European Journal of Pharmacology, 211 (1992) 351-358
3!
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52294
L-~-Giycerylphosphorylcholine antagonizes scopolamine-induced amnesia and enhances hippocampal cholinergic transmission in the rat S a n d r a Sigala, A s s u n t a I m p e r a t o a, P a o l a Rizzonelli, P a o l a Casolini a, Cristina Missalc and PierFranco Spano Institute of Pharmacology and Experimental Therapeutics, School of Medicine, University of Brescia, Brescia, Italy and a Institute of Medical Pharmacology, 2nd Chair, University 'La Sapienza ', Roma, Italy
Received 12 November 1991,accepted 26 November 1991
The effects of L-a-glycerylphosphorylcholine (a-GPC) on scopolamine-induced memory impairment and on brain acet~ choline (ACh) synthesis and release were investigated in rats. Oral administration of a-GPC 3 h before the behavioural te prevented the learning impairment induced by scopolamine given 30 min before the acquisition of a passive avoidance respons Similarly, retrograde amnesia induced by scopolamine, given immediately after acquisition training, was also completely reverse by the drug. These effectswere dose-dependent with a maximum at 300 mg/kg. The mechanism of action of this compound w~ investigated by measuring hippocampal ACh synthesis and release both in vivo by means of the microdialysis technique and vitro in tissue slices, a-GPC dose dependently increased ACh release with a maximum at 300 mg/kg. In addition, i.v. injection, [14C]a-GPC resulted in [a4C]ACh formation. The data suggest that the behavioural effects of a-GPC may be related to i property to increase hippocampal A C h synthesis and release. Scopolamine; Passive avoidance; Acetylcholine; Acetylcholine release; Hippocampus; Choline precursors
1. Introduction The role of the basal forebrain cholinergic system in cognitive processes is well established, particularly in association with the functional decline accompanying normal as well as pathological aging (for a rewiew see: Bartus et al., 1982). In aged rodents, impairments in learning and memory are associated with an age-dependent decline in forebrain cholinergic function (Fischer et al., 1989; Gibson and Peterson, 1981; Gilad et al., 1987; Rama Sastry et al., 1983; Sherman et al., 1981; Springer et al., 1987; Strong et al., 1980) and recent evidence indicates that cholinergic neurones in the nucleus basalis magnocellularis, the septal diagonal band area and the striatum undergo age-dependent atrophy (Hornberger et al., 1985; Biegon et al., 1986; Luine et al., 1986; AIb a n e s e et al., 1986). This hypothesis is also supported by experiments with various animal models of memory disruption. The muscarinic antagonist scopolamine indeed impairs re-
Correspondence to: C. Missale, Institute of Pharmacology and Experimental Therapeutics, University of Brescia, Via Valsabbina 19, 25124 Brescia, Italy. Tel. 39.30.399 6282, fax 39.30.3701157, 397802.
tention of passive avoidance and working memory i delayed response (Heise and Hudson, 1985) and radi maze experiments (Wirsching et al., 1984) with rat Analogous effects have been observed in human sul jects; scopolamine produced deficits of learning ar memory performance in young subjects in verbal task deficits which are qualitatively similar to those occu ring naturally in aged subjects (Drachman and Leavil 1974; Drachman et al., 1980). Memory dysfunctiol have also been observed in experimental animals wi~ neurotoxin lesions in the nucleus basalis magnocell~ laris (Miyamoto et al., 1985). Pharmacological therapies have been developed enhance cholinergic activity. Limited success, howeve has been obtained with cholinergic precursors. Cholil and cholinergic precursors have not consistently i: duced memory improvement; negative results, ho~ ever, could depend on the doses and time of treatmer more than on the lack of efficacy of these compoum (Blusztain and Wurtman, 1983). On the other han small but reliable improvements have been obtaine with acetylcholinesterase inhibitors or with dire acetylcholine (ACh) agonists (Heise 1987). In the present study we report the behavioural ar biochemical profile of L-a-glycerylphosphorylcholil (a-GPC), a compound which has the structural chara
352 teristics of a choline precursor. The transient amnesia induced by scopolamine in the passive avoidance test was used as a behavioural experimental model. The biochemical profile of the drug was investigated by studying its effects on both the in vitro and in vivo release of ACh and on ACh synthesis in the hippocampus, since changes noted in the short-term memory of the passive avoidance response have been related to hippocampal activity (Brizzee and Ordy, 1979; Schwegler et al., 1981). The results indicated that a - G P C prevented scopolamine-induced amnesia and that this effect was timeand dose-related with a significant increase of ACh production in the hippocampus,
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats (Charles River, Calco, Italy), weighing 200-250 g, were used. The animals were randomly caged at constant temperature (22°C) and humidity (60%) with a light cycle of 12 h (from 6.00 a.m. to 6.00 p.m.) and free access to food and water.
