- Email: [email protected]
Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning
Neurobiology of Learning and Memory 80 (2003) 315–322 www.elsevier.com/locate/ynlme
Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning L.K. Marriotta and D.L. Korolb,* a b
Arizona Research Laboratories, Division of Neural Systems, Memory and Aging, University of Arizona, Tucson, AZ, USA Department of Psychology and Neuroscience Program, University of Illinois, 603 E. Daniel St., Champaign, IL 61820, USA Received 31 July 2003; accepted 4 August 2003
Abstract Estrogen modulates learning and memory in ovariectomized and naturally cycling female rats, especially in tasks using spatial learning and navigation. Estrogen also modulates cholinergic function in various forebrain structures. Past studies have shown positive correlations between hippocampal ACh output and performance on hippocampus-dependent tasks. The present study examined whether estradiol replacement would potentiate hippocampal ACh release during place learning. In vivo microdialysis and HPLC were used to measure extracellular ACh levels in the hippocampus of ovariectomized female rats that had received s.c. injections of 17b-estradiol (10 lg) or sesame oil (vehicle treatment) 48 and 24 h prior to training on a place task. Estrogen did not alter baseline levels of extracellular ACh in the hippocampus. During training, hippocampal ACh increased in ovariectomized rats regardless of estrogen status. However, while estradiol did not enhance learning in this experiment, estradiol significantly potentiated the increase in hippocampal ACh release seen during place training. This represents the first demonstration of on-line assessment of ACh output in hippocampus during learning in female rats and suggests that estrogen-dependent modulation of ACh release during training might control activation of different neural systems used during learning. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Ovariectomy; Microdialysis; Gonadal steroids; Acetylcholine; Hippocampus; Estradiol; Spatial memory
1. Introduction Increases in circulating estradiol levels can modulate learning and memory on a wide range of tests across several species. Even though increases in estradiol have been found to impair learning and memory on certain tasks (Frye, 1995; Galea, Kavaliers, Ossenkopp, & Hampson, 1995; Korol et al., 1994; Korol & Kolo, 2002; Warren & Juraska, 1997), a large literature supports the idea that estrogen enhances memory on many tasks, especially those that tap working memory or hippocampal processing (See Dohanich, 2002 for review). It is likely that estrogen not only influences memory processing, but also affects the cognitive strategy used in a learning situation, perhaps through differential modulation of neural systems (Korol & Kolo, 2002; Korol & Manning, 2001). * Corresponding author. Fax: 1-217-244-5876. E-mail address: [email protected] (D.L. Korol).
1074-7427/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2003.08.003
The specific mechanisms by which estradiol enhances cognitive function are unknown, but are likely to include direct actions on specific brain structures and neurochemical systems. Circulating estradiol readily crosses the blood brain barrier (Kawata, 1995) and can be found to accumulate in neurons across many forebrain structures, such as the hippocampus, cortex, and basal forebrain nuclei. Many of these brain regions, including the hippocampus (Loy, Gerlach, & McEwen, 1988; Maggi, Susanna, Bettini, Mantero, & Zucchi, 1989; Shugrue & Mercenthaler, 2000), bed nucleus of the stria terminalis (Miller et al., 1999), diagonal band nuclei (Orensanz, Guillamon, Ambrosio, Segovia, & Azvara, 1982), and medial septal area (Fallon, Loughlin, & Ribak, 1983) contain classical estrogen receptors (ER) and DNA binding sites for estrogen. Of particular interest here, the brain regions that provide cholinergic innervation to the hippocampal formation and neocortex such as the medial septum,
316
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
diagonal band, and nucleus basalis magnocellularis (Everitt & Robbins, 1997) accumulate estrogen (Shugrue, Scrimo, & Merchenthaler, 2000) and are neurochemically sensitive to estrogen as seen by changes in synthesis, turnover, and release of acetylcholine. For example, estrogen administration to ovariectomized female rats has multiple effects on the cholinergic system, including increased levels of choline acetyltransferase (ChAT), ChAT mRNA expression and ChAT activity in the basal forebrain and high-affinity choline uptake in the hippocampal formation and cortex (Gibbs, 1996a, 1996b; Gibbs & Pfaff, 1992; Gibbs, Wu, Hersh, & Pfaff, 1994; Lapchak, Araujo, Quirion, & Beaudet, 1990; Luine, 1985; Simpkins et al., 1997; Singh, Meyer, Millard, & Simpkins, 1994). In addition, the number of ChATimmunoreactive neurons detected in the medial septum and vertical limb of the diagonal band of Broca (Gibbs & Pfaff, 1992) and stria terminalis (Miller et al., 1999) increases with estrogen replacement as does the number of neurons that are immunoreactive for both ChAT and ER in the medial septum, diagonal band nuclei, substantia innominata, ventral pallidum (Toran-Allerand et al., 1992), and stria terminalis (Miller et al., 1999). Estrogen replacement also increases potassium-evoked but not basal release of acetylcholine (ACh) in the hippocampus and cortex (Gabor, Nagle, Johnson, & Gibbs, 2003; Gibbs, Hashash, & Johnson, 1997), suggesting enhanced ACh release upon activation of the cholinergic input. Hippocampal cholinergic function has been implicated in learning and memory (see Gold, 2003 for review) with intrahippocampal ACh agonists enhancing (Kim & Levin, 1996) and ACh antagonists impairing spatial, contextual, and working memory tasks (Anagnostaras, Maren, & Fanselow, 1995; Carli, Luschi, & Samanin, 1995; Riekkinen & Riekkinen, 1997; Young, Bohenek, & Fanselow, 1995; Kim & Levin, 1996). Moreover, release of ACh in the hippocampus increases while male rats solve navigation tasks (Ragozzino, Unick, & Gold, 1996; Stancampiano, Cocco, Cugusi, Sarais, & Fadda, 1999) and this increase in ACh output is positively correlated with good performance on a variety of hippocampus-dependent tasks in male rats (Chang & Gold, 2003; Gold, 2003; McIntyre, Pal, Marriott, & Gold, 2002; Ragozzino & Gold, 1995). Together these findings suggest that ACh release may be a marker for activation and participation of the hippocampus in learning and memory. Acute estrogen administration also biases the learning strategy used by rats to solve a navigation task. For example, systemic administration of estradiol to ovariectomized female rats enhanced place learning, but impaired response learning on the similar mazes (Korol & Kolo, 2002). Thus, ACh and estrogen both contribute to regulating the participation of different neural systems in learning and memory. Recent
evidence indicates that estrogen may interact with cholinergic function in the hippocampus to modulate learning and memory (Fader, Hendricson, & Dohanich, 1998; Farr, Banks, & Morley, 2000; Packard & Teather, 1997a). Perhaps estradiol replacement potentiates hippocampal ACh release during performance of spatial tasks, thereby biasing strategy to favor place over response learning. The present study uses in vivo microdialysis to investigate whether estrogen replacement can augment ACh release in the hippocampus of ovariectomized female rats that are engaged in a place learning task.
2. Materials and methods 2.1. Subjects Fifteen virgin female Sprague–Dawley rats (Hilltop breeders, Scottdale, PA), 2–3 months of age, were housed individually with food and water available ad libitum. All animals were maintained on a 12-h light/ dark cycle and allowed to adjust to their new environment for 1 week following arrival. 2.2. Surgery Rats received atropine sulfate (0.4 mg/kg in 0.2 ml, i.p.) prior to anesthesia with sodium pentobarbital (50 mg/kg, i.p.) and were placed in a stereotaxic instrument with the incisor bar set at 5.0 mm above the interaural line. A plastic guide cannula (CMA/12; Carnegie Medicine, Stockholm) was lowered into the hippocampus at 3.45 mm posterior to bregma, 5.4 mm lateral, and 2.2 mm ventral from skull (from Pellegrino, Pellegrino, & Cushman, 1979). Three screws were used as anchors, and the cannula and screws were affixed to the skull with dental acrylic cement. Immediately after the cannula surgery was complete, rats were bilaterally ovariectomized with a dorsolateral approach. All sutured areas were cleaned with betadine one day after surgery. Rats were allowed to recover from surgery for at least one week prior to initiation of handling and food deprivation procedures. 2.3. Behavioral procedures Rats were trained on a place learning task 20–24 days following ovariectomy. The training apparatus was a four-arm black plexiglass plus-shaped maze. The maze rested on a table, elevated 88 cm from the floor, in the center of the room. The center platform was a 13 13 cm square; each arm was 13 cm wide, 45 cm long, with walls 7 cm high. The room contained various extra-maze visual cues including a desk, a large black curtain, a dimmed halogen light, and a lab bench with cabinets.
