Br. J. Pharmacol. (1993), 108, 387-392

a-Sialyl cholesterol reverses AF64A-induced deficit in passiveavoidance response and depletion of hippocampal acetylcholinein mice

'Eiichi Abe, Shigeo Murai, Yoshikatsu Masuda, Hiroko Saito & Tadanobu Itoh

Department of Pharmacology, School of Dentistry, Iwate Medical University, 19-1, Uchimaru, Morioka, 020, Japan

1 The effect of a-sialyl cholesterol (a-SC; a-D-N-acethylneuraminyl cholesterol) on disturbances of the

central cholinergic system induced by ethylcholine mustard aziridinium ion (AF64A) and by scopol-amine were studied by means of a step-down passive avoidance response and locomotor activities inmice. The levels of acetylcholine (ACh) in certain regions of the brain were measured to assess theneurochemical recovery promoted by a-SC.2 Treatment with AF64A (2.5, 5 and 10 nmol, i.c.v.) impaired the 24 h retention latencies of animals ina dose-dependent manner, and scopolamine (0.5 mg kg-', i.p.) also impaired the retention performance.Administration of a-SC (1 and 4mg kg-1, p.o.) once daily for 13 days improved the retentionperformance in AF64A-treated animals in a dose-dependent manner, but not in the scopolamine-treatedanimals.

3 Treatment with AF64A (2.5, 5 and 10 nmol, i.c.v.) and scopolamine (0.5 mg kg-', i.p.) increasedvertical and horizontal locomotor activities. a-SC dose-dependently attenuated the increase in locomotoractivies induced by 2.5 nmol of AF64A, but not the locomotor activities caused by 5 or 10 nmol ofAF64A, or scopolamine (0.5 mg kg', i.p.).4 The deficit retention performance of AF64A-treated animals was associated with depletion of AChlevels in the hippocampus, but not in the septum or cerebral cortex. Administration of a-SC to

AF64A-treated animals dose-dependently reversed the depletion of ACh levels in the hippocampus.

5 The results indicate that a-SC had significant effects after oral administration of AF64A-treatedanimals. The behavioural recovery promoted by a-SC may be based on the reversal of ACh depletion inthe hippocampus.

Keywords: a-Sialyl cholesterol; ethylcholine mustard aziridinium ion (AF64A); scopolamine; acetylcholine; hippocampus;passive avoidance response; locomotor activity

Introduction

x-Sialyl cholesterol (aX-SC; x-D-N-acetylneuraminyl chol-esterol) currently appears to be a neurotrophic factor. Recentneurobiological studies have shown that a-SC inducesneuritogenesis in a mouse neuroblastoma cell line (Tsuji etal., 1988). This effect may be necessary for the survival ofcells and for neurite growth, and also for the maintenance offunctions related to neurotransmitter production (Di Patre et

al., 1989). Other sialic acid-containing glycosphingolipids,gangliosides, especially GM, ganglioside, stimulate neuriteoutgrowth in vitro (Roisen et al., 1981; Tsuji et al., 1988),and facilitate recovery of high affinity choline uptake, cholineacetyltransferase activity in the cortex and active and passiveavoidance response after lesion of the nucleus basalis of rats(Pedata et al., 1984; Casamenti et al., 1985). Thus, it is ofinterest to study the effects of a-SC on animal models ofneurodegenerative disorders in the brain.Ethylcholine mustard aziridinium ion (AF64A) has been

proposed as a specific cholinergic neurotoxin, because intra-cerebroventricular (i.c.v.) administration of AF64A to ratsselectively destroys the central cholinergic system and reducesthe number of presynaptic cholinergic markers, such as high-affinity choline uptake, choline acetyltransferase activity,acetylcholine (ACh) release and ACh level (Leventer et al.,1987; Hortnagl et al., 1987). It also causes impairment ofmemory performances including a deficit of the avoidanceresponse in mice and rats (Pope et al., 1985; Gower et al.,1989). The reductions in cholinergic markers are long-lasting(Leventer et al., 1987). However, the density of brain mus-

carinic recognition sites was unchanged (Vickroy et al.,

Author for correspondence.

1985). The muscarinic receptor antagonist, scopolamine,blocks neurotransmission at central cholinergic muscarinicreceptors and transiently impairs the passive avoidance res-ponse in rats and mice (Flood & Cherkin, 1986; Verloes etal., 1988). These drugs have been used as a model of amnesiaand are useful for drug screening in animal models ofcholinergic dysfunction.