a - G P C (100-600 mg/kg) by oral gavage; 2 h and ! min after treatment rats were injected i.p. with scop, lamine (1 mg/kg) or vehicle. Thirty minutes later ra were placed individually on the plastic platform ar the behavioural test was performed as described pre~ ously. Retention was tested 24 h later. To test the effects of drugs on consolidation, ra were divided in groups of 10 and treated with or vehicle or oral a - G P C (100-600 mg/kg). Three hou later they were subjected to acquisition training ar immediately injected with i.p. vehicle or i.p. scop lamine (1 mg/kg). The consolidation test was pe formed as described previously 3 h later. After 24 rats were tested for retention. In a different experimental protocol groups of i rats were trained for acquisition of the passive avoi ance response. Immediately after acquisition they r ceived i.p. scopolamine (1 mg/kg) or i.p. vehicle a1 oral ~-GPC (100-300 m g / k g ) or oral vehicle. Tv hours later the behavioural test was repeated to veri consolidation of the acquired response. Twenty-fo~ hours later animals were tested for retention. Results are expressed as the percentage of anim~ acquiring and consolidating the behavioural respon (positive responses).
2.3. [3H]ACh release 2.2. Passive avoidance studies 2.2.1. Apparatus The step-down passive avoidance apparatus was a plexiglas rectangular box (30 x 30 × 40 cm high) with a steel rod grid floor (30 parallel steel rods, 0.3 cm diameter set 1.0 cm apart) and an isolated plastic platform (15 × 15 x 0.5 cm thick) set in one corner of the box. A constant and continuous current of 0.8 mA was applied to the grid floor by an external stimulator,
2.2.2. Experimental procedures Each rat was trained by gently placing it on the plastic platform; as soon as the animal stepped down from the platform an electric shock was delivered. The normal reaction of naive animals is to jump back onto the platform. The step-down latency was measured with a stop watch. Within 60 s naive rats acquired the passive avoidance response, refraining from stepping down into the grid floor, The consolidation test was done 3 h after training and the retention test after 24 h. Each rat was again placed on the platform and the step-down latency was measured. An upper cut-off time of 300 s was set. To test the effects of drugs on the acquisition of the passive avoidance responses rats were divided into groups of 10 at random. Three hours before acquisition training they received vehicle or different doses of
Groups of five rats received different doses of , GPC (100-600 m g / k g ) or vehicle by oral gavage. different times after administration (2, 3, 5, and 8 rats were killed by decapitation; brains were rapid removed and the hippocampus was dissected ai placed in Ca2+-free Earle's balanced salt soluti~ (EBSS) previously equilibrated with 0 2 : CO 2 (95 :,' The tissue was cross-chopped with a TC-2 Sorv~ tissue chopper and washed twice with Ca 2 +-free EBS Tissue slices were incubated in EBSS in the pro ence of 10 nM [3H]choline (86.7 Ci/mmol; Dupor NEN) at 37°C for 30 min under a stream of 0 2 : CC Slices were then washed twice with ice-cold EBSS eliminate excess [3H]choline and aliquots (5 n tissue/sample) were incubated at 37°C for 10 min the presence of 10/,LM hemicolinium. KC1 (15-45 mi~ was added and the incubation was continued for additional 10 min and then stopped by placing t] samples in ice. Samples were centrifuged at 1000 >~ for 2 min and aliquots of the supernatant were an~ ysed for radioactivity by using a beta-counter.