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Rats were food-restricted to approximately 80% of free-feeding body weight and handled daily (5 min/day) for 7 days. Animals were randomly assigned to one of two treatment groups: ovariectomy with estradiol (ovx + E2; n ¼ 8) and ovariectomy without hormone replacement (ovx + OIL; n ¼ 7, 2 eliminated as described below for a final n ¼ 5). At 48 and 24 h prior to behavioral testing, animals received s.c. injections of either 10 lg of 17-b estradiol-benzoate (Sigma) dissolved in 0.1 cc of sesame oil (ovx + E2) or vehicle alone (ovx + OIL). Twenty-four hours prior to training, rats received approximately 15 Frosted Cheerios in their home cages to decrease neophobia associated with the food reinforcement. Rats were trained to find food (one Frosted Cheerios pellet) at the end of the goal arm. The goal arm remained in a fixed position relative to the extra-maze cues. Start arms were randomized across the other three non-goal arms. Animals were trained to a choice accuracy of 9/10 trials correct, or to a maximum of 100 trials. An arm entry was recorded when all four paws passed a halfway mark in the arm. Following each arm choice, the animal was returned to the holding cage for 30 s while the maze was rotated 90° clockwise using pre-set stops. 2.4. Microdialysis procedure Twenty-four hours prior to training, a dialysis probe was inserted into the guide cannula for 5 min. This procedure minimizes obstruction due to gliosis during dialysis 24 h later (Benveniste, Drejer, Schousboe, & Diemer, 1987; Shuaib et al., 1990). On the day of training, a 3-mm dialysis probe (CMA/12; Carnegie Medicine) was inserted through the guide cannula and connected to plastic polyethylene tubing driven by a microinfusion system (CMA/100; Carnegie Medicine). The dialysis probe was continuously perfused at a rate of 2 ll/min with artificial cerebrospinal fluid: 128 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2 , 2.1 mM MgCl2 , 21 mM NaH2 PO4 , 1.3 mM Na2 HPO4 , 1.0 mM glucose, and brought to, pH 7.02, by NaOH, which contained 1 lM of the acetylcholinesterase inhibitor, neostigmine. We previously found that percent increases in release of ACh in the hippocampus during training are of comparable magnitude when neostigmine concentrations ranging from 0.1 to 6 lM are used (Chang, Savage, & Gold, 2003; Ragozzino et al., 1996; Stefani & Gold, 2001). The dialysis probe was inserted 105 min prior to behavioral training. The first 45 min of dialysis permitted equilibration between tissue and perfusion solution; these samples were discarded. Samples (24 ll) were then collected on ice every 12 min through the conclusion of the experiment. Four baseline samples were collected while rats remained in their holding cages. Immediately after collection of the baseline samples, rats were trained
317
on a place-learning version of the four-arm plus maze. Perfusate samples were collected every 12 min during training. The number of samples collected during maze learning varied with the number of trials to criterion. Four additional samples were collected after training while the rat remained in its holding cage. 2.5. Acetylcholine assay Using 20 ll of sample, ACh content was assessed using reverse-phase high performance liquid chromatography, with an enzymatic assay system and electrochemical detector. ACh was separated from free choline on a reverse-phase analytical column (Chromspher 5 C18, 100 3 mm, Chrompack, Middleburg, The Netherlands). An enzymatic postcolumn reactor unit containing acetylcholinesterase (EC 3.1.1.7; Sigma type VI-S) and choline oxidase (EC 1.1.3.17; Sigma) converted ACh ultimately to betaine and H2 O2 . Oxidation of H2 O2 was electrochemically detected by a platinum electrode held at +525 mV relative to a silver chloride reference electrode. Mobile phase (pH 8.0) containing 0.2 mM dibasic potassium phosphate, 1.0 mM tetramethylammonium hydroxide, 0.3 mM EDTA, and 0.005% Kathon CG (to prevent bacterial growth) was delivered at a rate of 0.6 ml/min by a solvent delivery system (ESA 580; ESA, Chelmsford, MA). ACh peaks were quantified by comparison to peak heights of ACh standard solutions prepared at the time of each assay. The detection limit for ACh was 50 fmol. 2.6. Histology Immediately following completion of microdialysis sample collection, rats were given a lethal dose of sodium pentobarbital followed by cardiac puncture to remove 3 cc of blood for subsequent analyses of circulating estradiol levels. Blood was kept on ice in a test tube containing 0.5 ml lithium heparin (Vacutainer Plus PST, Becton–Dickinson, Franklin Lakes, NJ) and centrifuged at 4 °C at 3500g for 12 min. The plasma was then collected and stored at )80 °C until estradiol levels were analyzed with radioimmunoassay (Diagnostic Research, Webster, TX). The assay has a sensitivity of 8 pg/ ml, sufficient to reveal differences between plasma levels of estradiol in our ovariectomized and hormone-treated rats. Moreover, the assay is quite specific to estradiol with cross-reactivity of 0.32% for estriole and 0.001% for testosterone. After blood collection, rats were perfused intracardially with 0.9% saline and a 10% formalin solution. Brains were removed and stored in a 30% sucrose/formalin solution. Brains were frozen at )20 °C, sectioned at 40 lm and stained with cresyl violet. Only rats with guide cannula placements in the hippocampus (Fig. 1) were included in the final analysis.
318
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Fig. 1. Photomicrograph showing the placement of the guide cannula in the hippocampus. Note that rats were excluded from analysis when histological characterization indicated misplacement of the microdialysis probe.
Vaginal smears were taken daily for seven days prior to behavioral testing to determine cycle stage and to validate the effectiveness of ovariectomy and hormone replacement. Vaginal smears were stained with HarrisÕ hematoxylin and eosin solutions and staged by microscopic examination. Each daily smear was assigned to one of three cytological stages: proestrus, estrus, and diestrus. 2.7. Data analyses Baseline ACh values were derived from the mean of the first four baseline samples taken prior to training. ACh values from samples collected during and after training were converted to percent difference scores using each ratÕs baseline values. Because the duration of training differed with number of trials to criterion, measures of ACh release during training included mean values during different periods of maze training, e.g., first half, second half, and final sample reflecting release as the rat reached criterion performance, and the postmaze period. Four samples were averaged for the postmaze period. Paired two-tailed student t tests were used to compare the mean change in values of ACh release between baseline and later samples within each group and unpaired two-tailed student t tests were used to compare baseline values and percent changes between treatment groups. Because of the 100-trial maximum, trials to criterion were compared using the non-parametric Mann–Whitney U test.
(t ¼ 3:9 for ovx + OIL, t ¼ 9:3, ovx + E2; p’s < :05). Estrogen treatment significantly enhanced ACh release while animals performed the place learning task (all maze samples averaged; t ¼ 2:2; p < :05). Specifically, in samples collected during training, ovx + OIL rats exhibited an average increase of 68 19% above baseline ACh release while the estrogen-treated rats demonstrated an increase of 121 14% ðp < :05Þ. This estrogen-induced potentiation of ACh release was evident during both the first (t ¼ 2:15; p ¼ :05) and second (t ¼ 2:26; p < :05) half of training (see Fig. 2). ACh release in the last sample, i.e., obtained as rats approached criterion performance, remained higher in estrogen- than in oil-treated rats (60 16% vs. 114 19%, oil vs estrogen, respectively, t ¼ 2:32; p < :05). Once rats reached criterion and were removed from the maze, extracellular ACh values dropped rapidly, nearing those at baseline. The averaged post-maze values were not different from baseline (ovx + OIL: t ¼ 1:27; p > :05; ovx + E2: t ¼ 1:35; p > :05) and did not differ by treatment group (t ¼ 0:28; p > :05). Rats in both treatment groups took comparable number of trials to reach criterion, i.e., 9 out of 10 correct (median ¼ 79 for oil- and 79 for estrogen-treated rats, U8;5 ¼ 14; p > :05). Plasma estradiol levels and cytological examinations confirmed the efficacy of the ovariectomy and of hormone replacement. Plasma estradiol levels were below the sensitivity level of the RIA for all oil-treated rats with the exception of two animals. These two animals had circulating estrogen levels of 25–26 pg/ml (comparable to diestrous levels), suggesting incomplete ovariectomy, and were thus excluded from the analysis. Estradiol treatment led to substantially elevated plasma levels of 104.4 14.1 pg/ml. While these values are high, they are not outside the range of values found at proestrus (Jarrar, Want, Cioffi, Bland, & Chaudry, 2000).
3. Results Estrogen treatment did not significantly alter baseline levels of extracellular ACh (t ¼ 0:41; p > :05). Mean ACh content in baseline samples was 4.2 1.1 pmol for the oil-treated group and 5.5 1.1 pmol for the estrogen-treated group. Rats in both groups showed increases in extracellular ACh levels during maze training
Fig. 2. Extracellular levels of ACh from hippocampus rose significantly during training (*). Estradiol treatment significantly potentiated this maze-related increase (+). The estrogen effects were evident during the first and second half of training as well as during the last sample reflecting the sample that learning criterion was reached. Also note that ACh levels returned to near baseline values during the 48-min, postmaze period.
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Fig. 3. Photomicrographs of vaginal smears stained with hematoxylin and eosin indicating diestrous (upper panel) and proestrous (lower panel) smears. Note that and diestrous smears are characterized primarily by the presence of leukocytes and proestrous smears by nucleated epithelial cells.
The intra-assay variance for samples from all animals was 5.4 0.7%. Results from vaginal smears indicated that ovx + OIL rats had diestrus smears (Fig. 3) highlighted by leukocytes, throughout the experimental period. Rats with estradiol replacement had diestrus smears until treatment, after which they demonstrated proestrus smears (Fig. 3) highlighted by nucleated endothelial cells. All ovx + E2 rats showed proestrus smears on the day of training.