In the present study, we used a passive avoidance task toinvestigate whether a-SC can reverse the effect of AF64A andprotect against the effect of scopolamine. Vertical andhorizontal locomotor activities were examined to determine iflocomotor dysfunction influenced the passive avoidance res-ponse. In parallel with behavioural studies, regional brainlevels of ACh were measured.

Methods

Animals

Male ddY mice (Nihon SLC, Hamamatsu) weighing between27-29 g at the beginning of the experiment were housed ingroups of 10-12 under conditions of controlled temperature(22 ± 1°C), humidity (55 ± 5%) and a 12 h light-dark cycle(lights on 07h 00min). The animals were allowed free accessto water and standard laboratory chow.

Drug administration

The experimental design and number of animals in eachgroup are summarized in Table 1 and Figures 1-6. In theAF64A-treatment experiments, mice were anaesthetized with

'." Macmillan Press Ltd, 1993

388 E. ABE et al.

pentobarbitone sodium (Nembutal; 50 mg kg-', i.p.) andwere mounted on a stereotaxic instrument (ST-7, Narishige,Japan). The animals were infused with either isotonic saline(pH 7.40) or AF64A (2.5, 5 or 10 nmol per ventricle) to atotal volume of 4.0 p1l in 4 min into the left cerebral ventricle.After the infusion, the needle was held in place for anadditional 3 min to allow for diffusion of the solution. Thesolution of AF64A diffused bilaterally into the ventricle andbilaterally reduced the ACh levels in the hippocampus.Stereotaxic coordinates were: P. 1 mm, L. - 0.1 mm frombregma and V. 2.5 mm below dura; the height of the incisorbar was - 13mm. In the groups receiving 5 and 10 nmol ofAF64A, 3 mice in each group died within 3 days of thesurgery. Five days after the surgery, the groups of AF64A-treated animals and sham-treated animals were given eithervehicle (distilled water, 10 ml kg-', p.o.) or a-SC (1 or4 mg kg-', p.o.) once daily for 13 days. On day 13, 2 h afterthe last dose of a-SC, animals were trained in an acquisitiontrial in a step-down passive avoidance test or locomotoractivity test.

In the scopolamine experiments, mice received either vehi-cle or a-SC (1 or 4 mg kg-', p.o.) once daily for 13 days. Onday 13, 30 min after the last dose of vehicle or a-SC, theanimals were treated with either saline (0.1 ml kg-', i.p.) orscopolamine (0.5 mg kg', i.p.) and 90 min later they weretrained in a passive avoidance acquisition trial or locomotoractivity test. On the testing day, all doses of a-SC or vehiclewere randomly administered. The administration period anddoses of a-SC were based on the results of pilot studies, sincea single dose of a-SC (1 or 4 mg kg-', p.o.) was ineffective onthe passive avoidance response and ACh levels in AF64A-treated animals. The time course of a-SC was 2 h after oraladministration, which was the time of the peak brain concen-tration (unpublished data supplied by MECT Co., Ltd.). Thedose, route of administration and pre-administration time ofscopolamine were according to the methods of Kameyama etal. (1986) and Verloes et al. (1988).

Passive avoidance response task

The apparatus consisted of a clear acrylic compartment(15 x 15 x 17 cm high) with a lid and an electrifiable grid ofstainless steel rods (4 mm in diameter, 8 mm between bars).A wooden platform (5 x 5 x 4 cm high) was situated at acorner of the grid floor. An electronic stimulator (SEN-310,Nihon Koden, Japan) and an isolator (SS-302J, NihonKoden) were used to deliver electric shocks.On day 13, each group of animals were trained in an

acquisition trial in a step-down passive avoidance task. Oneanimal was placed on the platform, and as soon as it had allfour feet on the grid floor, it received a foot-shock (1 mA,1 s) and was then immediately removed from the apparatus.The latency of the animal's descent from the platform wasrecorded as the baseline latency (acquisition latency). Theretention trial was carried out 24 h after the acquisition trialon day 14 (without drug administration). Each animal wasagain placed on the platform and the latency until it steppeddown to the floor was recorded, with a cut-off time of 300 s(retention latency). Any retention latencies statisticallyshorter than those of the respective control group were takenas indications of drug-induced impairment of retention ofmemory. All tests were performed between 09 h 00 min and13 h 00 min. A completely randomized design was used forthe allocation of treatment within the groups.

