natural oligomers of the amyloid-β protein specifically disrupt cognitive function
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The concept that cerebral accumulation of amyloid-β induces Alzheimer disease, the commonest cause of cognitive failure in older humans, remains controversial, in large part because of the difficulty in providing direct mechanistic evidence that an amyloid-β species impairs cognitive function in vivo. Alzheimer disease brain contains soluble and insoluble assemblies of amyloid-β, both of which have been hypothesized to underlie dementia1–7. Early evidence for amyloid-β-induced neurotoxicity in cell culture and in vivo was associated with insoluble, fibrillar forms of amyloid-β, such as those found in neuritic (amyloid) plaques4,8. In these studies, the neurotoxicity of amyloid-β persisted while aggregation was ongoing but diminished as the pro-cess of aggregation neared completion, suggesting that intermediate assemblies possessed neurotoxic activity. Studies in transgenic mice that overexpress the β-amyloid precursor protein (APP) have indicated that the accumulation of insoluble amyloid-β is accompanied by minimal neuron loss9,10 and that the correlation between total insoluble amy-loid-β and the degree of memory impairment is relatively weak11,12. In humans, a similarly weak correlation between insoluble amyloid-β deposits and dementia has been found13,14, which is exemplified by the existence of some cognitively normal individuals with high amounts of amyloid-β14–16. These observations indicate that species other than insoluble fibrillar amyloid-β may underlie cognitive deficits or neuro-degeneration in transgenic models of Alzheimer disease, and possibly in the very early stages of Alzheimer disease. They do not, however, exclude the possibility that fibrillar amyloid-β has some role in the
transgenic mouse model and in Alzheimer disease. Indeed, amyloid plaques in an APP transgenic model have recently been shown to dis-rupt the synchrony of neural inputs17.
Small, aqueously soluble oligomeric forms of amyloid-β possess a variety of biological activities, including the ability to inhibit hippocam-pal long-term potentiation (LTP) in rats in vivo18 and in hippocampal slices19,20. Whether LTP can be viewed as a direct surrogate of learning and memory is still debated, however21. Soluble oligomeric amyloid-β has also been hypothesized, but never directly proven, to mediate aspects of memory loss in APP transgenic mice12,22–24. We therefore addressed the unanswered question of whether cognitive deficits associated with amyloid-β may be directly caused by soluble amyloid-β oligomers.
Many previous studies have examined the neurotoxicity of synthetic amyloid-β, but an enduring barrier to understanding the pathogenicity of various amyloid-β species has been the difficulty of first specifying and then controlling their size and aggregation states. Recently, this problem was addressed by using natural human amyloid-β secreted into the conditioned culture medium of Chinese hamster ovary (CHO) cells that stably express a mutated form of APP that is known to be involved in Alzheimer disease. The conditioned medium from these 7PA2 cells contains monomeric amyloid-β and SDS-stable oligomeric amyloid-β without larger insoluble (amyloid) aggregates18. The con-centration of amyloid-β in 7PA2 conditioned medium is physiologically relevant (∼1–2 nM), that is, it is approximately the concentration found in normal human cerebrospinal fluid (CSF)25.
1Geriatric Research, Education and Clinical Center, Minneapolis Veterans Affairs Medical Center, Minneapolis, Minnesota 55417, USA. 2Department of Psychology, University of Minnesota, Minneapolis, Minnesota 55455, USA. 3Department of Neurology, Harvard Medical School and Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 4Laboratory for Neurodegenerative Research, Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Belfield, Dublin 4, Republic of Ireland. 5Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455, USA. 6Department of Neurology, 420 Delaware Street SE, MMC #295, University of Minnesota, Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to K.H.A. ([email protected]).