4. Discussion The present findings indicate that ACh levels in the hippocampus increase in female rats performing a placelearning task. These data are consistent with previous in vivo microdialysis studies in male rats suggesting that activation of an intact hippocampal system, as reflected
319
by an increase in ACh output, may modulate acquisition of different spatial tasks (McIntyre et al., 2002; Ragozzino & Gold, 1995; Ragozzino, Pal, Unick, Stefani, & Gold, 1998; Stancampiano et al., 1999). Moreover, these data also support previous work showing that cholinergic input to the hippocampal formation, as assessed by high affinity choline uptake activity, is activated during learning and memory (Marighetto, Durkin, Toumane, Lebrun, & Jaffard, 1989, 1994; Wenk, Hepler, & Olton, 1984). While ACh output increased in the hippocampus during maze learning regardless of estrogen status, estrogen replacement dramatically potentiated the increase in ACh release compared to hormone-deprived rats. Interestingly, estrogen replacement failed to alter baseline hippocampal ACh release, suggesting that estrogen treatment may enhance ACh release only when the system is otherwise activated such as during learning. This idea is supported by previous work demonstrating a similar potentiation of hippocampal ACh levels following short- and long-term estrogen treatment, but only after direct potassium-induced activation of the hippocampus (Gabor et al., 2003; Gibbs et al., 1997). That estrogen enhances ACh output fits in well with a rapidly growing literature supporting the view that estrogen regulates cholinergic function and the view that other neurobiological effects of estrogen may be regulated through cholinergic actions. Specifically, estradiol replacement increases ChAT and ChAT mRNA in the medial septal area (Gibbs, 1996b) and enhances ChAT activity in the CA1 region of the hippocampus (Luine, 1985). It is likely that estrogen acts through regulation of cholinergic function to modify dendritic spine density (Lam & Leranth, 2003) and NMDA receptor binding (Daniel & Dohanich, 2001). Furthermore, the ability of estrogen to disinhibit hippocampal CA1 pyramidal cells may partially depend on intact cholinergic input (Rudick, Gibbs, & Woolley, 2003). Together, these findings suggest that estrogen regulation of ACh may be involved in modulating neuronal excitability and the cellular and subcellular sequelae that ensue to alter neuronal plasticity and learning and memory. Consistent with this view, a subset (3–17%) of terminals in the dorsal hippocampus show colocalization of the vesicular acetylcholine transporter and ERa, supporting the possibility of a direct, non-genomic mechanism for the effects of estrogen on acetylcholine release (Towart et al., 2003). It is unlikely that the testing-induced increases in hippocampal ACh release found in the present study are due solely to increased locomotion. There was no correlation between the number of arm entries per 12-min sample and the amount of ACh released in the hippocampus (data not shown). In addition, previous studies indicate that unlike what has been found for
320
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
learning and memory, changes in hippocampal highaffinity choline uptake (Decker, Pelleymounter, & Gallagher, 1988; Wenk et al., 1984) or extracellular ACh levels measured by microdialysis (Ragozzino et al., 1996) are not secondary to locomotor activity. Our behavioral data indicate that acute estradiol administration failed to enhance place learning three weeks after ovariectomy. These data differ from those found previously in our laboratory in which the same estrogen regimen facilitated place or allocentric learning, but impaired response or egocentric learning (Korol & Kolo, 2002). Furthermore, we have shown that across the estrous cycle or with moderate durations of hormone replacement, i.e., 1 or 4 weeks, rats characterized by high circulating estrogen choose placelearning strategies over equally effective response strategies (Korol, Malin, Borden, Busby, & CouperLeo, 2003; Thomas, McElroy, & Korol, 2001). The lack of effects of estrogen on spatial learning found in the present experiment may be due to several factors including contamination of the training environment from the blockade of cues by the microdialysis tubing or investigator, and the disruption of surrounding tissue by the microdialysis probe. The latter effect may be exacerbated by the presence of increased levels of circulating estrogen, shown to enhance excitability and to decrease seizure threshold (Terasawa & Timiras, 1968; Wong & Moss, 1992), thereby potentially impairing performance to a greater degree in estrogen-treated rats. We are currently testing whether estrogen alters the balance of ACh release in hippocampus and striatum, with a predicted increase in hippocampal versus striatal ACh, and shifts learning towards the use of place strategies during acquisition. Evidence that ACh ratios in these structures are predictive of learning strategies has been shown in males (McIntyre, Marriott, & Gold, 2003). In addition to its effects on learning strategy, many studies have found that estradiol administration enhances memory. Performance on radial maze spatial working memory tasks is enhanced under various regimens of estrogen administration including acute (Sandstrom & Williams, 2001) or chronic replacement (Daniel, Fader, Spencer, & Dohanich, 1997). Relevant to the current findings, estrogen counteracts memory impairments induced by scopolamine, a cholinergic antagonist, when injected systemically (Dohanich, Fader, & Javorsky, 1994; Packard, Kohlmaier, & Alexander, 1996) or intrahippocampally (Fader et al., 1998; Packard & Teather, 1997b). Moreover, posttraining intrahippocampal estradiol injections in male rats enhanced performance on a hidden platform swim task that could be attenuated by concurrent peripheral administration of scopolamine (Packard et al., 1996). In sum, this study represents the first demonstration of on-line assessment of ACh output in hippocampus
during learning in hormone treated- and hormone-deprived female rats. The augmentation of hippocampal ACh output, during training but not at baseline, suggests that acute estrogen treatment may modulate the participation of different neural systems used during learning. Cholinergic function may be one marker or one mechanism of this modulation.
Acknowledgments This work was supported by a research grant from the National Science Foundation (IBN-0081061 DLK), with additional research support from Paul Gold, National Institute of Aging (AG 06748) and National Institute for Neurological Diseases and Stroke (NS 32914). We would like to thank Christa McIntyre for help with technical aspects of microdialysis and HPLC analysis, and Trish Pruis and Ed Roy for help with photomicrographs.
References Anagnostaras, S. G., Maren, S., & Fanselow, M. S. (1995). Scopolamine selectively disrupts the acquisition of contextual fear conditioning in rats. Neurobiology of Learning and Biology, 64, 191–194. Benveniste, H., Drejer, J., Schousboe, A., & Diemer, N. (1987). Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in rat. Journal of Neurochemistry, 49, 729–734. Carli, M., Luschi, R., & Samanin, R. (1995). (S)-WAY 100135, a 5HT1A receptor antagonist, prevents the impairment of spatial learning caused by intrahippocampal scopolamine. European Journal of Pharmacology, 283, 133–139. Chang, Q., & Gold, P. E. (2003). Switching from hippocampal to striatal memory systems during learning: Changes in patterns of acetylcholine release in rats. Journal of Neuroscience, 23, 3001– 3005. Chang, Q., Savage, L. M., & Gold, P. E. (2003). Use of acetylcholinesterase (AChE) inhibitors in the perfusate during brain microdialysis to measure acetylcholine (ACh) release during learning and memory. In Society for Neuroscience, 33rd Annual Meeting, New Orleans. Daniel, J. M., & Dohanich, G. P. (2001). Acetylcholine mediates the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus and the associated improvement in working memory. Journal of Neuroscience, 21, 6949–6956. Daniel, J. M., Fader, A. J., Spencer, A. L., & Dohanich, G. P. (1997). Estrogen enhances performance of female rats during acquisition of a radial arm maze. Hormones and Behavior, 32, 217–225. Decker, M. W., Pelleymounter, M. A., & Gallagher, M. (1988). Effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats. Journal of Neuroscience, 8, 90–99. Dohanich, G. P. (2002). Gonadal steroids, learning and memory. In D. W. Pfaff, A. P. Arnold, A. M. Etgen, S. E. Fahrbach, & R. T. Rubin (Eds.), Hormones, brain and behavior (pp. 265–327). San Diego: Academic Press. Dohanich, G. P., Fader, A. J., & Javorsky, D. J. (1994). Estrogen and estrogen-progesterone treatments counteract the effect of scopol-
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322 amine on reinforced T-maze alternation in female rats. Behavioral Neuroscience, 108, 988–992. Everitt, B. J., & Robbins, T. W. (1997). Central cholinergic systems and cognition. Annual Review of Psychology, 48, 649–684. Fader, A. J., Hendricson, A. W., & Dohanich, G. P. (1998). Estrogen improve performance of reinforced T-maze alternation and prevents the amnestic effects of scopolamine administered systemically or intrahippocampally. Neurobiology of Learning and Memory, 69, 225–240. Fallon, J. H., Loughlin, S. E., & Ribak, C. E. (1983). The islands of Calleja complex of rat basal forebrain. III. Histochemical evidence of a striatopallidal system. Journal of Comparative Neurology, 218, 91–120. Farr, S. A., Banks, W. A., & Morley, J. E. (2000). Estradiol potentiates acetylcholine and glutamate-mediated post-trial memory processing in the hippocampus. Brain Research, 864, 263–269. Frye, C. A. (1995). Estrus-associated decrements in a water maze task are limited to acquisition. Physiology and Behavior, 57, 5–14. Gabor, R., Nagle, R., Johnson, D. A., & Gibbs, R. B. (2003). Estrogen enhances potassium-stimulated acetylcholine release in the rat hippocampus. Brain Research, 962, 244–247. Galea, L. A., Kavaliers, M., Ossenkopp, K. P., & Hampson, E. (1995). Gonadal hormone levels and spatial learning performance in the Morris water maze in male and female meadow voles, Microtus pennsyvanicus. Hormones and Behavior, 29, 106–125. Gibbs, R. B. (1996a). Expression of estrogen receptor-like immunoreactivity by different subgroups of basal forebrain cholinergic neurons in gonadectomized male and female rats. Brain Research, 720, 61–68. Gibbs, R. B. (1996b). Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: Effects of estrogen and progesterone. Journal of Neuroscience, 16, 1049–1055. Gibbs, R. B., & Pfaff, D. W. (1992). Effects of estrogen and fimbriafornix transection on p75ngrf and ChAT expression in the medial septum and the diagonal band of Broca. Experimental Neurology, 116, 23–29. Gibbs, R. B., Wu, D., Hersh, L. B., & Pfaff, D. W. (1994). Effects of estrogen replacement on the relative levels of choline acetyltransferase, trkA, and nerve growth factor messenger RNAs in the basal forebrain and hippocampal formation of adult rats. Experimental Neurology, 129, 70–80. Gibbs, R. B., Hashash, A., & Johnson, D. (1997). Effects of estradiol on potassium-stimulated acetylcholine release in the hippocampus and overlying cortex of adult rats. Brain Research, 749, 143–146. Gold, P. E. (2003). Acetylcholine modulation of neural systems involved in learning and memory. Neurobiology of Learning and Memory, 80, 194–210 Jarrar, D., Want, P., Cioffi, W. G., Bland, K. I., & Chaudry, I. H. (2000). The female reproductive cycle is an important variable in the response to trauma-hemorrhage. American Journal of Physiology, 279, H1015–H1021. Kawata, M. (1995). Roles of steroid hormones and their receptors in structural organization in the nervous system. Neuroscience Research, 24, 1–46. Kim, J. S., & Levin, E. D. (1996). Nicotinic, muscarinic and dopaminergic actions in the ventral hippocampus and the nucleus accumbens: Effects on spatial working memory in rats. Brain Research, 725, 231–240. Korol, D. L., & Kolo, L. L. (2002). Estrogen-induced changes in place and response learning in young adult female rats. Behavioral Neuroscience, 116, 411–420. Korol, D. L., Malin, E. L., Borden, K. A., Busby, R.A., & CouperLeo, J.M., (2003). Shifts in preferred learning strategy across the estrous cycle in female rats, Submitted. Korol, D. L., & Manning, C. A. (2001). Effects of estrogen on cognition: Implications for menopause. In M. E. Carroll, & J. B.