Locomotor activities

Vertical and horizontal locomotor activities were measuredaccording to the method of Itoh et al. (1987). The apparatusconsisted of clear acrylic walls and a lid. An infraredphotocell was mounted at a height of 1.8 cm to measure

horizontal locomotor activity and 9 infrared photocells weremounted at a height of 6.5 cm to measure vertical locomotoractivity. The output from the photocells was amplified andentered directly into an EPSON PC 386M computer. Thelocomotor measurements were carried out at equivalent timesafter appropriate drug administration as described in theacquisition trial.On day 13, the sham and AF64A groups were tested 2 h

after the last doses of vehicle or a-SC (1 or 4 mg kg-', p.o.)were administered. In the scopolamine group, 30 min afterthe last doses of vehicle or a-SC, scopolamine (0.5 mg kg-',i.p.) was given and 90 min later the locomotor recordingswere started. Testing was performed in blocks, using 10 setsof cages, with treatments randomized between all animalswithin the experiment. Each mouse was placed singly in thecage and, 1 min later, the locomotor activities were auto-matically recorded by the number of light beam interruptionsdue to the animal's movements for 30 min. For purpose ofanalysis this was divided into 5 min periods. The apparatuswas thoroughly cleaned after each mouse was tested. Allanimals were used only once, and locomotor activities werealways investigated between 09 h 00 min and 13 h 00 min.

Acetylcholine assay

On day 14, animals which had undergone the passiveavoidance test were analyzed neurochemically. Since theeffect of scopolamine is transient, only the AF64A-treatedanimals underwent neurochemical analysis.

After the retention test, each animal was killed by a micro-wave beam focused on the head (0.7 s, 5 kW, TMW-6402C,Toshiba). Each brain was removed and dissected bilaterallyto obtain the cerebral cortex and hippocampus, and theregion of the septum was dissected as the septal complexaccording to the brain atlas of Slotnick & Leonard (1975).The individual tissues were rapidly frozen on dry ice, weighedand homogenized in 0.1 M perchloric acid (200 f1 per 10mgtissue) containing 0.1 mM EDTA and 100mM ethyl-homocholine (an internal standard) with an ultrasonic celldisruptor (Model 200, Branson) and centrifuged (12,000 g for20 min at 4°C). The supernatant was filtered (0.45 pm) andstored at - 80°C until analysis. The level of ACh wasmeasured by high performance liquid chromatography withelectrochemical detection as previously described (Murai etal., 1989).

Drugs

AF64A was freshly prepared by dissolving AF64-picrate(Mitsubishi Kasei, Yokohama) in physiological saline accord-ing to the technique of Fisher et al. (1982). The pH wasadjusted to 7.4 with solid NaHCO3 and the solution wasmaintained at room temperature for 1 h. The solution ofAF64A was then maintained on ice and used within 5 h ofpH adjustment. Scopolamine hydrobromide (Sigma, U.S.A.)was dissolved in physiological saline. a-Sialyl cholesterol(NM-71 1; MECT Co., Ltd, Tokyo) was dissolved in distilledwater. Pentobarbitone sodium (Nembutal; Abbott Lab.,U.S.A.) was administered in a volume of 0.01 ml per 10 gbody weight.

Data analysis

Results are expressed as means ± s.e.mean. Statistical ana-lysis was performed with computer software (SuperANOVA,Abacus Concepts, Inc., U.S.A.) for either Dunnett's multiplecomparison test (two-tailed) or Duncan's new multiple rangetest (one-tailed). Probability (P) values less than 0.05 wereconsidered significant.

NOOTROPIC PROPERTIES OF a-SIALYL CHOLESTEROL 389

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Figure 1 The effects of a-sialyl cholesterol (a-SC, 1 or 4 mg kg-1,p.o., once daily for 13 days) on (a) vertical and (b) horizontallocomotor activities in sham-treated mice. Each column representsthe mean movement counts accumulated in 5 min periods over30 min with vertical lines indicating the s.e.mean (n = 12 in eachgroup). Groups are: sham + vehicle (EJ); sham + ao-SC 1 mg kg- '( ); sham +a-SC 4mg kg-' (). There were no significantdifferences (Dunnett's test).

Results

Passive avoidance response

There were no significant differences in acquisition latencyamong the groups. The retention latencies of AF64A-treated(5 and 10 nmol, i.c.v.) animals were significantly lower thanthose of the respective control sham-treated animals.