Published online 19 December 2004; doi:10.1038/nn1372
Natural oligomers of the amyloid-β protein specifically disrupt cognitive functionJames P Cleary1,2, Dominic M Walsh3,4, Jacki J Hofmeister1, Ganesh M Shankar3, Michael A Kuskowski1,5, Dennis J Selkoe3 & Karen H Ashe1,6
A central unresolved problem in research on Alzheimer disease is the nature of the molecular entity causing dementia. Here we provide the first direct experimental evidence that a defined molecular species of the amyloid-β protein interferes with cognitive function. Soluble oligomeric forms of amyloid-β, including trimers and dimers, were both necessary and sufficient to disrupt learned behavior in a manner that was rapid, potent and transient; they produced impaired cognitive function without inducing permanent neurological deficits. Although β-amyloidosis has long been hypothesized to affect cognition, the abnormally folded protein species associated with this or any other neurodegenerative disease has not previously been isolated, defined biochemically and then specifically characterized with regard to its effects on cognitive function. The biochemical isolation of discrete amyloid-β moieties with pathophysiological properties sets the stage for a new approach to studying the molecular mechanisms of cognitive impairment in Alzheimer disease and related neurodegenerative disorders.
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To determine the effects of physiological amounts of naturally secreted human amyloid-β on complex learned behavior, we micro-injected 7PA2 conditioned medium intracerebroventricularly (i.c.v.) into rats. A persistent obstacle in such studies has been the lack of a sufficiently sensitive assessment procedure that is capable of measur-ing transient cognitive changes over time and treatment conditions. To overcome this problem, we used an operant task that enables one to assess subtle cognitive effects of very small doses of psychoactive drugs26. This procedure, the alternating lever cyclic ratio (ALCR) test, has proven to be one to two orders of magnitude more sensitive than previously published methods for measuring drug effects on cognitive
function27,28. Under ALCR, rats learn a complex sequence of lever-pressing requirements for food reinforcement in a two-lever experi-mental chamber. Rats must alternate between the two levers, switching to the other lever after pressing the first lever the required number of times to get a food pellet. The number of presses required for each food reward proceeds from low (2 presses) to high (56 presses), incorporat-ing intermediate values based on the quadratic function x2 – x. Errors are scored when the subject perseveres on a lever after a reward, that is, the rat does not alternate (perseveration error) or when a subject switches levers before completing the response requirement on that lever (switching error).
RESULTSNatural amyloid-β oligomers disrupt cognitive functionAdult (5-month-old) male Sprague-Dawley rats, which had been trained under ALCR until their error rates were stable, were surgically implanted with unilateral cannulae aimed at the right or left dorsal lateral ven-tricle in a randomized fashion. After recovery, a baseline of daily error rates was determined and reassessed monthly. All rats received all test substances (within-subject design) by means of once-weekly i.c.v. injec-tions, and error rates in the presence of each test substance were always compared with the same rat’s most recent baseline (non-test) rates.
The conditioned medium from 7PA2 cells contains all secretory products of human APP metabolism, including monomeric and oligomeric amyloid-β species18. Immunoprecipitation of 7PA2 con-ditioned medium with R1282, an antibody to amyloid-β (anti-amy-loid-β), showed considerable amounts of amyloid-β monomers and low-n oligomers (Fig. 1a). The first immunoprecipitation substantially reduced the amount of amyloid-β monomers and oligomers that could be precipitated in a second immunoprecipitation (Fig. 1a). An enzyme-linked immunosorbent assay (ELISA) for amyloid-β in the residual supernatant of 7PA2 conditioned medium after the second immuno-precipitation detected no amyloid-β (data not shown). This amyloid-β-free residual conditioned medium was used as the post-R1282 injectate. Conditioned medium from untransfected CHO cells contained essen-tially no amyloid-β as detected by either immunoprecipitation or west-ern blot (Fig. 1a) or ELISA (data not shown). Significant overall effects for switching errors (F3,54 = 3.37, P = 0.02; Fig. 1b) and perseveration errors (F3,54 = 2.77, P = 0.05; Fig. 1c) were obtained on the day of the injection of the conditioned medium (day 0) but not on day 1 or 3 after the injection. Subsequent matched-pair t-tests showed that 7PA2 conditioned medium increased switching errors to 146% of the baseline rate (t18 = 3.54; P = 0.002) and increased perseveration errors to 201% of the baseline rate (t18 = 3.81; P = 0.001). Neither CHO conditioned medium nor 7PA2 conditioned medium that was depleted of amyloid-β by two rounds of immunoprecipitation with R1282 produced any sig-nificant changes in error rates relative to baseline (Fig. 1b,c). Learned behavior was not significantly different from baseline at 24 h (day 1) or 72 h (day 3) after the injections. There were no significant differences in the response rates and in the number of reinforcers earned under any injection condition (see Supplementary Fig. 1 online). Thus, i.c.v. injection of naturally secreted human amyloid-β species consistently produced significant but transient disruptions of a complex learned behavior in rats.
