effect of beta amyloid peptides on neurons in hippocampal slice cultures

Neurobiology of Aging, Vol. 13, pp. 543-551, 1992 0197-4580/92 $ 5.00 + .00 Printed in the U.S.A. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Effect of Beta Amyloid Peptides on Neurons in Hippocampal Slice Cultures

A L F R E D T. M A L O U F

Department of Neurological Surgery RI-20, University of Washington Seattle, WA 98195

Received 11 May 1992; Accepted 15 June 1992

MALOUF, A. T. Effect of beta amyloid peptides on neurons in hippocampal slice cultures. NEUROBIOL AGING 13(5) 543- 551, 1992.--Several investigators have described the neurotrophic and neurotoxic effects of beta amyloid peptide fragments on dissociated hippocampal neurons in culture, in these prior studies, the peptides were added to dissociated cultures between day 0 and day 4 in vitro, before hippocampal neurons are fully mature. We have analyzed the neurotrophic and neurotoxic effects of beta amyloid fragments/31-28,/325-35 and/31-40 on hippocampal slice cultures, whose physiology and morphology resembles the intact hippocampus. Addition of/31-28 or/325-35 to the growth medium did not produce significant changes in dendritic length or number of branches. Nerve growth factor, previously reported to enhance the neurotoxic effects of B 1-40 on dissociated hippocampal neurons in culture, did not significantly enhance the neurotrophic effects of/31-28. To achieve high local concentrations of peptides and to avoid potential access problems in the cultures, we injected/31-28,/325-35, and/31-40 directly into the cultures. Amyloid-mediated neurotoxicity was not observed for/31-28 or/325-35, but/31-40 appeared to produce neurodegeneration around the site of injection.

Beta amyloid fragments Hippocampal slice cultures

THE cause of Alzheimer's Disease (AD) is unknown, but recent evidence suggests that beta amyloid peptide may be directly re- sponsible for the neurodegeneration observed in AD. Beta amyloid fragments have been shown to produce both neurotrophic and neurotoxic changes in dissociated hippocampal neurons maintained in monolayer cultures (22,23,25,26). When applied on the day of culture,/31-28,/325-35, and/31-40 prolong neuronal survival of dissociated hippocampal neurons (23,26), and /31-42 has been reported to increase neurite branching and length (22). When applied to dissociated hippocampal neurons on day 4 in vitro, {31-28,/325-35, and/31-40 were neurotoxic (26). These results suggest that beta arnyloid is neurotrophic to immature neurons and neurotoxic to mature neurons. However, hippocampal neurons in dissociated cultures require a minimum of 7 days in vitro before they resemble mature neurons (8). On day 4 in vitro, the dendrites of hippocampal neurons are still growing, which may make them more sensitive to beta amyloid-induced effects than mature neurons in situ.

The ability of beta amyloid fragments to alter neuronal viability, pathology, and physiological function may be better ap- proached using cultured brain slices (6). Slice cultures can be maintained in vitro for 2-8 weeks, allowing time for the neurons to mature. Analysis of cultured hippocampal slices demonstrates that their physiology, anatomy, and synaptic circuitry resembles that of intact hippocampus (5-7,15,20,21,27). Their similarity to brain tissue, compared to dissociated cultures, makes slice cultures a valuable preparation for studying brain function.

We have undertaken a number of studies designed to test the neurotrophic or neurotoxic properties of beta amyloid fragments B1-28,/325-35, and/31-40 on pyramidal neurons in hippocampal slice culture. In one set of experiments,/31-28 and/325-35

543

were added to the growth medium, and neurotoxicity in whole cultures or the dendritic morphology of individual pyramidal neurons was analyzed. In the second set of experiments, localized injections of/31-28, /325-35, or ¢31-40 were made into areas CA1 and CA3 of the cultures, and these areas were analyzed for nerve cell degeneration. These experiments did not yield con- clusive evidence that beta amyloid fragments are neurotrophic, but preliminary data suggest that/31-40 may be neurotoxic to hippocampal neurons in slice cultures.