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Figure 2 The effects of a-sialyl cholesterol (a-SC, I or 4 mg kg-',p.o., once daily for 13 days) on (a) vertical and (b) horizontallocomotor activities in AF64A (2.5 nmol, i.c.v.)-treated mice. Eachcolumn represents the mean movement counts accumulated in 5 minperiods over 30 min with vertical lines indicating the s.e.mean (n = 12in each group). Groups are: sham + vehicle (L ); AF64A + vehicle( _ ); AF64A + a-SC I mg kg'-I ( M ) and AF64A + a-SC 4 mgkg-' (). *P<0.05; **P<0.01 relative to appropriate groups(Duncan's test).

Scopolamine-treated animals similarly showed a significantlylower retention latency. The decrease in retention latencies inthe AF64A-treated animals were reversed by oc-SC (1 and4 mg kg-1) in a dose-dependent manner. There was a trend

Table 1 Step-down passive avoidance response in mice: effect of repeated administration of a-sialyl cholesterol (a-SC, I or 4 mg kg-1,p.o., once daily for 13 days) to sham-, AF64A (2.5, 5, 10 nmol, i.c.v.)- and scopolamine (0.5 mg kg', i.p.)-treated mice

Treatments (dose)

ShamShamShamAF64A (10 nmol)AF64A (1O nmol)AF64A (1O nmol)ShamAF64A (5 nmol)AF64A (5 nmol)AF64A (5 nmol)ShamAF64A (2.5 nmol)AF64A (2.5 nmol)AF64A (2.5 nmol)VehicleScopolamine (0.5 mg kg-')Scopolamine (0.5 mg kg-')Scopolamine (0.5 mg kg-')

+ Vehicle+ a-SC (1 mgkg-')+ a-SC (4 mg kg-')+ Vehicle+ a-SC (1 mgkg-')+ a-SC (4 mg kg-')+ Vehicle+ Vehicle+ a-SC (1 mgkg-')+ a-SC (4 mg kg-')+ Vehicle+ Vehicle+ a-SC (lmgkg-')+ a-SC (4mg kg-')+ Vehicle+ Vehicle+ a-SC (I mg kg-')+ a-SC (4 mg kg-')

nAcquisition latency

(s)

8 12.6 2.59 13.2±3.69 11.8 4.79 8.5±2.69 9.8±6.79 13.3±5.212 12.6 2.511 12.4±3.111 9.2±7.611 7.8±8.2

12 19.8±2.511 15.5±3.612 15.3+5.912 13.2 7.3

9 10.4 3.49 10.7 2.79 19.8±2.19 13.3 5.2

Retention latency(s)

203.1 + 32.6191.7 ± 32.1186.8 ± 38.084.0 ± 27.9tt102.2 44.6t155.0 + 25.8*

280.0 ± 12.579.0 ± 35.2tt138.6 ± 31.0t175.1 ± 22.4*

285.5 ± 21.8202.5 ± 28.7264.0 ± 15.2270.3 + 25.8

252.9 ± 22.665.8 i 20.9tt63.5 + 24.2tt57.3 ± 35.2tt

Each value represents the mean ± s.e.mean. n: numbers of animals.tP< 0.05; ttP< 0.01 compared with corresponding sham + vehicle group.*P <0.05 compared with corresponding AF64A + vehicle group (Duncan's test).

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390 E. ABE et al.

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Figure 3 The effects of a-sialyl cholesterol (a-SC, I or 4mg kg-1,p.o., once daily for 13 days) on (a) vertical and (b) horizontallocomotor activities in AF64A (5nmol, i.c.v.)-treated mice. Eachcolumn represents the mean movement counts accumulated in 5 minperiods over 30 min with vertical lines indicating the s.e.mean (n = 12in each group). For key to column shading see Figure 2 legend.*P<0.05; **P<0.01 relative to appropriate groups (Duncan's test).

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Figure 4 The effects of a-sialyl cholesterol (a-SC, 1 or 4mg kg-',p.o., once daily for 13 days) on (a) vertical and (b) horizontallocomotor activities in AF64A (10nmol, i.c.v.)-treated mice. Eachcolumn represents the mean movement counts accumulated in S minperiods over 30 min with vertical lines indicating the s.e.mean (n = 12in each group). For key to shading of columns see Figure 2 legend.*P<0.05; **P<0.01 relative to appropriate groups (Duncan's test).