321
Overmier (Eds.), Animal research and human health: Advancing human welfare through behavioral science (pp. 305–322). Washington, DC: American Psychological Association. Korol, D. L., Unick, K., Goosens, K., Crane, C., Gold, P. E., & Foster, T. C. (1994). Estrogen effects on spatial performance and hippocampal physiology in female rats. Society for Neuroscience Abstracts, 20, 1436. Lam, T. T., & Leranth, C. (2003). Role of the medial septum diagonal band of Broca cholinergic neurons in oestrogen-induced spine synapse formation on hippocampal CA1 pyramidal cells of female rats. European Journal of Neuroscience, 17, 1997–2005. Lapchak, P. A., Araujo, D. M., Quirion, R., & Beaudet, A. (1990). Chronic estradiol treatment alters central cholinergic function in the female rat: Effect on choline acetyltransferase activity, acetylcholine content, and nicotinic autoreceptor function. Brain Research, 525, 249–255. Loy, R., Gerlach, J. L., & McEwen, B. S. (1988). Autoradiographic localization of estraiol-binding neurons in the hippocampal formation and entorhinal cortex. Developmental Brain Research, 39, 245–251. Luine, V. N. (1985). Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats. Experimental Neurology, 89, 484–490. Maggi, A., Susanna, L., Bettini, E., Mantero, G., & Zucchi, I. (1989). Hippocampus: A target for estrogen action in the mammalian brain. Molecular Endocrinology, 3, 1165–1170. Marighetto, A., Durkin, T., Toumane, A., Lebrun, C., & Jaffard, R. (1989). Septal a-noradrenergic antagonism in vivo blocks the testing-induced activation of septohippocampal cholinergic neurones and prouces a concomitant deficit in working memory performance of mice. Pharmacology, Biochemistry and Behavior, 34, 553–558. Marighetto, A., Micheau, J., & Jaffard, R. (1994). Effects of intraseptally injected glutamatergic drugs on hippocampal sodium-dependent high-affinity choline uptake in ‘‘na€ıve’’ and ‘‘trained’’ mice. Pharmacology, Biochemistry and Behavior, 49, 689–699. McIntyre, C. K., Pal, S. N., Marriott, L. K., & Gold, P. E. (2002). Competition between memory systems: Acetylcholine release in hippocampus correlates negatively with good performance on an amygdala-dependent task. Journal of Neuroscience, 22, 1171– 1176. McIntyre, C. K., Marriott, L. K., & Gold, P. E. (2003). Patterns of brain acetylcholine release predict individual differences in preferred learning strategies in rats. Neurobiology of Learning and Memory, 79, 177–183. Miller, M. M., Hyder, S. M., Assayag, R., Panarella, S. R., Tousignant, P., & Franklin, K. B. (1999). Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain. Neuroscience, 91, 1143–1153. Orensanz, L. M., Guillamon, A., Ambrosio, E., Segovia, S., & Azvara, M. C. (1982). Sex differences in alpha-adrenergic receptors in the rat brain. Neuroscience Letters, 30, 275–278. Packard, M. G., Kohlmaier, J. R., & Alexander, G. M. (1996). Posttraining intrahippocampal estradiol injections enhance spatial memory in male rats: Interaction with cholinergic systems. Behavioral Neuroscience, 110, 626–632. Packard, M. G., & Teather, L. A. (1997a). Posttraining estradiol injections enhance memory in ovariectomized rats: Cholinergic blockade and synergism. Neurobiology of Learning and Memory, 68, 172–188. Packard, M. G., & Teather, L. A. (1997b). Intra-hippocampal estradiol infusion enhances memory in ovariectomized rats. Neuroreport, 8, 3009–3013. Pellegrino, L. J., Pellegrino, A. S., & Cushman, A. J. (1979). A stereotaxic atlas of the rat brain. New York: Plenum. Ragozzino, M. E., & Gold, P. E. (1995). Glucose injections into the medial septum reverse the effects of intraseptal morphine infusions
322
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
on hippocampal acetylcholine output and memory. Neuroscience, 68, 981–988. Ragozzino, M. E., Unick, K. E., & Gold, P. E. (1996). Hippocampal acetylcholine release during memory testing in rats: Augmentation by glucose. Proceedings of the National Academy of Sciences, 93, 4693–4698. Ragozzino, M. E., Pal, S. N., Unick, K. E., Stefani, M. R., & Gold, P. E. (1998). Modulation of hippocampal acetylcholine release and spontaneous alternation scores by intrahippocampal glucose injections. Journal of Neuroscience, 18, 1595–1601. Riekkinen, M., & Riekkinen, P. (1997). Dorsal hippocampal muscarinic acetylcholine and NMDA receptors disrupt water maze navigation. Neuroreport, 8, 645–648. Rudick, C. N., Gibbs, R. B., & Woolley, C. S. (2003). A role for the basal forebrain cholinergic system in estrogen induced disinhibition of hippocampal pyramidal cells. Journal of Neuroscience, 23, 4479– 4490. Sandstrom, N. J., & Williams, C. L. (2001). Memory retention is modulated by acute estradiol and progesterone replacement. Behavioral Neuroscience, 115, 384–393. Shuaib, A., Xu, K., Crain, B., Siren, A.-L., Feuerstein, G., Hallenbeck, J., & Davis, J. N. (1990). Assessment of damage from implantation of microdialysis probes in the rat hippocampus with silver degeneration staining. Neuroscience Letters, 112, 149–154. Shugrue, P. J., & Mercenthaler, I. (2000). Evidence for novel estrogen binding sites in the rat hippocampus. Neuroscience, 99, 605–612. Shugrue, P. J., Scrimo, P. J., & Merchenthaler, I. (2000). Estrogen binding and estrogen receptor characterization (ERa and ERb) in the cholinergic neurons of the rat basal forebrain. Neuroscience, 96, 41–49. Simpkins, J. W., Green, P. S., Gridley, K. E., Singh, M., de Fiebre, N. C., & Rajakumar, G. (1997). Role of estrogen replacement therapy in memory enhancement and the prevention of neuronal loss associated with AlzheimerÕs disease. American Journal of Medicine, 103(3A), 19S–25S. Singh, M., Meyer, E. M., Millard, W. J., & Simpkins, J. W. (1994). Ovarian steroid deprivation results in a reversible learning impairment and compromised cholinergic function in female Sprague– Dawley rats. Brain Research, 644, 305–312.
Stancampiano, R., Cocco, S., Cugusi, C., Sarais, L., & Fadda, F. (1999). Serotonin and acetylchoine release response in the rat hippocampus during a spatial memory task. Neuroscience, 89, 1135–1143. Stefani, M. R., & Gold, P. E. (2001). Intra-hippocampal infusions of K-ATP channel modulators influence spontaneous alternation performance: Relationships to acetylcholine release in the hippocampus. Journal of Neuroscience, 21, 609–614. Terasawa, E., & Timiras, P. S. (1968). Electrical activity during the estrous cycle of the rat: Cyclic changes in limbic structures. Endocrinology, 83, 207–216. Thomas, D. L., McElroy, M. W., & Korol, D. L. (2001). Learning strategy in the female rat shifts with chronic estradiol deprivation and replacement. Society for Neuroscience Abstracts, 27, 534.2. Toran-Allerand, C. D., Miranda, R. C., Bentham, W. D., Sohrabji, F., Brown, T. J., Hochberg, R. B., & MacLusky, N. J. (1992). Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain. Proceedings of the National Academy of Sciences, 89, 4668–4672. Towart, L. A., Alves, S. E., Znamensky, B., Hayashi, S., McEwen, B. S., & Milner, T. (2003). Subcellular relationships between cholinergic terminals and estrogen receptor-a in the dorsal hippocampus. Journal of Comparative Neurology, 463, 390–401. Warren, S. G., & Juraska, J. M. (1997). Spatial and nonspatial leaning across the rat estous cycle. Behavioral Neuroscience, 111, 259–266. Wenk, G. L., Hepler, D., & Olton, D. (1984). Behavior alters the uptake of [3 H]choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area. Behavioural Brain Research, 13, 129–138. Wong, M., & Moss, R. L. (1992). Long-term and short-term electrophysiological effects of estrogen on the synaptic properties of hippocampal CA1 neurons. Journal of Neuroscience, 12, 3217– 3225. Young, S. L., Bohenek, D. L., & Fanselow, M. S. (1995). Scopolamine impairs acquisition and facilitates consolidation of fear conditioning: Differential effects for tone vs context conditioning. Neurobiology of Learning and Memory, 63, 174–180.
Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning L.K. Marriotta and D.L. Korolb,* a b
Arizona Research Laboratories, Division of Neural Systems, Memory and Aging, University of Arizona, Tucson, AZ, USA Department of Psychology and Neuroscience Program, University of Illinois, 603 E. Daniel St., Champaign, IL 61820, USA Received 31 July 2003; accepted 4 August 2003
Abstract Estrogen modulates learning and memory in ovariectomized and naturally cycling female rats, especially in tasks using spatial learning and navigation. Estrogen also modulates cholinergic function in various forebrain structures. Past studies have shown positive correlations between hippocampal ACh output and performance on hippocampus-dependent tasks. The present study examined whether estradiol replacement would potentiate hippocampal ACh release during place learning. In vivo microdialysis and HPLC were used to measure extracellular ACh levels in the hippocampus of ovariectomized female rats that had received s.c. injections of 17b-estradiol (10 lg) or sesame oil (vehicle treatment) 48 and 24 h prior to training on a place task. Estrogen did not alter baseline levels of extracellular ACh in the hippocampus. During training, hippocampal ACh increased in ovariectomized rats regardless of estrogen status. However, while estradiol did not enhance learning in this experiment, estradiol significantly potentiated the increase in hippocampal ACh release seen during place training. This represents the first demonstration of on-line assessment of ACh output in hippocampus during learning in female rats and suggests that estrogen-dependent modulation of ACh release during training might control activation of different neural systems used during learning. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Ovariectomy; Microdialysis; Gonadal steroids; Acetylcholine; Hippocampus; Estradiol; Spatial memory
1. Introduction Increases in circulating estradiol levels can modulate learning and memory on a wide range of tests across several species. Even though increases in estradiol have been found to impair learning and memory on certain tasks (Frye, 1995; Galea, Kavaliers, Ossenkopp, & Hampson, 1995; Korol et al., 1994; Korol & Kolo, 2002; Warren & Juraska, 1997), a large literature supports the idea that estrogen enhances memory on many tasks, especially those that tap working memory or hippocampal processing (See Dohanich, 2002 for review). It is likely that estrogen not only influences memory processing, but also affects the cognitive strategy used in a learning situation, perhaps through differential modulation of neural systems (Korol & Kolo, 2002; Korol & Manning, 2001). * Corresponding author. Fax: 1-217-244-5876. E-mail address: [email protected] (D.L. Korol).
1074-7427/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2003.08.003
The specific mechanisms by which estradiol enhances cognitive function are unknown, but are likely to include direct actions on specific brain structures and neurochemical systems. Circulating estradiol readily crosses the blood brain barrier (Kawata, 1995) and can be found to accumulate in neurons across many forebrain structures, such as the hippocampus, cortex, and basal forebrain nuclei. Many of these brain regions, including the hippocampus (Loy, Gerlach, & McEwen, 1988; Maggi, Susanna, Bettini, Mantero, & Zucchi, 1989; Shugrue & Mercenthaler, 2000), bed nucleus of the stria terminalis (Miller et al., 1999), diagonal band nuclei (Orensanz, Guillamon, Ambrosio, Segovia, & Azvara, 1982), and medial septal area (Fallon, Loughlin, & Ribak, 1983) contain classical estrogen receptors (ER) and DNA binding sites for estrogen. Of particular interest here, the brain regions that provide cholinergic innervation to the hippocampal formation and neocortex such as the medial septum,
316
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
diagonal band, and nucleus basalis magnocellularis (Everitt & Robbins, 1997) accumulate estrogen (Shugrue, Scrimo, & Merchenthaler, 2000) and are neurochemically sensitive to estrogen as seen by changes in synthesis, turnover, and release of acetylcholine. For example, estrogen administration to ovariectomized female rats has multiple effects on the cholinergic system, including increased levels of choline acetyltransferase (ChAT), ChAT mRNA expression and ChAT activity in the basal forebrain and high-affinity choline uptake in the hippocampal formation and cortex (Gibbs, 1996a, 1996b; Gibbs & Pfaff, 1992; Gibbs, Wu, Hersh, & Pfaff, 1994; Lapchak, Araujo, Quirion, & Beaudet, 1990; Luine, 1985; Simpkins et al., 1997; Singh, Meyer, Millard, & Simpkins, 1994). In addition, the number of ChATimmunoreactive neurons detected in the medial septum and vertical limb of the diagonal band of Broca (Gibbs & Pfaff, 1992) and stria terminalis (Miller et al., 1999) increases with estrogen replacement as does the number of neurons that are immunoreactive for both ChAT and ER in the medial septum, diagonal band nuclei, substantia innominata, ventral pallidum (Toran-Allerand et al., 1992), and stria terminalis (Miller et al., 1999). Estrogen replacement also increases potassium-evoked but not basal release of acetylcholine (ACh) in the hippocampus and cortex (Gabor, Nagle, Johnson, & Gibbs, 2003; Gibbs, Hashash, & Johnson, 1997), suggesting enhanced ACh release upon activation of the cholinergic input. Hippocampal cholinergic function has been implicated in learning and memory (see Gold, 2003 for review) with intrahippocampal ACh agonists enhancing (Kim & Levin, 1996) and ACh antagonists impairing spatial, contextual, and working memory tasks (Anagnostaras, Maren, & Fanselow, 1995; Carli, Luschi, & Samanin, 1995; Riekkinen & Riekkinen, 1997; Young, Bohenek, & Fanselow, 1995; Kim & Levin, 1996). Moreover, release of ACh in the hippocampus increases while male rats solve navigation tasks (Ragozzino, Unick, & Gold, 1996; Stancampiano, Cocco, Cugusi, Sarais, & Fadda, 1999) and this increase in ACh output is positively correlated with good performance on a variety of hippocampus-dependent tasks in male rats (Chang & Gold, 2003; Gold, 2003; McIntyre, Pal, Marriott, & Gold, 2002; Ragozzino & Gold, 1995). Together these findings suggest that ACh release may be a marker for activation and participation of the hippocampus in learning and memory. Acute estrogen administration also biases the learning strategy used by rats to solve a navigation task. For example, systemic administration of estradiol to ovariectomized female rats enhanced place learning, but impaired response learning on the similar mazes (Korol & Kolo, 2002). Thus, ACh and estrogen both contribute to regulating the participation of different neural systems in learning and memory. Recent
evidence indicates that estrogen may interact with cholinergic function in the hippocampus to modulate learning and memory (Fader, Hendricson, & Dohanich, 1998; Farr, Banks, & Morley, 2000; Packard & Teather, 1997a). Perhaps estradiol replacement potentiates hippocampal ACh release during performance of spatial tasks, thereby biasing strategy to favor place over response learning. The present study uses in vivo microdialysis to investigate whether estrogen replacement can augment ACh release in the hippocampus of ovariectomized female rats that are engaged in a place learning task.
2. Materials and methods 2.1. Subjects Fifteen virgin female Sprague–Dawley rats (Hilltop breeders, Scottdale, PA), 2–3 months of age, were housed individually with food and water available ad libitum. All animals were maintained on a 12-h light/ dark cycle and allowed to adjust to their new environment for 1 week following arrival. 2.2. Surgery Rats received atropine sulfate (0.4 mg/kg in 0.2 ml, i.p.) prior to anesthesia with sodium pentobarbital (50 mg/kg, i.p.) and were placed in a stereotaxic instrument with the incisor bar set at 5.0 mm above the interaural line. A plastic guide cannula (CMA/12; Carnegie Medicine, Stockholm) was lowered into the hippocampus at 3.45 mm posterior to bregma, 5.4 mm lateral, and 2.2 mm ventral from skull (from Pellegrino, Pellegrino, & Cushman, 1979). Three screws were used as anchors, and the cannula and screws were affixed to the skull with dental acrylic cement. Immediately after the cannula surgery was complete, rats were bilaterally ovariectomized with a dorsolateral approach. All sutured areas were cleaned with betadine one day after surgery. Rats were allowed to recover from surgery for at least one week prior to initiation of handling and food deprivation procedures. 2.3. Behavioral procedures Rats were trained on a place learning task 20–24 days following ovariectomy. The training apparatus was a four-arm black plexiglass plus-shaped maze. The maze rested on a table, elevated 88 cm from the floor, in the center of the room. The center platform was a 13 13 cm square; each arm was 13 cm wide, 45 cm long, with walls 7 cm high. The room contained various extra-maze visual cues including a desk, a large black curtain, a dimmed halogen light, and a lab bench with cabinets.