METHOD

Slice Cultures

Hippocampal slice cultures were prepared by the roller tube method of Gahwiler (6). Briefly, hippocampi from 4-day rat brains were cut into 400 um thick transverse sections using a McElwain tissue chopper. Tissue slices were attached to 12 × 24 mm No. 1 glass coverslips (Gold Seal) by placing each slice in a drop of chicken plasma (Cocalico Biologicals) and adding a drop ofthrombin (Sigma, St. Louis, MO) to form a clot. The coverslips with attached slices were put directly into 15 ml conical culture tubes containing 1.5 ml of growth medium, the tubes were sealed, and continuously rotated at 6 revolutions/h in a dry heat incubator at 36°C. The growth medium contained 50% BME, 25% Earle's balanced salt solution (EBSS) and 25% horse serum (all components from GIBCO). After 10-14 days in vitro, the roller slices flattened from 400 um to between 50 and 80 ~m. Cultures retained the anatomical features and much of the synaptic organization of the intact hippocampus.

544 M A L O U [

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FIG. 1. Labelled pyramidal neuron from a cultured hippocampal slice. (A) This Neurobiotin-filled CA3 pyramidal neuron was from a culture treated with 30 ~M f l l -28 peptide added to the growth medium. (B) Computer-assisted camera lucida of the apical dendrites (arrow) of the neuron shown in A were traced in theee dimensions using a Eutectics image analysis computer to quantify dendritic length and number of branch points. Tracings were performed using a 100× oil objective. The cumulative data for traced neurons are shown in Fig. 2. Calibration - 100 ~m for A.

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FIG. 2./31-28 and ~'25-35 added to growth medium had no effect on dendritic arborization. Individual CA3 pyramidal neurons were filled and traced as described in the Method section. Data from beta amyloid treated culture (solid bars) are compared to control cultures (striped bars) in all panels. In A, control cultures were treated with 0.25% bovine serum albumin; in B-D, untreated control cultures were used; there was no significant difference between BSA and untreated controls. (A) The length of the apical dendrites from cultures treated for 2-14 days with 30 uM/31-28 were not different (top, p = 0.818) nor did they have significantly more branch points (bottom, p - 0.091 ) than control cultures. (B) Cultures were treated nerve growth factor (NGF) to attempt to enhance the potency of B I-28. Cultures treated for 2 days with 30 uM BI-28 plus 50 ng/ml NGF showed small increases in both apical dendritic length (top) and branch points (bottom), but these differences did not reach statistical significance (p = 0.085 and 0.153, respectively). (C) Cultures treated for 2-3 days with 100 uM/~1-28 plus 50 ng/ml NGF showed no changes in either apical dendritic length (top, p = 0,670) or branch number (bottom, p - 0.923). (D) Cultures treated with 100 uM/~25-35 for 2 days showed no changes in apical dendritic length (top, p = 0.915) or in total dendritic length (apical plus basal) (bottom, p = 0.54). Values are the mean _+ SEM; statistical significance was determined by t-test analysis using Statworks software on an Apple Macintosh computer.

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546 MALOUF

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FIG. 3. fll-28 and/525-35 added to the growth medium had no effect on neuronal viability in whole cultures. Cultures were treated with 100 ~'tl fll-28 (n = 3) or 100 uM fl25-35 (n = 6) for 2-6 days. The cell bodies of the granule cells in the dentate gyrus (DG) and pyramidal cells in areas CAI and CA3 were stained with cresyl violet. The cell body layers of fll-28- (B) and fl25-35- (C) treated cultures appeared to be intact when compared to untreated control cultures (A). Calibration = 400 tzm.