7PA2 conditioned medium that was selectively depleted of mono-meric amyloid-β by treatment with the protease insulin-degrading enzyme blocks the maintenance of hippocampal LTP in vivo, indicat-ing that amyloid-β oligomers, which cannot be digested by this protease and remain in the conditioned medium, may be responsible for the deficit in synaptic plasticity18. We therefore asked whether amyloid-β oligomers are also responsible for the deficits in learned behavior under
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Figure 1 Conditioned medium from cells secreting human amyloid-β oligomers and monomers disrupts learned behavior. (a) Conditioned medium from 7PA2 cells (lane 1) and untransfected sister CHO cells (lane 3) was immunoprecipitated with anti-amyloid-β R1282 and immunoblotted with 6E10 (epitope: amyloid-β residues 6–10). Lane 2 shows the result of a second R1282 immunoprecipitation of the supernatant from lane 1. The faint nonspecific band just above the amyloid-β trimer (T) confirms equal protein loading of the three samples. The amyloid-β concentration of 7PA2 conditioned medium used in lane 1 was 6,412 ± 767 pg/ml as measured by ELISA. D, dimer; M, monomer. (b,c) Mean (b) switching errors and (c) perseveration errors, expressed as a percentage of their respective baseline rates (100%), after rats received injections of 7PA2 conditioned medium, CHO conditioned medium or 7PA2 conditioned medium after two immunoprecipitations with R1282. Baseline error rates were calculated from mean errors over the previous ten testing sessions. Error bars, s.e.m.; *P < 0.05.
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ALCR that are induced by 7PA2 conditioned medium. Rather than inferring such causality by subtraction (after protease degradation), we attempted to link the assembly state of amy-loid-β directly with the observed behavioral deficit by physically separating monomeric and oligomeric amyloid-β species and then assess-ing their respective effects. To do this, we used size-exclusion chromatography of the 7PA2 conditioned medium under nondenaturing conditions to separate amyloid-β monomers from oligomers (Fig. 2a). We then injected a fraction that contained amyloid-β oligomers (dimers and trimers) but no monomers (fraction 7) and a fraction from the same size-exclusion chromatography that contained amyloid-β monomers but no oligomers (fraction 9; Fig. 2a) and assessed error rates under ALCR. Oligomers, but not monomers, of amyloid-β significantly increased both switching errors (Fig. 2b) and perseveration errors (Fig. 2c) on the day of the injection. Switching errors and perseveration errors increased to 173% (t18 = 2.72; P = 0.014) and 215% (t18 = 2.31; P = 0.033) of their baseline error rates, respectively, after injection of the oligomer-contain-ing fraction. Error rates returned to normal by day 3.
The approximately 55-kD protein that was present in fraction 7 is not a higher-molecular-weight aggregate of Aβ, as it is also readily apparent in CHO conditioned medium (Fig. 2a), it is not detected by amyloid-β
immunoprecipitation followed by autoradiography (Supplementary Fig. 2) and it is not removed by amyloid-β immunoprecipitation under condi-tions that abolish the ability of 7PA2 conditioned medium to interfere with ALCR performance (Supplementary Fig. 2). Therefore, the approximately 55-kD protein is not involved in disrupting cognitive function in these rats. Injection of fraction 9 from the same run, which contains amyloid-β monomers in amounts that are much larger than those of the oligomers in fraction 7, did not significantly affect error rates (Fig. 2b,c).
