptors (nAChRs) expressing the a7 subunit. Organotypic hippocampal slice cultures derived from neonatal rat were pre-treated for 72 h with corticosterone (100 nM) alone or with choline (0.1–10 mM), prior to a brief (1 h) NMDA exposure (5 AM). NMDA exposure produced significant cellular damage, reflected as increased fluorescence of the non-vital marker propidium iodide, in the CA1 region. While exposure to corticosterone alone did not produce damage, pre-treatment of cultures with corticosterone markedly exacerbated NMDA-induced toxicity. Pre-treatment with choline (z1 mM) alone or in combination with corticosterone markedly reduced subsequent NMDA toxicity, effects blocked by co-exposure to methyllycaconitine (100 nM), an antagonist active at nAChRs expressing the a7 subunit. These data suggest that even short-term exposure to high concentrations of GCs may adversely affect neuronal viability and that choline supplementation protects the brain from NMDA receptor-mediated damage, including that associated with hypercortisolemia. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Acetylcholine: nicotinic Keywords: Glucocorticoids; Excitotoxicity; Hippocampus; Neuroprotection; Cortisol

1. Introduction Abnormalities in function of the hypothalamic–pituitary– adrenal axis, manifested as hypercortisolemia, are associated with several clinical conditions, including late-gestation pregnancy, alcohol dependence, depression, Cushing’s syndrome, and Alzheimer’s disease [3,4,37,56]. Although acute elevations in circulating cortisol levels clearly affect * Corresponding author. Tel.: +1 859 257 6120; fax: +1 859 323 1979. E-mail address: [email protected] (M.A. Prendergast). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.08.008

glucose availability and reduce inflammation in response to a stressor [65], continuous exposure of the brain to high concentrations of glucocorticoids (GCs) may negatively affect neuronal function or viability. Indeed, individuals suffering from some of these clinical conditions reportedly display cognitive impairment [10,46,58,59] and decreases in the volume of multiple brain regions, including the hippocampus [13,35,59]. There is also mounting evidence that exposure to extra-physiological levels of GCs during gestation may negatively affect brain morphology, mood, sleep cycles, and behavior [33,69].

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Numerous studies suggest that exposure to high concentrations of GCs may directly produce neurotoxicity or potentiate subsequent insults, including exposure to NMDA [1,19,38,44]. However, the mechanisms underlying these effects are not completely understood. This effect of GCs may be related, in part, to an increased number or function of N-methyl-d-aspartate-type glutamate (NMDA) receptors [32,64,68], as well as, increased synthesis and release of excitatory amino acids [60] producing elevations in intracellular Ca2+ concentrations [17,28,29] and significant free radical formation [38]. GC-induced alterations of cholinergic receptor expression may also contribute to such effects. Indeed, chronic exposure to corticosterone reduced [125I]-a-BTX and highaffinity [3H]-nicotine binding in mouse hippocampus, likely reflecting a decrease in the number of nAChRs containing the a7 subunit and those containing an a4/h2 conformation [48,53]. Evidence of GC-induced alterations in the expression of some nAChRs is likely relevant to understanding the neuropathological effects of hypercortisolemia in that in vivo and in vitro studies demonstrate that nAChR agonists are neuroprotective against a variety of insults. Several reports have implicated the neuroprotective action of nicotine and other agonist to reflect function of a7-bearing nAChRs [11,14,27,43,44,52], though limited evidence suggests a role for those expressing the a4h2 subunits in some brain regions [16,30]. Interestingly, choline has been demonstrated to be a full and selective agonist at a7containing nAChRs [5,47], a partial agonist at a9, a4h4, a3h4 [49] and a co-agonist with acetylcholine at a4h2 nAChRs [76]. However, the ability of choline to desensitize or activate a7 nAChRs [6,5] and to modulate inhibitory synaptic transmission in rat hippocampal CA1 interneurons is approximately 10-fold less potent in comparison with acetylcholine [5,40,47]. Nonetheless, choline exposure protected neurons against neurotoxic effects of growth factor deprivation [26,39], exposure to high concentrations of dl-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid [62], and exposure to high doses of dizocilpine [22]. The purpose of the present studies was to assess the ability of choline pre-treatment to prevent NMDA receptor-mediated damage, both in the absence and presence of corticosterone exposure, which involves function of a7-bearing nAChRs. Although much is known regarding the beneficial effects of prenatal choline supplementation on later cognitive function and neuronal morphology, the relationship between choline treatment and deleterious effects of GC secretion has yet to be determined. Given evidence that a significant portion of fetus and newborns may experience periods of low oxygen and glucose levels [2,24,50], hypercortisolemia may potentiate these neurotoxic insults. Thus, choline supplementation at these critical times during development or birth may be of use in attenuating or preventing the consequences of hypercortisolemia and/or oxygen– glucose deprivation.