1. Exposure of Whole Cultures to Beta Am),loid Fragments

Peptide solutions./31-28 and/325-35 fragments used in these and other experiments were generously provided by Zymoge- netics, Inc. The peptides were synthesized on an Applied Bio- systems 431A peptide synthesizer, purified by HPLC, and subjected to electrospray mass spectroanalysis./31-28 was synthesized according to the amino acid sequence described by Wong et al. for cerebrovascular amyloid isolated from AD brain (24), and the sequence for/325-35 was taken from Yankner et al. (26)./31-28 was dissolved in growth medium at the concentrations indicated in the text. fl25-35 was dissolved in 35% acetonitrile plus 0.1% trifluoroacetic acid (ACN/TFA), and was diluted 1000X in growth medium for application to the cultures. Nerve growth factor (NGF 2.5S) was purchased from Boehringer Mannheim (cat. #100-700). NGF (250 #g) was dissolved in 2.5 ml Gey's balanced salt solution containing 10 mg/ml BSA, and was frozen at -70°C in 50 #1 aliquots. All slice cultures used in these experiments were 10-21 days in vitro at the time the peptides were added.

Cell labeling. Individual pyramidal cells were labeled using intracellular microelectrodes filled with 1% Lucifer Yellow (LY) in 1 M lithium chloride or 2% Neurobiotin in 1 M potassium acetate. LY was injected into cells by applying -0.5 to - 1.0 nA current for 4-8 min. Neurobiotin was injected into cells using

a +0.5 nA/100 ms current pulse, 50% duty cycle for 4-8 rain. The cultures were fixed with a solution of 3.5% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer for 3 h and then placed in 30% sucrose for 1-2 days prior to performing LY or Neurobiotin immunohistochemistry according to the method of Kunkel et al. (14).

Dendrite tracing. The dendritic length and number of branch points of fll-28-treated filled cells were quantified by tracing the apical dendrites in theee dimensions using the Eutectics image analysis system. The dendritic length of f125-35-treated filled cells was quantified by tracing both apical and basal dendrites in two dimensions using the Bioquant image analysis system. Equivalent dendritic length measurements were obtained using either the Bioquant or the Eutectics systems. All tracings were made using a 100× oil immersion objective.

H. Injection 0//31-28. [325-35. and/31-40

Peptide sohttions./31-28 ( 100/~m) was dissolved and injected in Gey's buffer. /325-35 was dissolved in either ACN/TFA or distilled water. The/325-35 peptide was then injected in undi- luted ACN/TFA (3.77 mm): alternatively, 1.06 mg peptide was dissolved in 100 ul ACN/TFA, diluted 100x in Gey's buffer, and injected at a final concentration of 100 u,~l./325-35 peptide dissolved in distilled water was injected at concentrations of 0.377

BETA AMYLOID ON HIPPOCAMPAL SLICE CULTURE 547

C

FIG. 4. Injection of/31-28 and ~25-35 dissolved in Gey's buffer. Approximately 0.1-0.3 ul of either 100 uM ¢q-28 (B, n - 9) or 100 •M {425-35 (C, n - 9) were made into area CA3 (arrows) of slice cultures. Control cultures (A) were injected with either Gey's buffer (n = 12) or 0.25% BSA dissolved in Gey's buffer (n - 3). The cultures were stained 7-9 days later with cresyl violet to determine if there was a loss of stained somata around the injection site. Calibration = 400 urn.

mM, 1.4 mM, or 2.8 mM./31-40 and the reverse peptide/340- 1, generously supplied by Dr. Bruce Yankner, were dissolved and injected in ACN/TFA at a concentration of 1.08 mM. In experiments where Substance P (SP) (Bachem) was coinjected with/31-40, SP was dissolved in distilled water at a concentration of 1.85 mM and diluted 10× in the/31-40 solution.