2.4. Microdialysis implantation and HPLC assay Rats were anaestetized with chloral hydrate (C g / k g i.p.) and implanted with dialysis tubes (out diameter = 290 tzm; Hospal-Dasco, Bologna, Italy)
3~
the level of the dorsal hippocampus or striatum according to the atlas of K6nig and Klippel. Surgery was carried out as described previously (Imperato and Di Chiara, 1985). A Ringer solution (4 mM KCI, 147 mM NaCI, 1.5 mM CaCI 2) with 100 nM neostigmine was pumped into the dialysis tubes at a constant rate of 2 /zl/min for ACh determination. ACh was estimated in 20-min samples of dialysate by high-performance liquid chromatography (HPLC) with electrochemical detection, according to Damsma et al. (1985). Experiments were performed 24 h after implantation of the dialysis tubes. The basal output of ACh, expressed as pmol/20 min (mean + S.E.M. of at least seven rats), was 8 ___0.65 and 12 + 0.9 for the dorsal hippocampus and the striatum, respectively. After 2 h perfusion, when ACh output had stabilized (the last three samples did not differ by more than 10%), a-GPC or saline was orally administered to rats in a final volume of 0.3 ml/100 g body weight.
a-GPC
An analysis of variance followed by a two-taile Student's t-test was used for statistical evaluation differences.
2.8. Drugs The sources of the drugs used in the present stu~ were as follows: a-GPC and [14C]a-GPC were kind provided by LPB, Milano, Italy; [3H]choline was fro: Dupont-NEN, Firenze, Italy; other drugs used in th study were from Sigma, St. Louis, MO, USA.
3. Results
3.1. Effects of a-GPC on the passive avoidance test The effects of different doses of a-GPC on tl scopolamine-induced disruption of acquisition of tl passive avoidance response in the rat are shown in fi
2.5. [14C]ACh synthesis 14C-Labelled
2. 7. Statistical analysis
(L-a-glycerylphosphoryl[1,2-
14C]choline) was directly administered into the carotid artery (i.v.). At different times after administration, rats were killed by decapitation and the frontal cortex, the striatum and the hippocampus were dissected, homogenized in 0.1 M HC104 and centrifuged at 3000 rpm for 40 min. The supernatants were injected onto a cation-exchange column and 1-min fractions were collected and analysed for radioactivity by using a betacounter. Previous calibration of the cation-exchange column showed that ACh eluted in the sixth fraction. The amount of [laC]ACh produced was then calculated and expressed as pg/mg protein,
2.6. [3H]Choline uptake Rats were divided into two groups at random. Three hours before they were killed, one group of rats received oral a-GPC (300 mg/kg) and the other oral vehicle. Rats were killed by decapitation. The brains were rapidly removed and the hippocampus was dissected and cross-cut as described previously. After two washes with Ca2+-free EBSS, slices (5 mg tissue/sampie) were incubated at 37°C for 10 min in the presence or in the absence of 10 izM hemicolinium. [3H]Choline (0.5-10 nM) was then added and the incubation was continued for an additional 10 min and then stopped by rapid filtration under reduced pressure through Whatman G F / B filters, using the Cell Harvester Apparatus (Biomedical Research and Development Laboratories, Gaithersburg, MA, USA). After treatment with 1 N NaOH filters were analysed for the radioactivity incorporated by using a beta-counter,
1A. Scopolamine was given at a dose of 1 mg/kg al the retention test was done 3 h after administration the basis of previous results showing that at this do and at this time the amnesic effect of the drug maximal (Memo et al., 1988). Scopolamine admini tered to naive rats 30 rain before training stron~ impaired acquisition of the behavioural response; on 20% of trained rats acquired the passive avoidan, response within 5 rain under these experimental conc tions, a-GPC given 3 h before training, while n modifying the behavioural response in naive rats (da not shown), strongly prevented the failure of acqui: tion elicited by scopolamine. This effect was dose-d pendent over the range of 50-600 mg/kg with a maz mum at 300 mg/kg. In particular, 50% of trained r~ receiving 100 mg/kg a-GPC and 90% of those receJ ing 300 and 600 mg/kg of the drug acquired t] passive avoidance response within 5 min. This w confirmed in the retention test performed 24 h lat~ all rats that had acquired the behavioural respon during the training session responded positively in t] retention test. The data shown in fig. 1B show the effects a-GPC on scopolamine-induced amnesia of the pre' ously acquired passive avoidance response. Scop lamine when given immediately after acquisition of t: behavioural response disrupted memory consolidati~ (Izquierdo, 1989), resulting in amnesia in the retentiq test performed 3 and 24 h later. Pretraining admin tration of a-GPC dose dependently prevented SCOlZ lamine-induced amnesia with a maximum effect at 3 mg/kg. The effects of post-training administration of a-GI on scopolamine-induced amnesia of the previously