Although heightened degradation by proteases in CSF of amyloid-β monomers versus oligomers that were injected i.c.v. might account for the absence of a monomer effect on ALCR performance, this is highly unlikely in view of the lack of degradation of monomers that were incubated in CSF for 2.5 h. Incubation of metabolically labeled 7PA2 conditioned medium at 37 °C in CSF for 2.5 h showed no diminution in the amount of amyloid-β monomers, and incubation of CSF for 2.5 h at 37 °C showed no degradation of the endogenous monomers present in CSF (Supplementary Fig. 3). In addition to this monomer stability, high amounts of naturally secreted mono-mers, when injected i.c.v. as was done in this study, do not alter hippocampal synaptic plasticity18. The data indicate that naturally secreted human amyloid-β oligomers, but not amyloid-β monomers or p3, the fragment released by α- and γ-secretase cleavage of APP, transiently disrupt learned behavior in rats.
Disrupted cognition dissociated from neurodegenerationThe transience of the effects of amyloid-β oligomers indicated that they may be acting pathophysiologically, in a manner that dissociates their
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Figure 2 Oligomers but not monomers of amyloid-β disrupt learned behavior. (a) Amyloid-β oligomers and monomers in 7PA2 conditioned medium were fractionated by size-exclusion chromatography. p3 is the fragment generated by α- and γ-secretase cleavage of APP. D, dimer; M, monomer; T, trimer. (b,c) Measurement of (b) switching errors and (c) perseveration errors after rats received 7PA2 amyloid-β oligomers (fraction 7) or monomers (fraction 9) by i.c.v. injection on day 0. Both types of errors decreased on subsequent days and were at baseline by day 3, indicating that the disruption of learned behavior by soluble amyloid-β oligomers is transient. Baseline error rates were means from the previous seven testing sessions. Error bars, s.e.m.; *P < 0.05.
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Figure 3 Rats continue to learn the behavioral task during chronic intermittent exposure to amyloid-β oligomers. (a,b) Mean baseline (a) switching errors and (b) perseveration errors showed downward, nonsignificant trends (P values, 0.05–0.15; paired Student’s t-test), immediately before versus after receiving four rounds of 7PA2 (n = 6) or CHO (n = 7) conditioned medium given i.c.v. at 1-week intervals. Error bars, s.e.m.
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disruptive effects on cognitive function from the neurodegenerative processes that are characteristic of Alzheimer disease. If so, then peri-odic injections of amyloid-β-containing conditioned medium might not result in progressive deterioration of performance across time. To test this hypothesis, we injected 7PA2 conditioned medium or CHO conditioned medium once a week for four consecutive weeks into rats trained under the ALCR procedure and compared their error rates dur-ing the 10 d before and after this injection regimen. Rats receiving mul-tiple i.c.v. injections of oligomer-containing 7PA2 conditioned medium did not differ in performance (after recovery from their day 0 deficits each week) from rats receiving multiple i.c.v. injections of CHO condi-tioned medium that was devoid of oligomers (Fig. 3). Rats receiving the four 7PA2 conditioned medium injections not only maintained their overall performance but also showed a tendency to improve during the 4-week course of the injection regimen (Fig. 3).
Remarkable potency of natural amyloid-β oligomersThe amyloid-β oligomers were remarkably potent at concentrations of amyloid-β monomer in the conditioned medium that are similar to those found in human CSF. To put their potency into perspective, we compared ALCR errors after i.c.v. injection of 20 µl of 7PA2 condi-tioned medium, containing about 30 fmol (128 pg) amyloid-β , with errors after low-to-moderate i.c.v. doses of the well-known amnestic drug scopolamine. Typical i.c.v. scopolamine doses that are used to disrupt cognitive function in rats are 3–50 µg29,30. We injected 2 µg and 5 µg of scopolamine, containing about 4 and 11 nmol, respectively, into the lateral ventricles of rats that were responding under ALCR. The dis-ruption in learned behavior that was due to amyloid-β fell between the two doses of scopolamine (Fig. 4), indicating that the effects of soluble
amyloid-β oligomers in 7PA2 conditioned medium were at least four to five orders of magnitude more potent on a molar basis than the typical amnestic dose of i.c.v. scopolamine in the rat. The onset and duration of action of amyloid-β oligomers were also different from those of scopolamine. Compared with a 30-min pretesting injection time, 7PA2 conditioned medium was found to be more effective when injected 2 h pretesting (data not shown). In contrast, scopolamine worked well when injected 30 min before the session, whereas its effects had decayed substantially by 2 h postinjection (data not shown).