2. Materials and methods 2.1. Hippocampal culture preparation Preparation of hippocampal cultures followed procedures described by Stoppini et al. (1991). Whole brains from 8day-old male Sprague–Dawley rat pups (Harlan, Indianapolis, IN) were aseptically removed and placed into dissection medium (4 8C). Dissecting medium is made of Minimum Essential Medium plus 2 mM l-glutamine, 25 mM HEPES, and 50 AM penicillin/streptomycin solutions. Bilateral hippocampi were removed and placed into culture medium (4 8C) made of dissecting 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 Engineering, Gomshall, UK), each hippocampus was coronally sectioned at 200 Am and placed into fresh culture medium. Each unilateral hippocampus yielded approximately 12 slices (~24 slices per animal). The slices used for all studies were selected based on the visible morphology of the slice, which does differ across the septotemporal axis of the hippocampus. To avoid use of slices with significant variations in morphology, only those slices that were indistinguishable in size and shape were used in these studies. In doing so, slices from the most anterior and posterior portions of the hippocampus were discarded. Three slices were transferred onto an individual MillicellCM 0.4 Am biopore membrane insert (Millipore, Bedford, MA) and then placed in 35 mm 6-well culture plates (yielding 18 slices/animal) containing 1 ml of pre-incubated cell culture medium. Excess medium on top of slices was aspirated to ensure cultures remained exposed to the atmosphere of 5% CO2/95% air. Cultures were kept at 37 8C in an incubator and were allowed to become attached to membrane inserts following 5 days in culture medium. Gibco BRL (Gaithersburg, MD) supplied all culture medium solutions with the exception of Heat-Inactivated Horse Serum (Sigma, St. Louis, MO). Care of all animals was carried out in accordance with the National Institute of Health 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. 2.2. NMDA challenge At 8 days in vitro (DIV), a portion of all cultures (n=18 slices/group) were 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. Each experimental group contained three slices from six different animals. Cultures were then placed in new plates

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containing 1 ml of Ca2+ Locke’s buffer on the top and bottom alone or with the addition of 5 AM NMDA (Sigma) and 10 AM glycine (MP Biomedicals, Aurora, OH) and returned to an incubator at 37 8C for 1 h to produce excitotoxicity (after Ref. [44]). We exposed additional cultures to NMDA in buffer with 20 AM (+)-MK-801 maleate (MP Biomedicals), 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 Ag/ml of the fluorescent dye propidium iodide (Molecular Probes, Eugene, OR), a marker of non-vital cells, and returned to the incubator for 24 h.

the presence or absence of corticosterone for 72 h. This concentration of MLA was selected because the pharmacology and distribution of [3H]methyllycaconitine binding corresponds with [125I]-a-bungarotoxin binding in rodent brain suggesting activity at a7-containing nAChRs at low nanomolar concentrations [15,70]. This concentration of DHhE was selected for this study based on its ability to block nicotine-induced elevated free intracellular Ca2+ levels in HEK293 cells expressing the h2 subunit [12]. Further, 10 AM DHhE was selected because it reduced the open probability of nAChRs expressing h subunits in rat hippocampal CA1 stratum radiatum interneurons [57]. Determination of neuronal damage as indicated by uptake of propidium iodide was measured 24 h later.