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m g / k g with a maximum at 300 m g / k g (controls: 70 ~ increase; a - G P C 200% increase). T h e time course of the effects of a - G P C on pota sium-stimulated [3H]ACh release in hippocampal slip is shown in fig. 3B. T h e potentiating effect of a - G P ( given at the maximally effective dose of 300 m g / k was evident 2 h after administration, reached its max m u m after 3 h and was still detectable, a l t h o u g h lower levels, when the drug was administered 5 before the animals were killed. In contrast 8 h aft, a - G P C administration potassium-stimulated [ 3 H ] A ( release reached control levels. Unlike a - G P C , phosphatydylcholine did not affe hippocampal [3H]ACh release w h e n administerd different doses and at different times before the a~ mals were killed. Basal and stimulated [3H]ACh r lease was similar in hippocampal slices obtained fro controls and treated rats (fig. 4). 3.3. Effects o f a - G P C on in vivo A C h release
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Fig. 1. Effects of pre-training administration of a-GPC on scopolamine-induced memory impairment in the passive avoidance test. (A) rats were treated with saline or a-GPC (50-600 mg/kg p.o.) 3 h before training. Two hours and 30 min after a-GPC rats received i . p . scopolamine (1 mg/kg) or i.p. vehicle and 30 rain later the rats were trained as described in Materials and methods. Rats were tested for retention of the acquired response 24 h later. (B) Rats were treated with saline or a-GPC (100-600 mg/kg p.o.) and 3 h later were trained as described in Materials and methods. Immediately after acquisition of the passive avoidance response rats received i.p. scopolamine (1 mg/kg) and the retention test was done 3 and 24 h later. • P < 0.001 vs. scopolamine. Each point represents the mean+_ S.E.M. of three independent experiments with 10 rats.
Figure 5 shows that oral a - G P C e n h a n c e d bas A C h release in the striatum as well as in the hippocar pus in a d o s e - d e p e n d e n t way. T h e threshold dose w 75 m g / k g for both areas. A C h release was increase further by a - G P C given at 150 m g / k g , reaching i m a x i m u m after administration of 300 m g / k g . Intere~, ingly, the highest dose was slightly m o r e effective A
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a - G P C dose dependently antagonized scopolamine-induced amnesia with a maximal effect at 300 m g / k g .
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Figure 3 A shows the effects of a single administration of a - G P C (300 m g / k g p.o.), given 3 h before the animals were killed, on [3H]ACh release induced by a depolarizing concentration of potassium (30 m M ) in rat hippocampal slices. Basal [3H]ACh release was not modified in a - G P C - t r e a t e d rats. In contrast, [3H]ACh release evoked by 30 m U potassium was greater in hippocampal slices obtained from a - G P C - t r e a t e d animals than in those from controls. T h e facilitating effect of a - G P C was d o s e - d e p e n d e n t over the range 5 0 - 6 0 0
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quired response are shown in fig. 2. In this protocol scopolamine and a - G P C were given together within 2 min after acquisition and the retention tests were performed either 3 h (fig. 2A) or 24 h (fig. 2B) later.
3.2. Effects o f a - G P C on [ 3 H ] A C h release
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Fig. 2. Effect of post-training administration of a-GPC on SCOl lamine-induced memory disruption of the passive avoidance sponse. Immediately after acquisition of the passive avoidance sponse rats received i.p. scopolamine (1 mg/kg) or i.p. vehicle p.o. a-GPC (100-600 mg/kg) or p.o. vehicle. Rats were tested retention of the behavioural response 3 h (A) or 24 h (B) later. B represent the means+_S.E.M, of three independent experiments 10 rats in each group. ** P < 0.001 vs. controls; * P < 0.001 scopolamine. ( [] ) Controls; ( [] ) scopolamine; ( [] ) scopolamine + GPC 100 mg/kg; ([]) scopolamine + a-GPC 300 mg/kg; ([]) scol lamine + a-GPC 600 mg/kg.