Injection of beta amyloid fragments into cultures. All slice cultures used in these experiments were 10-21 days in vitro at the time the peptides were injected. Peptides were loaded into glass microelectrodes with tip diameters of 1-2 #m. The cultures were removed from the 15 ml conical culture tube and placed in a 35 mm culture dish containing growth medium supple- mented with 10 mM HEPES buffer. The dish was placed on an inverted Nikon TMS microscope in a sterile hood, and the tip of the pipette was placed into area CA1 or CA3 of the hippocampal slice culture using a Narishigi micromanipulator. For /31-28 and/325-35, mild pressure was applied to the pipette for 5-10 s, and approximately 0.1-0.3 #1 of peptide solution was injected into each injection site within a culture. The advantages of this injection procedure were:

1. we could clearly visualize the peptide solution as it spread into the tissue, thereby confirming the success of each injection, and

2. this procedure produced little damage at the injection site,

/31-40 and /340-1 were injected over the course of 1 min to ensure that these peptides diffused at least 200 ~m from the site of injection. To minimize the possible washout of peptide upon return of the culture to the roller drum, all injected cultures were left stationary in the 35 mm dishes for 1-2 h following injections.

Determination ~fneuronal to-dcity. One to 7 days after injection of the beta amyloid peptide, the cultures were removed from the growth medium and incubated in 0.5 ug/ml propidium iodide (PI) for 1 h. PI is a fluorescent probe that labels the nuclei of dead cells. Cultures were observed using a Leitz fluorescence microscope. The cultures were then fixed in 4% paraformaldehyde and stained with cresyl violet to stain cell bodies in the pyramidal cell layer. Loss of cresyl violet stained cell bodies coincided with PI fluorescence.

Tau immunoreactivity has been used to identify neuronal degeneration following in situ injections of 131-40 (13). To assess neuronal damage produced by injection of/31-40 into slice cultures, the cultures were fixed, treated with alkaline phosphatase (15 units/ml Boehringer Mannheim), and incubated with a mouse monoclonal antibody to tau- 1 ( 1:2000 dilution, Boehrin- ger Mannheim). The ABC method (Vector Laboratories) was used for detection oftau- 1. Following tau- 1 immunohistochemistry, the cultures were counterstained with cresyl violet to stain neuronal cell bodies.

548 MALOUF

RESULTS

Eff~'ct ~['/31-28 and/325-35 Applied to the Growth Medium

The neurotrophic or neurotoxic effects of beta amyloid were examined following the addition of/31-28 to the culture medium. The dendrites of individual pyramidal neurons in slice cultures were then analyzed for changes in dendritic length or number of branch points. Growth medium containing 30 uM/31-28 was applied to the slice cultures for 2-14 days. The cultures were placed in an electrophysiology recording chamber, and individual CA3 pyramidal neurons were filled with either LY or Neuro- biotin, and their apical dendrites were traced using the Eutectics image analysis system to quantify dendritic length and number of branches (Fig. 1). There was no statistically significant difference in total dendritic length or number of branch points in cultures treated with 30 ~M/31-28 compared to control cultures treated with 0.25% bovine serum albumin (BSA) (Fig. 2A). Ap- plication of 100 ~M /31-28 was also ineffective in producing changes in the length or branching of pyramidal cell dendrites.

Slice cultures were treated with/31-28 plus 50 ng/ml NGF to increase the potency of/31-28 (25). When individual CA3 neurons were analyzed, cultures treated with 30 #M/31-28 plus NGF showed small increases in both apical dendritic length and branch points, but these differences did not reach statistical significance (Fig. 2B). There was also no difference in dendritic length or branch points for neurons in cultures treated with 100 ~M/31-28 plus NGF (Fig. 2C). NGF alone produced no effect (data not shown).

/31-28 is one of the least potent of the amyloid fragments (26), which may account for its lack of effect on slice cultures. Therefore,/325-35, one of the most potent amyloid fragments, was applied to slice cultures (26). However, individual CA3 pyramidal neurons in slice cultures showed no significant difference from untreated controls in apical or total (apical plus basal) dendritic length following exposure to 100 uM/325-35 in the growth medium for 2 days (Fig. 2D).

In addition to examining individual neurons, we also examined/31-28- and/325-35-induced neurotoxicity in whole cultures. Slice cultures were treated with amyloid peptides for 2-6 days. Upon visual examination, there was no difference in PI fluorescent labelling of dead cells in cultures exposed to/31-28 or/325-35 and untreated controls. These cultures were then fixed and stained with cresyl violet to visualize neuronal cell bodies.