2.3. Corticosterone pre-treatment

2.5. Measurement of excitotoxicity

We designed the present set of experiments to examine potentiation of NMDA-mediated damage by corticosterone pre-treatment in rat hippocampus. At 5DIV, cultures (n=18 slices/group) were randomly transferred to culture plates containing 1 ml in each well of either standard culture medium or 100 nM corticosterone (Sigma) in standard culture medium. All six-well culture plates were then returned to the incubator for 72 h. For all experiments, corticosterone stock solutions were prepared in 100% dimethyl sulfoxide (DMSO; Sigma) and diluted in normal culture medium to a final concentration of 1% 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 GC exposure in an incubator, cultures were then either assessed for neuronal damage or subjected to 1 h of NMDA exposure, as described previously. To determine neuronal damage, cultures were placed in 1 ml of fresh culture medium with the addition of propidium iodide and returned to the incubator for 24 h. Twenty-four hours later, delayed neuronal damage was measured by determination of propidium iodide uptake in the dentate gyrus, CA3, and CA1 regions of all cultures.

Cell damage, as measured by propidium iodide staining, was detected by fluorescent microscopy 24 h after NMDA exposure. The use of propidium iodide as a marker of cell death results in reproducible findings between experiments and significant correlations between the uptake of propidium iodide and other reliable measures of cell death (for review, see Ref. [75]). Propidium iodide uptake likely reflects staining of necrotic or end-stage apoptotic cells [71]. Uptake of propidium iodide was visualized using a 4X objective on a Nikon TE200 microscope (Nikon, Melville, NY) fitted for fluorescence detection (Mercury-arc lamp) connected to a personal computer via a CCD camera (Gel Expert, Nucleotech, San Mateo, CA). 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. Intensity 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 granule layer of the dentate gyrus, and pyramidal layer of the CA3 and CA1 regions of each individual explant using NIH Image. Area of optical intensity accounted for differences in regional variation across slices and did not include either the hilus or subicular complex. A background optical intensity was also measured for each explant from the visual field surrounding the explant. This background measurement was subtracted individually from those obtained for the explant subregions before statistical analysis to account for potential daily variation in camera performance 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 intensity. 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

2.4. Choline Pre-Treatment A final series of studies was conducted to examine the possible neuroprotective effects of choline exposure against corticosterone’s exacerbation of NMDA-induced toxicity. Choline chloride (Fluka BioChemika, Milwaukee, WI; 0.1– 10 mM; n=18 slices/group) was applied to cultures in the presence or absence of 100 nM corticosterone for a 72-h pre-treatment period prior to the 1-h exposure to NMDA. To assess the mediating roles of nAChR subtype activity on the neuroprotective actions of choline (10 mM), methyllycaconitine citrate (MLA; Tocris, Ballwin, MO; 100 nM), an a7 antagonist, or dihydro-h-erythroidine hydrobromide (DHhE; Sigma; 10 AM), an antagonist active at nAChRs containing the h2 subunit, were co-exposed with choline in

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was background intensity for that slice; and C was the mean fluorescence for a particular region in control slices. 2.6. Statistical analysis Raw data were first analyzed using two-way analyses of variance (ANOVA) to compare propidium iodide uptake in different treatment groups by sex (treatment  sex) in each hippocampal subregion. 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 test. The level of significance was set at Pb0.05.