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Figure 5 The effects of a-sialyl cholesterol (a-SC, 1 or 4mg kg-',p.o., once daily for 13 days) on (a) vertical and (b) horizontallocomotor activities in scopolamine (0.5mg kg-', i.p.)-treated mice.Each column represents the mean movement counts accumulated in5 min periods over 30 min with vertical lines indicating the s.e.mean(n = 9 in each group). Groups are: vehicle + vehicle (L );scopolamine + vehicle ( M ); scopolamine + a-SC 1 mg kg-' ( M )and scopolamine + a-SC 4mg kg-' (m). *P<0.05; **P<0.01relative to appropriate groups (Duncan's test).

towards a decreased retention latency in the group thatreceived 2.5 nmol of AF64A. a-SC (1 of 4 mg kg-') did notattenuate the scopolamine-induced deficit of retention perfor-mance, however (Table 1).

Locomotor activities

Administration of a-SC (1 or 4 mg kg-', p.o.) to sham-treated mice did not significantly change either vertical andhorizontal locomotor activities during the test (Figure 1). Themice treated with AF64A (2.5, 5 and 10 nmol, i.c.v.) showeda significant increase in both vertical and horizontallocomotor activities and increasing doses of AF64A pro-longed the duration of its action. a-SC dose-dependentlyattenuated the increase in locomotor activities induced byAF64A at a dose of 2.5 nmol, but not at doses of 5 or10 nmol (Figures 2-4). Scopolamine (0.5 mg kg-', i.p.)similarly increased both vertical and horizontal locomotoractivities, but a-SC did not attenuate the increase inlocomotor activities (Figure 5).

Regional acetylcholine levels

AF64A significantly decreased ACh levels in the hippocam-pus in a dose-dependent manner, but not in the septum andcerebral cortex. The corresponding ACh levels (mean +s.e.mean) in the sham-treated group were: 38.2 ± 1.1 in theseptum and 25.5 ± 0.8 in the cerebral cortex. The administra-tion of a-SC (4 mg kg-', p.o.) to AF64A-treated animals

I

NOOTROPIC PROPERTIES OF a-SIALYL CHOLESTEROL 391

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Figure 6 Acetylcholine (ACh) levels in the hippocampus: effect ofrepeated administration of a-sialyl cholesterol (a-SC, 1 or 4 mg kg- ',

p.o., one daily for 13 days) to sham- and AF64A (2.5, 5 or 10 nmol,i.c.v.)-treated mice. Each column represents the mean with s.e.mean

indicated by the vertical lines (n = 8-12, as indicated in Table 1).The doses of drugs are indicated under the columns. Groups are:

sham + vehicle ( Z ); sham + a-SC 1 mg kg- ( _ ); sham + a-SC4mg kg- ' ( M ); AF64A + vehicle ( E); AF64A + a-SC 1 mg kg-'( M ) and AF64A + a-SC 4mg kg-' ( O ). ttP<O.01 comparedwith corresponding sham + vehicle group. *P<0.05; **P<0.01compared with corresponding- AF64A + vehicle group.

significantly reversed the depletion of hippocampal ACh. Incontrast, administration of a-SC (1 or 4mg kg-') to sham-treated animals did not change the level of ACh in thehippocampus (Figure 6).

Discussion

The present study provides the first evidence that the deficitin behavioural performance and depletion of ACh inducedby AF64A can be reversed by a-SC.AF64A and scopolamine significantly decreased retention

latency in the passive avoidance response. These results are

consistent with earlier studies of AF64A (Pope et al., 1985;Yamazaki et al., 1991) and scopolamine (Kameyama et al.,1986; Verloes et al., 1988) reported using rats and mice.AF64A (Yamazaki et al., 1991), scopolamine (Kameyama etal., 1986) or a-SC (unpublished observation) did not changethe threshold footshock intensity, suggesting that changes insensitivity to the electric shock does not contribute tochanges in the passive avoidance response. Increasedlocomotor activities were observed in both AF64A- andscopolamine-treated animals. These effects possibly con-

tributed to the decrease in acquisition and retention latencies.Acquisition latencies in the groups given AF64A at higherdoses (5 and 10 nmol, i.c.v.) were considerably lower than inthe sham group. a-SC was ineffective in reversing the increasein locomotor activities of the AF64A (5 and 10 nmol, i.c.v.)-treated group, and there was no trend of decreasing acquisi-tion latency in the scopolamine group. Thus, it seems thatthese nonspecific drug effects did not contribute to the pas-sive avoidance response, and the reversal of decreased reten-tion latencies by a-SC was related to improving the deficitmemory capacity of AF64A-treated animals.