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As illustrated in fig. 7, i.v. injection of [14C]a-GP (4 m g / k g ) i n d u c e d the f o r m a t i o n of radioactive A C h i the frontal cortex, s t r i a t u m a n d h i p p o c a m p u s . T~ effect was d e t e c t a b l e 10 m i n after the injection [ t 4 C ] a - G P C a n d [14C]ACh was still p r e s e n t 12 h late
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4. Discussion
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T h e basal f o r e b r a i n cholinergic system plays a maj~ role in m e m o r y f u n c t i o n in b o t h h u m a n s a n d expe~ m e n t a l a n i m a l s (for a review see: Bartus et al., 198~ a n d it has b e e n suggested that cholinergic impairme: may be responsible for the m e m o r y d i s t u r b a n c e assoc ated with old age in r o d e n t s (Lippa et al., 1980; Strot et al., 1980). In line with those, anticholinergic dru~
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3.5. Effectsof a-GPCon [14C]AChsynthesis
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Fig. 3. Effects of a-GPC on potassium-stimulated [3H]ACh release in rat hippocampal slices. (A) five groups of five rats each were treated with either saline or different doses (50-600 mg/kg p.o.) of a-GPC and killed by decapitation 3 h later. Hippocampal slices were preloaded with [3H]choline and 30 mM potassium-stimulated [3H]ACh release was determined as described in Materials and methods. (B) rats were treated with saline or a-GPC (300 mg/kg p.o.) and killed by decapitation at different times (2-8 h) after drug administration. Hippocampal slices were preloaded with [3H]choline and both basal and 30 mM potassium-stimulated [3HIACh release were measured as described in Materials and methods. Points are the means_+S.E.M. of three independent experiments run in triplicate. * P < 0.001 vs. controls (©) Controls; (o) a-GPC,
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52294
L-~-Giycerylphosphorylcholine antagonizes scopolamine-induced amnesia and enhances hippocampal cholinergic transmission in the rat S a n d r a Sigala, A s s u n t a I m p e r a t o a, P a o l a Rizzonelli, P a o l a Casolini a, Cristina Missalc and PierFranco Spano Institute of Pharmacology and Experimental Therapeutics, School of Medicine, University of Brescia, Brescia, Italy and a Institute of Medical Pharmacology, 2nd Chair, University 'La Sapienza ', Roma, Italy
Received 12 November 1991,accepted 26 November 1991
The effects of L-a-glycerylphosphorylcholine (a-GPC) on scopolamine-induced memory impairment and on brain acet~ choline (ACh) synthesis and release were investigated in rats. Oral administration of a-GPC 3 h before the behavioural te prevented the learning impairment induced by scopolamine given 30 min before the acquisition of a passive avoidance respons Similarly, retrograde amnesia induced by scopolamine, given immediately after acquisition training, was also completely reverse by the drug. These effectswere dose-dependent with a maximum at 300 mg/kg. The mechanism of action of this compound w~ investigated by measuring hippocampal ACh synthesis and release both in vivo by means of the microdialysis technique and vitro in tissue slices, a-GPC dose dependently increased ACh release with a maximum at 300 mg/kg. In addition, i.v. injection, [14C]a-GPC resulted in [a4C]ACh formation. The data suggest that the behavioural effects of a-GPC may be related to i property to increase hippocampal A C h synthesis and release. Scopolamine; Passive avoidance; Acetylcholine; Acetylcholine release; Hippocampus; Choline precursors
1. Introduction The role of the basal forebrain cholinergic system in cognitive processes is well established, particularly in association with the functional decline accompanying normal as well as pathological aging (for a rewiew see: Bartus et al., 1982). In aged rodents, impairments in learning and memory are associated with an age-dependent decline in forebrain cholinergic function (Fischer et al., 1989; Gibson and Peterson, 1981; Gilad et al., 1987; Rama Sastry et al., 1983; Sherman et al., 1981; Springer et al., 1987; Strong et al., 1980) and recent evidence indicates that cholinergic neurones in the nucleus basalis magnocellularis, the septal diagonal band area and the striatum undergo age-dependent atrophy (Hornberger et al., 1985; Biegon et al., 1986; Luine et al., 1986; AIb a n e s e et al., 1986). This hypothesis is also supported by experiments with various animal models of memory disruption. The muscarinic antagonist scopolamine indeed impairs re-
Correspondence to: C. Missale, Institute of Pharmacology and Experimental Therapeutics, University of Brescia, Via Valsabbina 19, 25124 Brescia, Italy. Tel. 39.30.399 6282, fax 39.30.3701157, 397802.