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Rats were food-restricted to approximately 80% of free-feeding body weight and handled daily (5 min/day) for 7 days. Animals were randomly assigned to one of two treatment groups: ovariectomy with estradiol (ovx + E2; n ¼ 8) and ovariectomy without hormone replacement (ovx + OIL; n ¼ 7, 2 eliminated as described below for a final n ¼ 5). At 48 and 24 h prior to behavioral testing, animals received s.c. injections of either 10 lg of 17-b estradiol-benzoate (Sigma) dissolved in 0.1 cc of sesame oil (ovx + E2) or vehicle alone (ovx + OIL). Twenty-four hours prior to training, rats received approximately 15 Frosted Cheerios in their home cages to decrease neophobia associated with the food reinforcement. Rats were trained to find food (one Frosted Cheerios pellet) at the end of the goal arm. The goal arm remained in a fixed position relative to the extra-maze cues. Start arms were randomized across the other three non-goal arms. Animals were trained to a choice accuracy of 9/10 trials correct, or to a maximum of 100 trials. An arm entry was recorded when all four paws passed a halfway mark in the arm. Following each arm choice, the animal was returned to the holding cage for 30 s while the maze was rotated 90° clockwise using pre-set stops. 2.4. Microdialysis procedure Twenty-four hours prior to training, a dialysis probe was inserted into the guide cannula for 5 min. This procedure minimizes obstruction due to gliosis during dialysis 24 h later (Benveniste, Drejer, Schousboe, & Diemer, 1987; Shuaib et al., 1990). On the day of training, a 3-mm dialysis probe (CMA/12; Carnegie Medicine) was inserted through the guide cannula and connected to plastic polyethylene tubing driven by a microinfusion system (CMA/100; Carnegie Medicine). The dialysis probe was continuously perfused at a rate of 2 ll/min with artificial cerebrospinal fluid: 128 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2 , 2.1 mM MgCl2 , 21 mM NaH2 PO4 , 1.3 mM Na2 HPO4 , 1.0 mM glucose, and brought to, pH 7.02, by NaOH, which contained 1 lM of the acetylcholinesterase inhibitor, neostigmine. We previously found that percent increases in release of ACh in the hippocampus during training are of comparable magnitude when neostigmine concentrations ranging from 0.1 to 6 lM are used (Chang, Savage, & Gold, 2003; Ragozzino et al., 1996; Stefani & Gold, 2001). The dialysis probe was inserted 105 min prior to behavioral training. The first 45 min of dialysis permitted equilibration between tissue and perfusion solution; these samples were discarded. Samples (24 ll) were then collected on ice every 12 min through the conclusion of the experiment. Four baseline samples were collected while rats remained in their holding cages. Immediately after collection of the baseline samples, rats were trained
317
on a place-learning version of the four-arm plus maze. Perfusate samples were collected every 12 min during training. The number of samples collected during maze learning varied with the number of trials to criterion. Four additional samples were collected after training while the rat remained in its holding cage. 2.5. Acetylcholine assay Using 20 ll of sample, ACh content was assessed using reverse-phase high performance liquid chromatography, with an enzymatic assay system and electrochemical detector. ACh was separated from free choline on a reverse-phase analytical column (Chromspher 5 C18, 100 3 mm, Chrompack, Middleburg, The Netherlands). An enzymatic postcolumn reactor unit containing acetylcholinesterase (EC 3.1.1.7; Sigma type VI-S) and choline oxidase (EC 1.1.3.17; Sigma) converted ACh ultimately to betaine and H2 O2 . Oxidation of H2 O2 was electrochemically detected by a platinum electrode held at +525 mV relative to a silver chloride reference electrode. Mobile phase (pH 8.0) containing 0.2 mM dibasic potassium phosphate, 1.0 mM tetramethylammonium hydroxide, 0.3 mM EDTA, and 0.005% Kathon CG (to prevent bacterial growth) was delivered at a rate of 0.6 ml/min by a solvent delivery system (ESA 580; ESA, Chelmsford, MA). ACh peaks were quantified by comparison to peak heights of ACh standard solutions prepared at the time of each assay. The detection limit for ACh was 50 fmol. 2.6. Histology Immediately following completion of microdialysis sample collection, rats were given a lethal dose of sodium pentobarbital followed by cardiac puncture to remove 3 cc of blood for subsequent analyses of circulating estradiol levels. Blood was kept on ice in a test tube containing 0.5 ml lithium heparin (Vacutainer Plus PST, Becton–Dickinson, Franklin Lakes, NJ) and centrifuged at 4 °C at 3500g for 12 min. The plasma was then collected and stored at )80 °C until estradiol levels were analyzed with radioimmunoassay (Diagnostic Research, Webster, TX). The assay has a sensitivity of 8 pg/ ml, sufficient to reveal differences between plasma levels of estradiol in our ovariectomized and hormone-treated rats. Moreover, the assay is quite specific to estradiol with cross-reactivity of 0.32% for estriole and 0.001% for testosterone. After blood collection, rats were perfused intracardially with 0.9% saline and a 10% formalin solution. Brains were removed and stored in a 30% sucrose/formalin solution. Brains were frozen at )20 °C, sectioned at 40 lm and stained with cresyl violet. Only rats with guide cannula placements in the hippocampus (Fig. 1) were included in the final analysis.
318
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Fig. 1. Photomicrograph showing the placement of the guide cannula in the hippocampus. Note that rats were excluded from analysis when histological characterization indicated misplacement of the microdialysis probe.
Vaginal smears were taken daily for seven days prior to behavioral testing to determine cycle stage and to validate the effectiveness of ovariectomy and hormone replacement. Vaginal smears were stained with HarrisÕ hematoxylin and eosin solutions and staged by microscopic examination. Each daily smear was assigned to one of three cytological stages: proestrus, estrus, and diestrus. 2.7. Data analyses Baseline ACh values were derived from the mean of the first four baseline samples taken prior to training. ACh values from samples collected during and after training were converted to percent difference scores using each ratÕs baseline values. Because the duration of training differed with number of trials to criterion, measures of ACh release during training included mean values during different periods of maze training, e.g., first half, second half, and final sample reflecting release as the rat reached criterion performance, and the postmaze period. Four samples were averaged for the postmaze period. Paired two-tailed student t tests were used to compare the mean change in values of ACh release between baseline and later samples within each group and unpaired two-tailed student t tests were used to compare baseline values and percent changes between treatment groups. Because of the 100-trial maximum, trials to criterion were compared using the non-parametric Mann–Whitney U test.
(t ¼ 3:9 for ovx + OIL, t ¼ 9:3, ovx + E2; p’s < :05). Estrogen treatment significantly enhanced ACh release while animals performed the place learning task (all maze samples averaged; t ¼ 2:2; p < :05). Specifically, in samples collected during training, ovx + OIL rats exhibited an average increase of 68 19% above baseline ACh release while the estrogen-treated rats demonstrated an increase of 121 14% ðp < :05Þ. This estrogen-induced potentiation of ACh release was evident during both the first (t ¼ 2:15; p ¼ :05) and second (t ¼ 2:26; p < :05) half of training (see Fig. 2). ACh release in the last sample, i.e., obtained as rats approached criterion performance, remained higher in estrogen- than in oil-treated rats (60 16% vs. 114 19%, oil vs estrogen, respectively, t ¼ 2:32; p < :05). Once rats reached criterion and were removed from the maze, extracellular ACh values dropped rapidly, nearing those at baseline. The averaged post-maze values were not different from baseline (ovx + OIL: t ¼ 1:27; p > :05; ovx + E2: t ¼ 1:35; p > :05) and did not differ by treatment group (t ¼ 0:28; p > :05). Rats in both treatment groups took comparable number of trials to reach criterion, i.e., 9 out of 10 correct (median ¼ 79 for oil- and 79 for estrogen-treated rats, U8;5 ¼ 14; p > :05). Plasma estradiol levels and cytological examinations confirmed the efficacy of the ovariectomy and of hormone replacement. Plasma estradiol levels were below the sensitivity level of the RIA for all oil-treated rats with the exception of two animals. These two animals had circulating estrogen levels of 25–26 pg/ml (comparable to diestrous levels), suggesting incomplete ovariectomy, and were thus excluded from the analysis. Estradiol treatment led to substantially elevated plasma levels of 104.4 14.1 pg/ml. While these values are high, they are not outside the range of values found at proestrus (Jarrar, Want, Cioffi, Bland, & Chaudry, 2000).
3. Results Estrogen treatment did not significantly alter baseline levels of extracellular ACh (t ¼ 0:41; p > :05). Mean ACh content in baseline samples was 4.2 1.1 pmol for the oil-treated group and 5.5 1.1 pmol for the estrogen-treated group. Rats in both groups showed increases in extracellular ACh levels during maze training
Fig. 2. Extracellular levels of ACh from hippocampus rose significantly during training (*). Estradiol treatment significantly potentiated this maze-related increase (+). The estrogen effects were evident during the first and second half of training as well as during the last sample reflecting the sample that learning criterion was reached. Also note that ACh levels returned to near baseline values during the 48-min, postmaze period.
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
Fig. 3. Photomicrographs of vaginal smears stained with hematoxylin and eosin indicating diestrous (upper panel) and proestrous (lower panel) smears. Note that and diestrous smears are characterized primarily by the presence of leukocytes and proestrous smears by nucleated epithelial cells.
The intra-assay variance for samples from all animals was 5.4 0.7%. Results from vaginal smears indicated that ovx + OIL rats had diestrus smears (Fig. 3) highlighted by leukocytes, throughout the experimental period. Rats with estradiol replacement had diestrus smears until treatment, after which they demonstrated proestrus smears (Fig. 3) highlighted by nucleated endothelial cells. All ovx + E2 rats showed proestrus smears on the day of training.