3. Results 3.1. NMDA challenge Initial studies were designed to examine the regional effects of NMDA exposure on cell damage in 8DIV cultures. Relative to control cultures, NMDA exposure resulted in significant damage in the CA1 region, but not the CA3 or dentate gyrus regions [ F(6, 158)=5.290, pb0.001, post-hoc pb0.05]. Additional analyses revealed that coexposure to a non-toxic concentration of MK-801 (20 AM) during NMDA administration significantly reduced NMDAassociated damage observed in the CA1 region to near control levels (Fig. 1).

Fig. 2. Choline pre-treatment reduced subsequent damage induced by 1 h NMDA exposure. Both 1.0 and 10.0 mM choline exposure significantly reduced NMDA-induced CA1 damage. This effect was blocked by concurrent administration with 100 nM methyllycaconitine, but not with 10.0 AM DHhE. Data expressed as percentage of untreated control (meanFS.E.M.). Dashed line represents control value. *Pb0.05 vs. untreated control and corticosterone. **Pb0.05 vs. NMDA. n=18 slices/ group.

3.2. Corticosterone pre-treatment Hippocampal cultures pre-treated with 100 nM corticosterone, a concentration not itself damaging, for 72 h prior to NMDA exposure resulted in a potentiation of NMDAmediated CA1 damage [ F(10, 260)=12.661, pb0.001, posthoc pb0.05]. Whereas exposure of cultures to NMDA produced an approximate 60% increase in cell damage, NMDA-induced toxicity in cultures pre-treated with corticosterone demonstrated an approximate 160% increase in cell damage in the CA1 region (Fig. 1). Co-exposure with 20 AM MK-801 during NMDA treatment resulted in a marked attenuation of CA1 damage in cultures pre-treated with corticosterone, to near control values. 3.3. Choline Pre-Treatment

Fig. 1. Potentiation of damage in corticosterone pre-treated hippocampal cultures for 72 h prior to 1 h of 5 AM NMDA exposure. In the CA1 pyramidal neuronal layer, NMDA exposure produced a significant increase in cell damage, which was potentiated by prior exposure to 100 nM corticosterone. No significant propidium iodide uptake was observed in the dentate and CA3 regions. Co-exposure to MK-801 and NMDA for 1 h significantly reduced potentiation of damage by corticosterone to levels of control cultures. Data expressed as percentage of untreated control (meanFS.E.M.). Dashed line represents control value. *Pb0.05 vs. untreated control. ** and #Pb0.05 vs. NMDA. ##Pb0.05 vs. 100 nM corticosterone. n=18 slices/group.

A separate series of studies were conducted to examine the effects of choline pre-treatment against NMDA-induced damage. In hippocampal cultures exposed to choline (0.1– 10 mM) for 72 h prior to NMDA administration, we observed a concentration-dependent neuroprotective effect of choline [ F(10, 186)=7.761, pb0.001, post-hoc pb0.05]. Cellular damage was significantly reduced by approximately 30% and 70%, respectively, in the CA1 region in cultures pre-treated with 1 and 10 mM choline compared to NMDA-challenged cultures (Fig. 2). Co-exposure of explants to 100 nM MLA resulted in a significant attenuation of the neuroprotective effect by 10 mM choline. This protective effect of choline, however, was not reduced by co-exposure to 10 AM DHhE administration during choline exposure. When co-administered in the absence of choline, neither MLA nor DHhE significantly altered NMDA-mediated CA1 damage (data not shown).

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AM), significantly mitigated the protective effects of choline pre-treatment. Representative images of fluorescent propidium iodide uptake in hippocampal cultures exposed to corticosterone and choline for 72 h are presented in Fig. 4.

4. Discussion

Fig. 3. Pre-treatment with choline for 72 h protection against exacerbation of NMDA-mediated CA1 damage by corticosterone exposure. Both 1.0 and 10.0 mM choline exposure significantly ameliorated potentiation of CA1 damage when co-exposed with 100 nM corticosterone. This effect was blocked by concurrent administration with 100 nM methyllycaconitine, but not with 10.0 AM DHhE. Data expressed as percentage of untreated control (meanFS.E.M.). Dashed line represents control value. *Pb0.05 vs. untreated control and corticosterone. **Pb0.05 vs. NMDA. n=18 slices/group.