tention of passive avoidance and working memory i delayed response (Heise and Hudson, 1985) and radi maze experiments (Wirsching et al., 1984) with rat Analogous effects have been observed in human sul jects; scopolamine produced deficits of learning ar memory performance in young subjects in verbal task deficits which are qualitatively similar to those occu ring naturally in aged subjects (Drachman and Leavil 1974; Drachman et al., 1980). Memory dysfunctiol have also been observed in experimental animals wi~ neurotoxin lesions in the nucleus basalis magnocell~ laris (Miyamoto et al., 1985). Pharmacological therapies have been developed enhance cholinergic activity. Limited success, howeve has been obtained with cholinergic precursors. Cholil and cholinergic precursors have not consistently i: duced memory improvement; negative results, ho~ ever, could depend on the doses and time of treatmer more than on the lack of efficacy of these compoum (Blusztain and Wurtman, 1983). On the other han small but reliable improvements have been obtaine with acetylcholinesterase inhibitors or with dire acetylcholine (ACh) agonists (Heise 1987). In the present study we report the behavioural ar biochemical profile of L-a-glycerylphosphorylcholil (a-GPC), a compound which has the structural chara
352 teristics of a choline precursor. The transient amnesia induced by scopolamine in the passive avoidance test was used as a behavioural experimental model. The biochemical profile of the drug was investigated by studying its effects on both the in vitro and in vivo release of ACh and on ACh synthesis in the hippocampus, since changes noted in the short-term memory of the passive avoidance response have been related to hippocampal activity (Brizzee and Ordy, 1979; Schwegler et al., 1981). The results indicated that a - G P C prevented scopolamine-induced amnesia and that this effect was timeand dose-related with a significant increase of ACh production in the hippocampus,
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats (Charles River, Calco, Italy), weighing 200-250 g, were used. The animals were randomly caged at constant temperature (22°C) and humidity (60%) with a light cycle of 12 h (from 6.00 a.m. to 6.00 p.m.) and free access to food and water.
a - G P C (100-600 mg/kg) by oral gavage; 2 h and ! min after treatment rats were injected i.p. with scop, lamine (1 mg/kg) or vehicle. Thirty minutes later ra were placed individually on the plastic platform ar the behavioural test was performed as described pre~ ously. Retention was tested 24 h later. To test the effects of drugs on consolidation, ra were divided in groups of 10 and treated with or vehicle or oral a - G P C (100-600 mg/kg). Three hou later they were subjected to acquisition training ar immediately injected with i.p. vehicle or i.p. scop lamine (1 mg/kg). The consolidation test was pe formed as described previously 3 h later. After 24 rats were tested for retention. In a different experimental protocol groups of i rats were trained for acquisition of the passive avoi ance response. Immediately after acquisition they r ceived i.p. scopolamine (1 mg/kg) or i.p. vehicle a1 oral ~-GPC (100-300 m g / k g ) or oral vehicle. Tv hours later the behavioural test was repeated to veri consolidation of the acquired response. Twenty-fo~ hours later animals were tested for retention. Results are expressed as the percentage of anim~ acquiring and consolidating the behavioural respon (positive responses).
2.3. [3H]ACh release 2.2. Passive avoidance studies 2.2.1. Apparatus The step-down passive avoidance apparatus was a plexiglas rectangular box (30 x 30 × 40 cm high) with a steel rod grid floor (30 parallel steel rods, 0.3 cm diameter set 1.0 cm apart) and an isolated plastic platform (15 × 15 x 0.5 cm thick) set in one corner of the box. A constant and continuous current of 0.8 mA was applied to the grid floor by an external stimulator,
2.2.2. Experimental procedures Each rat was trained by gently placing it on the plastic platform; as soon as the animal stepped down from the platform an electric shock was delivered. The normal reaction of naive animals is to jump back onto the platform. The step-down latency was measured with a stop watch. Within 60 s naive rats acquired the passive avoidance response, refraining from stepping down into the grid floor, The consolidation test was done 3 h after training and the retention test after 24 h. Each rat was again placed on the platform and the step-down latency was measured. An upper cut-off time of 300 s was set. To test the effects of drugs on the acquisition of the passive avoidance responses rats were divided into groups of 10 at random. Three hours before acquisition training they received vehicle or different doses of
Groups of five rats received different doses of , GPC (100-600 m g / k g ) or vehicle by oral gavage. different times after administration (2, 3, 5, and 8 rats were killed by decapitation; brains were rapid removed and the hippocampus was dissected ai placed in Ca2+-free Earle's balanced salt soluti~ (EBSS) previously equilibrated with 0 2 : CO 2 (95 :,' The tissue was cross-chopped with a TC-2 Sorv~ tissue chopper and washed twice with Ca 2 +-free EBS Tissue slices were incubated in EBSS in the pro ence of 10 nM [3H]choline (86.7 Ci/mmol; Dupor NEN) at 37°C for 30 min under a stream of 0 2 : CC Slices were then washed twice with ice-cold EBSS eliminate excess [3H]choline and aliquots (5 n tissue/sample) were incubated at 37°C for 10 min the presence of 10/,LM hemicolinium. KC1 (15-45 mi~ was added and the incubation was continued for additional 10 min and then stopped by placing t] samples in ice. Samples were centrifuged at 1000 >~ for 2 min and aliquots of the supernatant were an~ ysed for radioactivity by using a beta-counter.