4. Discussion The present findings indicate that ACh levels in the hippocampus increase in female rats performing a placelearning task. These data are consistent with previous in vivo microdialysis studies in male rats suggesting that activation of an intact hippocampal system, as reflected
319
by an increase in ACh output, may modulate acquisition of different spatial tasks (McIntyre et al., 2002; Ragozzino & Gold, 1995; Ragozzino, Pal, Unick, Stefani, & Gold, 1998; Stancampiano et al., 1999). Moreover, these data also support previous work showing that cholinergic input to the hippocampal formation, as assessed by high affinity choline uptake activity, is activated during learning and memory (Marighetto, Durkin, Toumane, Lebrun, & Jaffard, 1989, 1994; Wenk, Hepler, & Olton, 1984). While ACh output increased in the hippocampus during maze learning regardless of estrogen status, estrogen replacement dramatically potentiated the increase in ACh release compared to hormone-deprived rats. Interestingly, estrogen replacement failed to alter baseline hippocampal ACh release, suggesting that estrogen treatment may enhance ACh release only when the system is otherwise activated such as during learning. This idea is supported by previous work demonstrating a similar potentiation of hippocampal ACh levels following short- and long-term estrogen treatment, but only after direct potassium-induced activation of the hippocampus (Gabor et al., 2003; Gibbs et al., 1997). That estrogen enhances ACh output fits in well with a rapidly growing literature supporting the view that estrogen regulates cholinergic function and the view that other neurobiological effects of estrogen may be regulated through cholinergic actions. Specifically, estradiol replacement increases ChAT and ChAT mRNA in the medial septal area (Gibbs, 1996b) and enhances ChAT activity in the CA1 region of the hippocampus (Luine, 1985). It is likely that estrogen acts through regulation of cholinergic function to modify dendritic spine density (Lam & Leranth, 2003) and NMDA receptor binding (Daniel & Dohanich, 2001). Furthermore, the ability of estrogen to disinhibit hippocampal CA1 pyramidal cells may partially depend on intact cholinergic input (Rudick, Gibbs, & Woolley, 2003). Together, these findings suggest that estrogen regulation of ACh may be involved in modulating neuronal excitability and the cellular and subcellular sequelae that ensue to alter neuronal plasticity and learning and memory. Consistent with this view, a subset (3–17%) of terminals in the dorsal hippocampus show colocalization of the vesicular acetylcholine transporter and ERa, supporting the possibility of a direct, non-genomic mechanism for the effects of estrogen on acetylcholine release (Towart et al., 2003). It is unlikely that the testing-induced increases in hippocampal ACh release found in the present study are due solely to increased locomotion. There was no correlation between the number of arm entries per 12-min sample and the amount of ACh released in the hippocampus (data not shown). In addition, previous studies indicate that unlike what has been found for
320
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
learning and memory, changes in hippocampal highaffinity choline uptake (Decker, Pelleymounter, & Gallagher, 1988; Wenk et al., 1984) or extracellular ACh levels measured by microdialysis (Ragozzino et al., 1996) are not secondary to locomotor activity. Our behavioral data indicate that acute estradiol administration failed to enhance place learning three weeks after ovariectomy. These data differ from those found previously in our laboratory in which the same estrogen regimen facilitated place or allocentric learning, but impaired response or egocentric learning (Korol & Kolo, 2002). Furthermore, we have shown that across the estrous cycle or with moderate durations of hormone replacement, i.e., 1 or 4 weeks, rats characterized by high circulating estrogen choose placelearning strategies over equally effective response strategies (Korol, Malin, Borden, Busby, & CouperLeo, 2003; Thomas, McElroy, & Korol, 2001). The lack of effects of estrogen on spatial learning found in the present experiment may be due to several factors including contamination of the training environment from the blockade of cues by the microdialysis tubing or investigator, and the disruption of surrounding tissue by the microdialysis probe. The latter effect may be exacerbated by the presence of increased levels of circulating estrogen, shown to enhance excitability and to decrease seizure threshold (Terasawa & Timiras, 1968; Wong & Moss, 1992), thereby potentially impairing performance to a greater degree in estrogen-treated rats. We are currently testing whether estrogen alters the balance of ACh release in hippocampus and striatum, with a predicted increase in hippocampal versus striatal ACh, and shifts learning towards the use of place strategies during acquisition. Evidence that ACh ratios in these structures are predictive of learning strategies has been shown in males (McIntyre, Marriott, & Gold, 2003). In addition to its effects on learning strategy, many studies have found that estradiol administration enhances memory. Performance on radial maze spatial working memory tasks is enhanced under various regimens of estrogen administration including acute (Sandstrom & Williams, 2001) or chronic replacement (Daniel, Fader, Spencer, & Dohanich, 1997). Relevant to the current findings, estrogen counteracts memory impairments induced by scopolamine, a cholinergic antagonist, when injected systemically (Dohanich, Fader, & Javorsky, 1994; Packard, Kohlmaier, & Alexander, 1996) or intrahippocampally (Fader et al., 1998; Packard & Teather, 1997b). Moreover, posttraining intrahippocampal estradiol injections in male rats enhanced performance on a hidden platform swim task that could be attenuated by concurrent peripheral administration of scopolamine (Packard et al., 1996). In sum, this study represents the first demonstration of on-line assessment of ACh output in hippocampus
during learning in hormone treated- and hormone-deprived female rats. The augmentation of hippocampal ACh output, during training but not at baseline, suggests that acute estrogen treatment may modulate the participation of different neural systems used during learning. Cholinergic function may be one marker or one mechanism of this modulation.
Acknowledgments This work was supported by a research grant from the National Science Foundation (IBN-0081061 DLK), with additional research support from Paul Gold, National Institute of Aging (AG 06748) and National Institute for Neurological Diseases and Stroke (NS 32914). We would like to thank Christa McIntyre for help with technical aspects of microdialysis and HPLC analysis, and Trish Pruis and Ed Roy for help with photomicrographs.
References Anagnostaras, S. G., Maren, S., & Fanselow, M. S. (1995). Scopolamine selectively disrupts the acquisition of contextual fear conditioning in rats. Neurobiology of Learning and Biology, 64, 191–194. Benveniste, H., Drejer, J., Schousboe, A., & Diemer, N. (1987). Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in rat. Journal of Neurochemistry, 49, 729–734. Carli, M., Luschi, R., & Samanin, R. (1995). (S)-WAY 100135, a 5HT1A receptor antagonist, prevents the impairment of spatial learning caused by intrahippocampal scopolamine. European Journal of Pharmacology, 283, 133–139. Chang, Q., & Gold, P. E. (2003). Switching from hippocampal to striatal memory systems during learning: Changes in patterns of acetylcholine release in rats. Journal of Neuroscience, 23, 3001– 3005. Chang, Q., Savage, L. M., & Gold, P. E. (2003). Use of acetylcholinesterase (AChE) inhibitors in the perfusate during brain microdialysis to measure acetylcholine (ACh) release during learning and memory. In Society for Neuroscience, 33rd Annual Meeting, New Orleans. Daniel, J. M., & Dohanich, G. P. (2001). Acetylcholine mediates the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus and the associated improvement in working memory. Journal of Neuroscience, 21, 6949–6956. Daniel, J. M., Fader, A. J., Spencer, A. L., & Dohanich, G. P. (1997). Estrogen enhances performance of female rats during acquisition of a radial arm maze. Hormones and Behavior, 32, 217–225. Decker, M. W., Pelleymounter, M. A., & Gallagher, M. (1988). Effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats. Journal of Neuroscience, 8, 90–99. Dohanich, G. P. (2002). Gonadal steroids, learning and memory. In D. W. Pfaff, A. P. Arnold, A. M. Etgen, S. E. Fahrbach, & R. T. Rubin (Eds.), Hormones, brain and behavior (pp. 265–327). San Diego: Academic Press. Dohanich, G. P., Fader, A. J., & Javorsky, D. J. (1994). Estrogen and estrogen-progesterone treatments counteract the effect of scopol-
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322 amine on reinforced T-maze alternation in female rats. Behavioral Neuroscience, 108, 988–992. Everitt, B. J., & Robbins, T. W. (1997). Central cholinergic systems and cognition. Annual Review of Psychology, 48, 649–684. Fader, A. J., Hendricson, A. W., & Dohanich, G. P. (1998). Estrogen improve performance of reinforced T-maze alternation and prevents the amnestic effects of scopolamine administered systemically or intrahippocampally. Neurobiology of Learning and Memory, 69, 225–240. Fallon, J. H., Loughlin, S. E., & Ribak, C. E. (1983). The islands of Calleja complex of rat basal forebrain. III. Histochemical evidence of a striatopallidal system. Journal of Comparative Neurology, 218, 91–120. Farr, S. A., Banks, W. A., & Morley, J. E. (2000). Estradiol potentiates acetylcholine and glutamate-mediated post-trial memory processing in the hippocampus. Brain Research, 864, 263–269. Frye, C. A. (1995). Estrus-associated decrements in a water maze task are limited to acquisition. Physiology and Behavior, 57, 5–14. Gabor, R., Nagle, R., Johnson, D. A., & Gibbs, R. B. (2003). Estrogen enhances potassium-stimulated acetylcholine release in the rat hippocampus. Brain Research, 962, 244–247. Galea, L. A., Kavaliers, M., Ossenkopp, K. P., & Hampson, E. (1995). Gonadal hormone levels and spatial learning performance in the Morris water maze in male and female meadow voles, Microtus pennsyvanicus. Hormones and Behavior, 29, 106–125. Gibbs, R. B. (1996a). Expression of estrogen receptor-like immunoreactivity by different subgroups of basal forebrain cholinergic neurons in gonadectomized male and female rats. Brain Research, 720, 61–68. Gibbs, R. B. (1996b). Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: Effects of estrogen and progesterone. Journal of Neuroscience, 16, 1049–1055. Gibbs, R. B., & Pfaff, D. W. (1992). Effects of estrogen and fimbriafornix transection on p75ngrf and ChAT expression in the medial septum and the diagonal band of Broca. Experimental Neurology, 116, 23–29. Gibbs, R. B., Wu, D., Hersh, L. B., & Pfaff, D. W. (1994). Effects of estrogen replacement on the relative levels of choline acetyltransferase, trkA, and nerve growth factor messenger RNAs in the basal forebrain and hippocampal formation of adult rats. Experimental Neurology, 129, 70–80. Gibbs, R. B., Hashash, A., & Johnson, D. (1997). Effects of estradiol on potassium-stimulated acetylcholine release in the hippocampus and overlying cortex of adult rats. Brain Research, 749, 143–146. Gold, P. E. (2003). Acetylcholine modulation of neural systems involved in learning and memory. Neurobiology of Learning and Memory, 80, 194–210 Jarrar, D., Want, P., Cioffi, W. G., Bland, K. I., & Chaudry, I. H. (2000). The female reproductive cycle is an important variable in the response to trauma-hemorrhage. American Journal of Physiology, 279, H1015–H1021. Kawata, M. (1995). Roles of steroid hormones and their receptors in structural organization in the nervous system. Neuroscience Research, 24, 1–46. Kim, J. S., & Levin, E. D. (1996). Nicotinic, muscarinic and dopaminergic actions in the ventral hippocampus and the nucleus accumbens: Effects on spatial working memory in rats. Brain Research, 725, 231–240. Korol, D. L., & Kolo, L. L. (2002). Estrogen-induced changes in place and response learning in young adult female rats. Behavioral Neuroscience, 116, 411–420. Korol, D. L., Malin, E. L., Borden, K. A., Busby, R.A., & CouperLeo, J.M., (2003). Shifts in preferred learning strategy across the estrous cycle in female rats, Submitted. Korol, D. L., & Manning, C. A. (2001). Effects of estrogen on cognition: Implications for menopause. In M. E. Carroll, & J. B.