Additional explants were co-exposed with choline (0.1– 10 mM) and 100 nM corticosterone for 72 h. Pre-treatment with choline (1–10 mM) was associated with a significant reduction in potentiation of NMDA-induced CA1 damage by corticosterone exposure [ F(14, 413)=7.258, pb0.001, posthoc pb0.05; Fig. 3]. At the highest choline concentration (i.e. 10 mM), a complete reduction in exacerbation of damage by corticosterone was observed in the CA1 region. Concurrent pre-treatment with MLA (100 nM), but not with DHhE (10

Postnatal day (PD) 1 begins the development of hippocampal granule cell mossy fibers, the majority of which fully mature by PD21 [8]. This period of mossy fiber development corresponds with a stage of low circulating corticosterone levels and reduced response to stressors, termed the stress hypo- or non-responsive period [55]. Organotypic hippocampal cultures taken at PD8 and cultured for 5-8DIV, such as in this report, are undergoing developmental changes [45,61]. Previous research suggests that corticosterone may detrimentally affect hippocampal cytoarchitecture and function. For example, neonatal GC treatment is associated with alterations in hippocampal granule cell neurogenesis [9,20,21]. Maternal separation, which is associated with elevated plasma corticosterone levels, decreased hippocampal mossy fiber density and increased spatial memory impairments in adult animals [23]. In addition, restraint stress or exogenous corticosterone exposure decreased the number of branch points and the length of CA3 dendrites in adult rodent and non-human primate hippocampus [18,34,67,72]. These findings suggest that corticosterone exposure especially during a developmental period may aversely affect viability or function of the hippocampus.

Fig. 4. Representative hippocampal images of a(n): (A) control culture, (B) culture exposed to 5 AM 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 mM choline prior to NMDA, (E) culture co-exposed to corticosterone, choline, and 100 nM methyllycaconitine prior to NMDA, and (F) culture co-exposed to corticosterone, choline, and 10.0 AM DHhE prior to NMDA.

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In this report, we demonstrated that 1 h of NMDA exposure produced toxicity in the CA1 region of hippocampal cultures and that pre-treatment with corticosterone markedly sensitizes hippocampal cultures to NMDA toxicity. Exposure to corticosterone alone did not produce overt toxicity. Further, pre-treatment of cultures with choline attenuated NMDA-induced toxicity in a concentrationdependent manner. The present findings are consistent with those previously published in that corticosterone and dexamethasone potentiate NMDA-induced toxicity [1,44,63]. Though the present studies did not examine expression of NMDA receptor, the deleterious effects on neuronal viability by corticosterone exposure may be related, in part, to increased number or function of NMDA receptors [32,64,68], as well as increased release of excitatory amino acids [60], producing typical excitotoxicity. Pre-treatment with choline concentration-dependently attenuated CA1 damage induced by NMDA exposure. Seventy-two-hour pre-treatment with 10 mM choline reduced NMDA-mediate CA1 damage by approximately 70% whereas choline pre-treatment completely blocked potentiation of damage by corticosterone exposure in the CA1 region. In both instances, the protective effect of choline observed in these studies was reversed by coadministration with MLA, an antagonist active at a7bearing nAChRs, but not by DHhE, an antagonist active at nAChRs possessing the h2 subunit. Findings from this report are somewhat consistent with other data in suggesting that choline treatment/supplementation may protect the CNS from insult [26,62]. In concordance with this report, both studies demonstrated that co-administration with MLA significantly attenuated the protective effects of choline. We must note, however, that MLA has been reported to antagonize function of a3- and a6-containing nAChRs [41]; while these subtypes of nAChRs are not found in abundance in the hippocampus, their role in observed effects of choline cannot be discounted. Given that choline was protective against NMDA toxicity in the absence of corticosterone exposure, these data do not readily suggest a direct interaction between choline and corticosterone exposure on hippocampal functioning and viability. These data are of import in demonstrating a significant functional interaction with choline preventing damaging effects of excess NMDA receptor function and, perhaps most importantly, hypercortisolemia. We previously demonstrated that nicotine up-regulates hippocampal expression of the Ca2+ buffering protein calbindin-D28k via activity of nAChRs possessing the a7 subunit [43,51], an effect closely correlated with the ability of nicotine to protect hippocampal slice cultures from NMDA-induced damage. While it is unclear if increased expression of calbindin may contribute to the protect effect of choline, it certainly is possible that choline functions like nicotine in this regard. In that choline exposure produced an increase in [125I]-aBTX binding, at least in PC-12 cells [25] and corticosterone treatment is known to reduce [125I]-