DISCUSSIONHere we demonstrate significant cognitive deficits that are directly attributable to a naturally secreted assembly form of human amyloid-β. The active amyloid-β species are oligomeric, not monomeric or fibril-lar, and their effects are characterized by rapid onset, high potency and transience. Our combined biochemical and cognitive analyses provide the first direct behavioral data supporting the emerging hypothesis that diffusible oligomers of amyloid-β are responsible for important compo-nents of neural dysfunction leading up to or associated with Alzheimer disease31–33. The rapid onset and transience of the oligomer effects are indicative of a pathophysiological action, similar to that seen with amnestic drugs such as scopolamine. Such pathophysiological activity is usually independent of permanent neuronal injury and, indeed, our experimental paradigm does not suggest a role for any neurodegenera-tive processes in the deleterious cognitive effects produced transiently by femtomole quantities of soluble amyloid-β oligomers.
In previous studies, intracerebral injections of synthetic amyloid-β that included mixtures of amyloid-β fibrils, protofibrils, oligomers and monomers in indeterminate proportions exerted deleterious effects on learned behavior in rats34–36. However, the deficits were reported to develop over weeks and, once present, they persisted or worsened even without further injections. Because intracerebral injections are usually made during stereotaxic surgery (rather than long after such surgery as in our paradigm), any immediate acute effects of the injectate on learn-ing or memory typically go unstudied. Two studies attempted to address potential acute effects of very high concentrations of synthetic amyloid-β species. In one study, increased maze entry errors were observed after i.c.v. injections of 2.0 µl of 1 mM synthetic amyloid-β(1–40) that were given immediately before maze testing37. In another study, no effect of intrahippocampal injections of 0.5 µl of 1 mM synthetic amyloid-β(1–40) that were administered 30 min before testing were observed in a radial arm maze test38. The concentrations and total amounts of synthetic amyloid-β administered were several orders of magnitude higher than those of the natural oligomers used in our work. Also, the duration between administration and testing was different. Because the assembly sizes of the synthetic amyloid-β that was injected were not defined in either study, the specific forms of amyloid-β being tested could not be deduced, in contrast to the present work.
The finding that natural, low-molecular-weight oligomers produce transient deficits in cognitive function contrasts with studies showing sustained deficits in learned behavior in rats after intrahippocampal injection with mixtures of synthetic soluble and fibrillar amyloid-β at high concentrations28,34. It is possible that neurons in primate or human brains might be more susceptible to permanent damage from amyloid-β than those of the rat, because primate neurons are more sensitive than rodent neurons to the neurodegenerative effects of fibrillar amyloid-β in vivo39. This issue does not, however, detract from our observation that soluble amyloid-β oligomers can produce transient brain dysfunction in the absence of neurological damage. This is an important concept, because it may provide an explanation for the observation that subtle brain dysfunction can be detected in
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Figure 4 The disruptive effect of amyloid-β oligomers on learned behavior is potent. (a,b) The acute increases in (a) day 0 switching errors and (b) day 0 perseveration errors (as a percentage of baseline errors) that result from about 30 fmol amyloid-β in 7PA2 conditioned medium (measured by ELISA) are comparable to those induced by 4 and 11 nmol scopolamine injected i.c.v. (for all groups, n = 18). Because the effects of scopolamine decayed more rapidly than those of 7PA2 conditioned medium and were undetectable 2 h after i.c.v. injection, the data for scopolamine were collected at 30 min postinjection. Error bars, s.e.m.
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certain individuals who are genetically at risk for Alzheimer disease but remain neurologically stable for many years before the expected onset of disease40,41.