2.4. Microdialysis implantation and HPLC assay Rats were anaestetized with chloral hydrate (C g / k g i.p.) and implanted with dialysis tubes (out diameter = 290 tzm; Hospal-Dasco, Bologna, Italy)
3~
the level of the dorsal hippocampus or striatum according to the atlas of K6nig and Klippel. Surgery was carried out as described previously (Imperato and Di Chiara, 1985). A Ringer solution (4 mM KCI, 147 mM NaCI, 1.5 mM CaCI 2) with 100 nM neostigmine was pumped into the dialysis tubes at a constant rate of 2 /zl/min for ACh determination. ACh was estimated in 20-min samples of dialysate by high-performance liquid chromatography (HPLC) with electrochemical detection, according to Damsma et al. (1985). Experiments were performed 24 h after implantation of the dialysis tubes. The basal output of ACh, expressed as pmol/20 min (mean + S.E.M. of at least seven rats), was 8 ___0.65 and 12 + 0.9 for the dorsal hippocampus and the striatum, respectively. After 2 h perfusion, when ACh output had stabilized (the last three samples did not differ by more than 10%), a-GPC or saline was orally administered to rats in a final volume of 0.3 ml/100 g body weight.
a-GPC
An analysis of variance followed by a two-taile Student's t-test was used for statistical evaluation differences.
2.8. Drugs The sources of the drugs used in the present stu~ were as follows: a-GPC and [14C]a-GPC were kind provided by LPB, Milano, Italy; [3H]choline was fro: Dupont-NEN, Firenze, Italy; other drugs used in th study were from Sigma, St. Louis, MO, USA.
3. Results
3.1. Effects of a-GPC on the passive avoidance test The effects of different doses of a-GPC on tl scopolamine-induced disruption of acquisition of tl passive avoidance response in the rat are shown in fi
2.5. [14C]ACh synthesis 14C-Labelled
2. 7. Statistical analysis
(L-a-glycerylphosphoryl[1,2-
14C]choline) was directly administered into the carotid artery (i.v.). At different times after administration, rats were killed by decapitation and the frontal cortex, the striatum and the hippocampus were dissected, homogenized in 0.1 M HC104 and centrifuged at 3000 rpm for 40 min. The supernatants were injected onto a cation-exchange column and 1-min fractions were collected and analysed for radioactivity by using a betacounter. Previous calibration of the cation-exchange column showed that ACh eluted in the sixth fraction. The amount of [laC]ACh produced was then calculated and expressed as pg/mg protein,
2.6. [3H]Choline uptake Rats were divided into two groups at random. Three hours before they were killed, one group of rats received oral a-GPC (300 mg/kg) and the other oral vehicle. Rats were killed by decapitation. The brains were rapidly removed and the hippocampus was dissected and cross-cut as described previously. After two washes with Ca2+-free EBSS, slices (5 mg tissue/sampie) were incubated at 37°C for 10 min in the presence or in the absence of 10 izM hemicolinium. [3H]Choline (0.5-10 nM) was then added and the incubation was continued for an additional 10 min and then stopped by rapid filtration under reduced pressure through Whatman G F / B filters, using the Cell Harvester Apparatus (Biomedical Research and Development Laboratories, Gaithersburg, MA, USA). After treatment with 1 N NaOH filters were analysed for the radioactivity incorporated by using a beta-counter,
1A. Scopolamine was given at a dose of 1 mg/kg al the retention test was done 3 h after administration the basis of previous results showing that at this do and at this time the amnesic effect of the drug maximal (Memo et al., 1988). Scopolamine admini tered to naive rats 30 rain before training stron~ impaired acquisition of the behavioural response; on 20% of trained rats acquired the passive avoidan, response within 5 rain under these experimental conc tions, a-GPC given 3 h before training, while n modifying the behavioural response in naive rats (da not shown), strongly prevented the failure of acqui: tion elicited by scopolamine. This effect was dose-d pendent over the