aBTX binding in mouse brain [48,53], choline may be postulated to produce a restoration of cholinergic btoneQ that contributes in an uncharacterized manner to neuronal viability. There is another possible explanation for the mechanism underlying the observed neuroprotection by choline exposure in this study. One report demonstrated that 100 mM choline exposure produced a voltage-dependent blockade of glutamate-activated single-channel cesium currents recorded from Xenopus oocytes expressing recombinant NMDA receptors assembled from NR1 and NR2A subunits [66]. If choline reduced hippocampal toxicity by directly inhibiting Ca2+ flux through NMDA receptors, then we would expect a profile of neuroprotection similar to that of MK801 exposure. However, unlike MK-801 administration, choline pre-treatment failed to completely block CA1 toxicity. Perhaps more pertinent, when administered during the 1 h NMDA challenge, choline failed to attenuate potentiation of damage by corticosterone exposure even at a 10-mM concentration (data not shown). Based on these findings, it may be more likely that choline exposure is producing a neuroprotective effect by opposing the reduction of nAChR function induced by corticosterone treatment and/or increasing expression of calbindin-D28k. 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 density of NMDA receptors in this region [36] or to their resistance to block by Mg+ ex vivo [54]. Additionally, there is recent evidence, suggesting that the sensitivity of the CA1 region to excitotoxic insult may be related, in part, to overactivation of intrinsic hippocampal excitatory pathways and resulting bnetwork excitationQ terminating in the CA1 region of isolated explants [31,42]. While we and others have previously reported the greater sensitivity of the CA1 region to excitotoxic insult, as compared to 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 [73]. However, this same group did report that corticosterone potentiated the toxic effects of the HIV-1 glycoprotein gp120 in the CA1 region of explants [74]. Clearly, the region-specific effects of glucocorticoids in the hippocampus vary markedly with the nature of a concurrent insult. Perhaps most significantly, these are the first data to demonstrate that choline treatment or supplementation may prevent the development of neuronal injury produced by hypercortisolemia. In that we derived this tissue preparation from neonatal rat hippocampus, these data may be most relevant to understating one benefit of choline supplementation during pregnancy and the early postnatal period. Late gestation and the very early postnatal period in humans are typically associated with modest hypercortisolemia in the fetus/child [37] and this may clearly affect neuronal

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viability. Though much is known regarding the beneficial effects of prenatal choline supplementation on later cognitive function and neuronal morphology, specific evidence of a functional interaction between choline treatment and detrimental effects of elevated GC secretion has not been previously examined. This may be further relevant given evidence that a significant portion of fetus and newborns may experience periods of hypoxia and/or hypoglycemia [2,24,50], neurotoxic insults that may be exacerbated if hypercortisolemia persist. Thus, choline supplementation at these critical times, in particular, may be of use in attenuating or preventing the consequences of perinatal hypercortisolemia and/or hypoxia/hypoglycemia.

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The authors wish to thank Dr. Jim Pauly for critically reading the manuscript. The authors would also like to thank John A. Blanchard III and Robert C. Holley for their assistance with this study.

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