Amyloid-β-induced cognitive deficits may occur before or in the absence of frank neurodegeneration in APP transgenic mice that model features of Alzheimer disease12,42–44. Our data strengthen the notion that memory deficits in these mice may be caused by amyloid-β oligo-mers. A central message of our data is that subtle brain dysfunction that occurs in presymptomatic stages of Alzheimer disease, such as that which is observed with highly sensitive functional imaging tech-niques40,41, might also be related to amyloid-β oligomer effects and might therefore be reversible with appropriate interventions before widespread neuronal degeneration.
Our demonstration that cognitive deficits are directly attributable to low amounts of naturally assembled amyloid-β oligomers indicates that trimers, the predominant oligomer present in fraction 7 (Fig. 2a), may be particularly active. These results are consistent with recent findings that memory deficits in the Tg2576 APP mouse model of Alzheimer dis-ease45 are dependent on endogenous amyloid-β assemblies comprised of multiples of trimers (S. Lesne and K.H.A., unpublished data). Regardless of which types of soluble amyloid-β oligomers are disrupting cogni-tive function, preventing them from accumulating by inhibiting their assembly, promoting their clearance or reducing monomer production are all rational therapeutic goals. Indeed, our linkage of acute behavioral deficits with soluble oligomers provides a mechanistic explanation for the rapid restoration of cognitive function that followed administration of anti-amyloid-β antibodies in behaviorally impaired APP transgenic mice23,24. Finally, the acute biological activity of naturally secreted amy-loid-β oligomers implies a neuropharmacological site of action, thus far unknown, which might also be an excellent therapeutic target.
METHODSImmunoprecipitations and amyloid-β detection. Medium was harvested, cleared of cells and then immunoprecipitated with the polyclonal antibody R1282. Samples were then electrophoresed on 10–20% tricine gels and trans-ferred onto 0.2-µm nitrocellulose membranes at 400 mA for 2 h. Filters were boiled for 10 min in PBS and blocked overnight at 4 °C with 5% fat-free milk in 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween 20 (TBS-T). After washing the membranes in TBS-T, the monoclonal antibody 6E10 (1 µg/ml) was used to probe the blots25. Bound antibody was visualized using horseradish peroxidase–conjugated anti-mouse immunoglobulin (1:40,000; Jackson Immunoresearch Laboratories, Inc.) and ECL + Detection (Amersham Pharmacia Biotech). Amyloid-β concentrations in conditioned media were mea-sured by ELISA as previously described46.
Size-exclusion chromatography. Conditioned medium from either 7PA2 or CHO cells was concentrated about 10-fold using YM-3 Centriprep filters (Amicon, Millipore) and was subjected to size-exclusion chromatography. Concentrated conditioned medium (1 ml) was injected onto a Superdex 75 (10/30 HR) col-umn (Amersham Biosciences), and 1-ml fractions were eluted with 50 mM ammonium acetate, pH 8.5, at a flow rate of 1 ml/min. Half of each fraction was frozen within 1–2 min of collection and was stored at –80 °C pending injection into rats. The other half was lyophilized and resuspended in 2× sample buffer (20 µl), boiled for 10 min and electrophoresed on a 26-well 10–20% tris-tricine Criterion gel (BioRad). Protein was transferred onto 0.2-µm nitrocellulose and immunoblotted with 2G3 (1 µg/ml). Monoclonal antibody 2G3 was raised to amyloid-β(33–40) and specifically recognizes amyloid-β species ending at residue 40; it was provided by P. Seubert and D. Schenk (Elan Pharmaceuticals). Bound 2G3 was detected as described above.