The pyramidal and granule cell layers of all cultures treated with /31-28 or/325-35 remained intact (Fig. 3). In contrast, when a known neurotoxin such as glutamate ( 10 mM) was added to the growth medium for 24 h, there was strong PI labelling of dentate granule cells and pyramidal cells, and a clear loss ofcresyl violet stained somata. The absence of PI staining, together with the presence of an intact pyramidal cell layer, strongly suggests that neither/31-28 nor/325-35 were neurotoxic to neurons in the slice cultures when added to the growth medium.

Direct Injection qf /31-28, /325-35, and/31-40

Unlike dissociated hippocampal neurons in culture, the nerve cells in slice cultures are covered by a layer of astrocytes that could limit the access of beta amyloid peptides to neurons. To overcome this potential problem, and to achieve high concentrations of these peptides in localized areas within the slice, beta amyloid peptides were injected directly into slice cultures. /31- 28 or/325-35, dissolved in buffer (Fig. 4), ACN/TFA (Fig. 5) or water (Fig. 6), were injected into the cultures. Neurotoxicity was assessed by PI fluorescence and by cresyl violet staining. Beta amyloid appeared to produce neuronal damage when injected in water (Fig. 6C) but not in buffer or ACN/TFA (Figs. 4C, 5B). However, injection of bovine serum albumin (BSA) in water also produced damage (Fig. 6A).

To determine if longer beta amyloid fragments were more potent on slice culture than/31-28 or/325-35, we obtained the /31-40 and the reverse sequence/340-1 peptides from Dr. Bruce Yankner. This/31-40 peptide has been shown to be neurotoxic in dissociated hippocampal cultures and when stereotaxically injected into intact rat brain, while/340-1 or vehicle alone (ACN/ TFA) were inactive (1L26). /31-40 or /340-1 (1.08 mM) was dissolved in ACN/TFA and injected over the course of 1 min to allow the peptides to diffuse at least 200 #m away from the point of injection. Seven days after injection, tau-I immunoreactivity showed heavily stained axonal fibers surrounding the injection sites, but there were no immunoreactive somata (Fig. 7). The tau-I immunoreactivity surrounding the hole in Fig. 7B indicates that there was a high concentration of/31-40 in this area, and suggests that this damage was due to the actions of the/31-40 peptide. The hole was not caused by physical disrup- tion of the tissue at the time of injection, since the injection site can be observed intact at high magnification (not shown). The

A

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B

FIG. 5. Injection of/325-35 dissolved in 35% acetonitrile plus 0.1% trifluoracetic acid (ACN/TFA). ~25-35 (3.77 mM) was dissolved and injected in ACN/TFA (B, n = 3) and control cultures were injected with ACN/TFA alone (A, n = 3). All injections were made into area CA3 (arrows). After 6 days, the cultures were stained with cresyl violet to assess loss of neuronal cell bodies surrounding the site of injection. No damage was observed beyond the smalt holes made by the pipette at the injection site (arrows). Calibration - 400 urn.

BETA A M Y L O I D ON H I P P O C A M P A L SLICE C U L T U R E 549

B

FIG. 6. Injection of ¢~25-35 dissolved in distilled water. (A) Control cultures were injected with distilled water alone (n = 4) or 0.4 mg/ml BSA in distilled water (n = 7). (B) ~25-35 was dissolved in distilled water at concentrations of 0.377-2.8 mM, and injected into areas CAI and CA3 (arrows, n = 13). After 3-7 days, the cultures were stained with cresyl violet to assess loss of neuronal cell bodies surrounding the injection sites. Twelve of 13 cultures injected with ~25-35 showed damage in areas CA I and CA3 (B), but this damage does not appear to be different from that seen with control injections (A). Calibration - 400 ~m.