321
Overmier (Eds.), Animal research and human health: Advancing human welfare through behavioral science (pp. 305–322). Washington, DC: American Psychological Association. Korol, D. L., Unick, K., Goosens, K., Crane, C., Gold, P. E., & Foster, T. C. (1994). Estrogen effects on spatial performance and hippocampal physiology in female rats. Society for Neuroscience Abstracts, 20, 1436. Lam, T. T., & Leranth, C. (2003). Role of the medial septum diagonal band of Broca cholinergic neurons in oestrogen-induced spine synapse formation on hippocampal CA1 pyramidal cells of female rats. European Journal of Neuroscience, 17, 1997–2005. Lapchak, P. A., Araujo, D. M., Quirion, R., & Beaudet, A. (1990). Chronic estradiol treatment alters central cholinergic function in the female rat: Effect on choline acetyltransferase activity, acetylcholine content, and nicotinic autoreceptor function. Brain Research, 525, 249–255. Loy, R., Gerlach, J. L., & McEwen, B. S. (1988). Autoradiographic localization of estraiol-binding neurons in the hippocampal formation and entorhinal cortex. Developmental Brain Research, 39, 245–251. Luine, V. N. (1985). Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats. Experimental Neurology, 89, 484–490. Maggi, A., Susanna, L., Bettini, E., Mantero, G., & Zucchi, I. (1989). Hippocampus: A target for estrogen action in the mammalian brain. Molecular Endocrinology, 3, 1165–1170. Marighetto, A., Durkin, T., Toumane, A., Lebrun, C., & Jaffard, R. (1989). Septal a-noradrenergic antagonism in vivo blocks the testing-induced activation of septohippocampal cholinergic neurones and prouces a concomitant deficit in working memory performance of mice. Pharmacology, Biochemistry and Behavior, 34, 553–558. Marighetto, A., Micheau, J., & Jaffard, R. (1994). Effects of intraseptally injected glutamatergic drugs on hippocampal sodium-dependent high-affinity choline uptake in ‘‘na€ıve’’ and ‘‘trained’’ mice. Pharmacology, Biochemistry and Behavior, 49, 689–699. McIntyre, C. K., Pal, S. N., Marriott, L. K., & Gold, P. E. (2002). Competition between memory systems: Acetylcholine release in hippocampus correlates negatively with good performance on an amygdala-dependent task. Journal of Neuroscience, 22, 1171– 1176. McIntyre, C. K., Marriott, L. K., & Gold, P. E. (2003). Patterns of brain acetylcholine release predict individual differences in preferred learning strategies in rats. Neurobiology of Learning and Memory, 79, 177–183. Miller, M. M., Hyder, S. M., Assayag, R., Panarella, S. R., Tousignant, P., & Franklin, K. B. (1999). Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain. Neuroscience, 91, 1143–1153. Orensanz, L. M., Guillamon, A., Ambrosio, E., Segovia, S., & Azvara, M. C. (1982). Sex differences in alpha-adrenergic receptors in the rat brain. Neuroscience Letters, 30, 275–278. Packard, M. G., Kohlmaier, J. R., & Alexander, G. M. (1996). Posttraining intrahippocampal estradiol injections enhance spatial memory in male rats: Interaction with cholinergic systems. Behavioral Neuroscience, 110, 626–632. Packard, M. G., & Teather, L. A. (1997a). Posttraining estradiol injections enhance memory in ovariectomized rats: Cholinergic blockade and synergism. Neurobiology of Learning and Memory, 68, 172–188. Packard, M. G., & Teather, L. A. (1997b). Intra-hippocampal estradiol infusion enhances memory in ovariectomized rats. Neuroreport, 8, 3009–3013. Pellegrino, L. J., Pellegrino, A. S., & Cushman, A. J. (1979). A stereotaxic atlas of the rat brain. New York: Plenum. Ragozzino, M. E., & Gold, P. E. (1995). Glucose injections into the medial septum reverse the effects of intraseptal morphine infusions
322
L.K. Marriott, D.L. Korol / Neurobiology of Learning and Memory 80 (2003) 315–322
on hippocampal acetylcholine output and memory. Neuroscience, 68, 981–988. Ragozzino, M. E., Unick, K. E., & Gold, P. E. (1996). Hippocampal acetylcholine release during memory testing in rats: Augmentation by glucose. Proceedings of the National Academy of Sciences, 93, 4693–4698. Ragozzino, M. E., Pal, S. N., Unick, K. E., Stefani, M. R., & Gold, P. E. (1998). Modulation of hippocampal acetylcholine release and spontaneous alternation scores by intrahippocampal glucose injections. Journal of Neuroscience, 18, 1595–1601. Riekkinen, M., & Riekkinen, P. (1997). Dorsal hippocampal muscarinic acetylcholine and NMDA receptors disrupt water maze navigation. Neuroreport, 8, 645–648. Rudick, C. N., Gibbs, R. B., & Woolley, C. S. (2003). A role for the basal forebrain cholinergic system in estrogen induced disinhibition of hippocampal pyramidal cells. Journal of Neuroscience, 23, 4479– 4490. Sandstrom, N. J., & Williams, C. L. (2001). Memory retention is modulated by acute estradiol and progesterone replacement. Behavioral Neuroscience, 115, 384–393. Shuaib, A., Xu, K., Crain, B., Siren, A.-L., Feuerstein, G., Hallenbeck, J., & Davis, J. N. (1990). Assessment of damage from implantation of microdialysis probes in the rat hippocampus with silver degeneration staining. Neuroscience Letters, 112, 149–154. Shugrue, P. J., & Mercenthaler, I. (2000). Evidence for novel estrogen binding sites in the rat hippocampus. Neuroscience, 99, 605–612. Shugrue, P. J., Scrimo, P. J., & Merchenthaler, I. (2000). Estrogen binding and estrogen receptor characterization (ERa and ERb) in the cholinergic neurons of the rat basal forebrain. Neuroscience, 96, 41–49. Simpkins, J. W., Green, P. S., Gridley, K. E., Singh, M., de Fiebre, N. C., & Rajakumar, G. (1997). Role of estrogen replacement therapy in memory enhancement and the prevention of neuronal loss associated with AlzheimerÕs disease. American Journal of Medicine, 103(3A), 19S–25S. Singh, M., Meyer, E. M., Millard, W. J., & Simpkins, J. W. (1994). Ovarian steroid deprivation results in a reversible learning impairment and compromised cholinergic function in female Sprague– Dawley rats. Brain Research, 644, 305–312.
Stancampiano, R., Cocco, S., Cugusi, C., Sarais, L., & Fadda, F. (1999). Serotonin and acetylchoine release response in the rat hippocampus during a spatial memory task. Neuroscience, 89, 1135–1143. Stefani, M. R., & Gold, P. E. (2001). Intra-hippocampal infusions of K-ATP channel modulators influence spontaneous alternation performance: Relationships to acetylcholine release in the hippocampus. Journal of Neuroscience, 21, 609–614. Terasawa, E., & Timiras, P. S. (1968). Electrical activity during the estrous cycle of the rat: Cyclic changes in limbic structures. Endocrinology, 83, 207–216. Thomas, D. L., McElroy, M. W., & Korol, D. L. (2001). Learning strategy in the female rat shifts with chronic estradiol deprivation and replacement. Society for Neuroscience Abstracts, 27, 534.2. Toran-Allerand, C. D., Miranda, R. C., Bentham, W. D., Sohrabji, F., Brown, T. J., Hochberg, R. B., & MacLusky, N. J. (1992). Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain. Proceedings of the National Academy of Sciences, 89, 4668–4672. Towart, L. A., Alves, S. E., Znamensky, B., Hayashi, S., McEwen, B. S., & Milner, T. (2003). Subcellular relationships between cholinergic terminals and estrogen receptor-a in the dorsal hippocampus. Journal of Comparative Neurology, 463, 390–401. Warren, S. G., & Juraska, J. M. (1997). Spatial and nonspatial leaning across the rat estous cycle. Behavioral Neuroscience, 111, 259–266. Wenk, G. L., Hepler, D., & Olton, D. (1984). Behavior alters the uptake of [3 H]choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area. Behavioural Brain Research, 13, 129–138. Wong, M., & Moss, R. L. (1992). Long-term and short-term electrophysiological effects of estrogen on the synaptic properties of hippocampal CA1 neurons. Journal of Neuroscience, 12, 3217– 3225. Young, S. L., Bohenek, D. L., & Fanselow, M. S. (1995). Scopolamine impairs acquisition and facilitates consolidation of fear conditioning: Differential effects for tone vs context conditioning. Neurobiology of Learning and Memory, 63, 174–180.