In parallel with the deficit in passive avoidance response,AF64A significantly decreased ACh levels in the hippocam-pus, but not in the other regions examined, which is inagreement with other reports (Leventer et al., 1985;Yamazaki et al., 1991). The reason for selectivity in thehippocampus after i.c.v. administration of AF64A is not wellunderstood. It has been shown that intrastriatal (Sandberg etal., 1984) and intracortical (Mouton et al., 1989) injection ofAF64A decreased the levels of ACh in the area of theinjection site. Accordingly, the selectivity of AF64A for

the hippocampus may be due to the infusion site, since thehippocampus borders upon the ventricle (Vickroy et al.,1985).Although AF64A at a dose of 2.5 nmol significantly

decreased the ACh level in the hippocampus and increasedlocomotor activities, it did not impair the passive avoidanceresponse. These results suggest that the minimum effectivedose of AF64A which impairs the passive avoidance responseis higher than that causing ACh depletion. Supporting thishypothesis, impairment of the working memory in the radialmaze and T-water maze tasks was observed in rats receivingAF64A at doses ranging from 2 to 6 nmol, i.c.v., but AChlevels in the hippocampus decreased at a dose from 0.6 nmol,i.c.v. (Gower et al., 1989). The minumum effective dose ofAF64A to impair 24 h retention in the passive avoidanceresponse was 3.75 nmol, i.c.v. in rats (Yamazaki et al., 1991).

Decreases in noradrenaline, dopamine and glutamate levelsaccompanied by a reduction in ACh levels have beenobserved in the rat hippocampus after i.c.v. administration ofAF64A (Hortnagl et al., 1987; 1991). However, these non-cholinergic alterations are secondary to functional changes inthe cholinergic system (Hortnagl et al., 1987; 1991), andAF64A disrupts only presynaptic cholinergic substrates invitro (Sandberg et al., 1985; Vickroy et al., 1985). Therefore,a key factor for the improvement of the deficit in retentionlatencies and the increase in locomotor activities of theAF64A-treated animals by ax-SC involves reversal of AChdepletion in the hippocampus. The precise actions of a-SC onthe cholinergic system are unknown. However, a-SC may actthrough repair of the dysfunction of cholinergic system and/or through subsequent stimulation of sprouting and of otherreparative effects by neurones that were not damaged byAF64A.The scopolamine-induced deficit in behavioural perfor-

mance was not effectively attenuated by a-SC. The effects ofscopolamine in the behavioural performance (amnesia andlocomotor stimulation) are due to the blockade of muscarinicreceptors in the brain (Verloes et al., 1988; Pepeu et al., 1989;Toide, 1989). These observations provide the additional sug-gestion that a-SC does not affect muscarinic receptors, eitherdirectly or indirectly.

Sialic acid-containing glycosphingolipids, gangliosides, alsoact as neurotrophic factors (Tsuji et al., 1988). GM, gang-lioside alleviates memory impairment and prevents decreasesin choline acetyltransferase activity and in high affinitycholine uptake in the cerebral cortex after electrolytic oribotenic acid lesions of the nucleus basalis in rats (Casamentiet al., 1985; Di Patre et al., 1989). These observationsindicate that the neurotrophic action of gangliosides can actagainst neuronal dysfunction in the brain and againstmemory impairments. However, gangliosides have a limitedtherapeutic benefit in patients with neurodegenerative braindisorders, because only low concentrations of these com-pounds can cross the blood-brain barrier (Orland et al.,1979). In man, less than 0.05% of peripherally administeredGM1 ganglioside is taken up into the brain (Svennerholm,1991). Unlike GM, ganglioside, orally administered a-SCeasily crosses the blood-brain barrier (unpublished observa-tions). It may prove to be suitable for the treatment ofhuman disorders requiring central activity after peripheraladministration. Hence, the pharmacological availability ofax-SC may offer a therapeutic potential for drugs aimed atalleviating cognitive disorders associated with brain choliner-gic abnormalities.

The authors would like to thank Ms Mayumi Miyata for her tech-nical assistance. The generous gift of AF64A-picrate by Dr AkihiroTobe (Mitsubishi Kasei, Tokyo) is gratefully acknowledged. a-SCwas supplied by MECT CO., Ltd.

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References

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(Received December 9, 1991Revised September 21, 1992

Accepted September 29, 1992)