absence of tau-1 immunoreact ive somata may have been due to the low activity of the alkaline phosphatase (Boehringer Mannheim), since cresyl violet staining revealed degeneration at all seven sites injected with /41-40 (Fig. 8A, B). Figure 8B shows the same culture as in Fig. 7B. The absence of pyramidal cell somata on both sides of the hole indicate that /41-40 produced widespread degeneration in this culture. Neurotoxicity was not blocked when substance P (SP) was coinjected with/31- 40 (Fig. 8C), as was reported in dissociated cultures (26) and in vivo (13). The/440-1 reverse peptide was also neurotoxic in five of seven injection sites, but the damage from ¢340-1 was not as extensive as that produced by ~1-40. The most extensive degeneration produced by/440-1 peptide is shown in Fig. 8D. ACN/ TFA alone produced no damage.

DISCUSSION

In these studies, we examined the effects of ¢¢1-28, ~25-35, and/31-40 on hippocampal slice cultures. ~1-28 and/425-35

did not produce consistent neurotrophic or neurotoxic changes in slice cultures when applied in the growth medium or when injected directly into the cultures. In contrast, preliminary data suggest that /41-40 may be neurotoxic when injected into the cultures.

There are a number of possible explanations for the insen- sitivity of neurons in slice cultures to the effects of/41-28 and /325-35 applied to the growth medium. First, when added to the growth medium, access of the peptides to the neurons may be restricted. The roller slice cultures are about 70-80 um thick, and have a layer ofastrocytes that cover the nerve cells. Although the slice cultures are easily penetrated by soluble compounds ( 15), beta amyloid fragments are insoluble in physiological buffers. Contact with salt solutions induces the formation of insoluble fibrils (1,3,4,9-11,17), and it is possible that these fibrils do not efficiently penetrate into the slice culture. However, in one report, intraventricular injections of/31-28 produced amnestic effects in mice: the peptide presumably entering the brain by passing

FIG. 7. Tau-I immunoreactivity following injection of/31-40. (A) Dark tau-1 immunoreactive processes were observed surrounding the injection site (arrows), but no immunoreactive somata were observed near the injection site. (B) In this culture, the area surrounding the injection site (asterisk) did not survive (large hole). The large hole near the injection site is surrounded by a tau-I immunoreactive dense fiber plexus (arrows), while the small hole, which is further from the injection site, is not surrounded by fibers. Calibration = 100 um (A) and 200 um (B).

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FIG. 8. Injection of/31-40 and/340-1 produced neurodegeneration. Following tau-I immunohistochemistry, the cultures were counterstained with cresyl violet. Injection sites are marked by arrows, and the loss of pyramidal neurons is outlined by dashed lines in A-D. /31-40 produced neurodegeneration at all seven injection sites (n = four cultures), but the amount of degeneration was variable. (A) Injection of/31-40 produced moderate degeneration at both injection sites in this culture. (B) Injection of/31-40 produced extensive neurodegeneration at both injection sites in this culture. This is the same culture shown in Fig. 7B. A loss of pyramidal cells was observed on either side of the hole in area CA3 (bottom), suggesting that /31-40 produced complete cellular necrosis near the injection site in addition to neuron specific degeneration further from the injection site. Extensive neurodegeneration was also observed at the second injection site in area CA1. (C) Substance P (0.185 mM) did not inhibit the neuronal damage produced by/31-40 in five of six injections (n = three cultures). (D) Injection of/340-1 produced neurodegeneration in .five o f seven sites (n - 4 cultures). The largest area of degeneration produced by/340-1 was observed in area CA3 of this culture (bottom). Injection of/340-1 into area CA I (top) produced a less degeneration. Calibration = 200 um.

through the wall of the ventricles (2). The constant rotation (6 rev/h) of the roller slice cultures, while contributing to thinning, may also exacerbate any solubility or penetration problems. The rotation of the slice cultures may result in deposition of the insoluble amyloid fibrils on the walls of the culture tube rather than onto neurons as in dissociated cultures (18,19). In some experiments, exposure of cultures to amyloid peptides for 1-3 days may have been too short to observe neurotrophic or neurotoxic changes. Although amyloid-induced effects have been reported in dissociated cultures after 24 h, recent studies using older cultures indicate that 7 days may be required for beta amyloid fragments to produce their full effects.