ALCR paradigm. Twenty-four male Sprague-Dawley rats, approximately 120 days old and weighing 300–350 g at the beginning of the experiment, were maintained at 90% of their free-feeding weights during the experimental stud-ies. During the course of the study, data from five rats were eliminated from
analysis because of blocked cannulae or extremely high error variability (s.e.m. > mean). Behavioral training and testing was carried out in two-lever rat test chambers (model E10; Coulbourn Instruments, Inc.) that were enclosed in sound-attenuating compartments. Each food reinforcer consisted of a 45-mg pellet (F0021; Bioserv Holton Inc.) that was delivered to a tray situated midway between the levers. A food tray light flash and an audible pellet-dispenser click signaled food delivery. Control of experimental contingencies and data collec-tion was accomplished using specialized computer software and interface (MED PC; Med Associates).
For ALCR training, behavioral sessions were carried out 5 d per week. Rats were first trained to press both levers for food reinforcement and were sub-sequently reinforced for a lever press only if they switched levers after each reinforcer delivery. The ratio of required responses per reinforcer delivery was slowly increased to 10:1 across 26 daily sessions. At this point, the ALCR was introduced using the following sequential response per reinforcer ratios: 1, 3, 5, 8, 10, 15, 15, 10, 8, 5, 3, 1. This cycle was repeated six times during each daily session. The response per reinforcer ratio cycle was slowly increased to the terminal values of 2, 6, 12, 20, 30, 42, 56, 56, 42, 30, 20, 12, 6 and 2 required responses per reinforcer, and the cycle was repeated six times each session. Sessions ended when the rat completed six cycles or after 2 h (mean session time ∼40 min). Perseveration errors were accumulated until the subject pressed the correct lever, whereas switching errors were counted as a single occurrence for each premature switch to the incorrect lever. Rats received 40 sessions of training under ALCR before surgery.
After recovery from surgery, mean baseline error rates (switching and perse-veration) under ALCR were established for individual rats. To establish baseline error rates, errors were averaged across at least six sessions, with the restriction that the highest and lowest error rates were removed before calculating the mean. Behavioral test sessions were carried out Monday through Friday each week, with injections always given on Tuesday. Only one test injection session was carried out each week. After the injection, the cannula was capped with a stylet, and the rat was placed in a holding box for 2 h before the ALCR session (30 min for rats receiving scopolamine). On noninjection days, rats were subjected to sham injections, under which the same procedure was followed but no injectate was actually given. In addition, 20 µl of saline (0.9%) was injected at least once each week to help keep the cannulae patent. All experiments were done in accordance with guidelines of the Institutional Animal Care and Use Committee of the Minneapolis Veterans Affairs Medical Center.
Injected test substances. Rats were injected with saline (0.9%), CHO condi-tioned medium, 7PA2 conditioned medium, 7PA2 conditioned medium that had been twice immunoprecipitated with R1282, the product from a size-exclusion chromatogram that contained either amyloid-β monomers or amyloid-β oligo-mers or scopolamine hydrobromide (2.0 µg and 5.0 µg, as the salt). Injection volumes of conditioned medium and products from size-exclusion chromato-grams were constant at 20 µl. Injection volumes of scopolamine were 10 µl. All injection-to-session intervals were 2 h, except in the case of scopolamine injections (30 min).
Statistics. Because of the large differences between mean switching errors and mean perseveration errors, these error rates were analyzed separately. Because all animals received each compound, we used a repeated-measures analysis of variance (ANOVA) as the inferential statistical method of analysis. Errors that were due to injection of compounds were analyzed together with the appropriate mean baseline error rates on injection day (day 0) and for several days after. If the repeated measures ANOVA F value was significant, post hoc comparisons of errors under the injectates were compared with the appropriate baseline errors using matched-pair t-tests. Statistical analyses were carried out using raw error data.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTSWe thank S. Lesné, J. Huard and M. Podlisny for critical discussions. We are grateful to J. Fadeeva and V. Betts for technical assistance. These studies were funded by the Minnesota Medical Foundation (K.H.A.), the Mayo Medical Foundation (K.H.A.), the US National Institutes of Health (K.H.A., D.J.S.) and the Wellcome Trust (D.M.W.). K.H.A. is a member of the Scientific Advisory Board of Acumen Pharmaceuticals.
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COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Neuroscience website for details).
Received 21 September; accepted 10 November 2004Published online at http://www.nature.com/natureneuroscience/
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