To eliminate physical barriers and to achieve very high local concentrations of beta amyloid peptides, we injected the peptides directly into the cultures. No neurotoxicity was observed when /31-28 and/325-35 were dissolved in buffer. Injections of/325- 35 dissolved in water produced clear neuronal damage, but BSA in water appeared to produce a similar degree of damage, making it difficult to interpret these results. To determine whether our

cultures were more sensitive to longer beta amyloid fragments, we injected/31-40 from Dr. Bruce Yankner 's laboratory. Pre- vious studies demonstrated that this/31-40 peptide was neurotoxic when applied to cultured neurons in vitro and when injected into intact rat brain. Injections of 1.08 m M ~1-40 into hippocampal slice cultures consistently produced neurodegeneration well beyond the injection site. Injection of reverse peptide/340-1 produced degeneration in five of seven cases, but the area of degeneration was more limited than with the/31-40 peptide. Thus, the largest area of degeneration produced by the f140- 1 peptide was about equivalent to the smallest area produced by/31-40. This differential neurotoxicity did not appear to be due to variability in injection volume, because we noted any observed discrepancies in the diffusion pattern of injected peptide solutions. Given the minor toxicity produced by/340-1 and by BSA, it appears that any protein injected into the cultures at high concentrations will produce a small amount of nonspecific neuronal damage. The extensive degeneration produced by the /31-40 peptide, when compared to the restricted toxicity pro-

BETA A M Y L O I D ON H I P P O C A M P A L SLICE C U L T U R E 551

duced by the/340-1 peptide, suggests that /31-40 is neurotoxic to CAI and CA3 pyramidal neurons in hippocampal slice cultures. However, a detailed dose-response curve for/31-40 neurotoxicity has not been made in these cultures. Injecting lower concentrations of these peptides may eliminate the nonspecific neurotoxicity produced by/340-1, and may facilitate the inter- pretation of the/31-40's neuroactive properties in the slice cultures. The failure of substance P to block #1-40 neurotoxicity may also be related to the high concentration of/31-40 injected.

In summary, we have examined the neuroactive properties of three beta amyloid fragments on hippocampal slice cultures using a variety of experimental conditions. The absence of con- vincing and reproducible neurotoxic responses following injection of/31-28 or/325-35 into slice cultures is difficult to explain. However, it is possible that mature neurons in both dissociated ( 12,16) and slice cultures are less sensitive to the effects of these peptides than immature neurons in dissociated cultures. Prelim- inary data suggests that the/31-40 peptide is neurotoxic to neurons in the slice cultures, but these results are complicated by the minor toxicity of the/340-1 peptide. The method of assessing

neurodegeneration may also play a critical role in evaluating the activity of beta amyloid peptides. If low concentrations of beta amyloid compromise the health of neurons but are not fatal, ALZ-50 immunoreactivity may more reproducibly identify these types of changes than assessments based on visual obser- vation of dissociated cultures orcresyl violet stained neurons in slice cultures. The variable results obtained in our studies using different beta amyloid fragments and a variety of conditions suggest that these peptides may have a complex mechanism of action. Understanding the conditions under which they are neuroactive will be critical to identifying the role of beta amyloid in the etiology of Alzheimer's Disease.

ACKNOWLEDGEMENTS

I thank Loan Nguyen, Mark Harrigan, and Carol Robbins for their technical assistance, Drs. Dennis Kunkel and Philip Scfiwartzkroin for their helpful comments, Zymogenetics, Inc. for the gift of/31-28 and /325-35 peptides, and Dr. Bruce Yankner for the/31-40 and/340-1 peptides. This study was supported by a grant from the Alzheimer's Disease and Related Disorders Association.

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effect of beta amyloid peptides on neurons in hippocampal slice cultures

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