range of 50-600 mg/kg with a maz mum at 300 mg/kg. In particular, 50% of trained r~ receiving 100 mg/kg a-GPC and 90% of those receJ ing 300 and 600 mg/kg of the drug acquired t] passive avoidance response within 5 min. This w confirmed in the retention test performed 24 h lat~ all rats that had acquired the behavioural respon during the training session responded positively in t] retention test. The data shown in fig. 1B show the effects a-GPC on scopolamine-induced amnesia of the pre' ously acquired passive avoidance response. Scop lamine when given immediately after acquisition of t: behavioural response disrupted memory consolidati~ (Izquierdo, 1989), resulting in amnesia in the retentiq test performed 3 and 24 h later. Pretraining admin tration of a-GPC dose dependently prevented SCOlZ lamine-induced amnesia with a maximum effect at 3 mg/kg. The effects of post-training administration of a-GI on scopolamine-induced amnesia of the previously
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m g / k g with a maximum at 300 m g / k g (controls: 70 ~ increase; a - G P C 200% increase). T h e time course of the effects of a - G P C on pota sium-stimulated [3H]ACh release in hippocampal slip is shown in fig. 3B. T h e potentiating effect of a - G P ( given at the maximally effective dose of 300 m g / k was evident 2 h after administration, reached its max m u m after 3 h and was still detectable, a l t h o u g h lower levels, when the drug was administered 5 before the animals were killed. In contrast 8 h aft, a - G P C administration potassium-stimulated [ 3 H ] A ( release reached control levels. Unlike a - G P C , phosphatydylcholine did not affe hippocampal [3H]ACh release w h e n administerd different doses and at different times before the a~ mals were killed. Basal and stimulated [3H]ACh r lease was similar in hippocampal slices obtained fro controls and treated rats (fig. 4). 3.3. Effects o f a - G P C on in vivo A C h release
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Fig. 1. Effects of pre-training administration of a-GPC on scopolamine-induced memory impairment in the passive avoidance test. (A) rats were treated with saline or a-GPC (50-600 mg/kg p.o.) 3 h before training. Two hours and 30 min after a-GPC rats received i . p . scopolamine (1 mg/kg) or i.p. vehicle and 30 rain later the rats were trained as described in Materials and methods. Rats were tested for retention of the acquired response 24 h later. (B) Rats were treated with saline or a-GPC (100-600 mg/kg p.o.) and 3 h later were trained as described in Materials and methods. Immediately after acquisition of the passive avoidance response rats received i.p. scopolamine (1 mg/kg) and the retention test was done 3 and 24 h later. • P < 0.001 vs. scopolamine. Each point represents the mean+_ S.E.M. of three independent experiments with 10 rats.
Figure 5 shows that oral a - G P C e n h a n c e d bas A C h release in the striatum as well as in the hippocar pus in a d o s e - d e p e n d e n t way. T h e threshold dose w 75 m g / k g for both areas. A C h release was increase further by a - G P C given at 150 m g / k g , reaching i m a x i m u m after administration of 300 m g / k g . Intere~, ingly, the highest dose was slightly m o r e effective A
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Figure 3 A shows the effects of a single administration of a - G P C (300 m g / k g p.o.), given 3 h before the animals were killed, on [3H]ACh release induced by a depolarizing concentration of potassium (30 m M ) in rat hippocampal slices. Basal [3H]ACh release was not modified in a - G P C - t r e a t e d rats. In contrast, [3H]ACh release evoked by 30 m U potassium was greater in hippocampal slices obtained from a - G P C - t r e a t e d animals than in those from controls. T h e facilitating effect of a - G P C was d o s e - d e p e n d e n t over the range 5 0 - 6 0 0
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quired response are shown in fig. 2. In this protocol scopolamine and a - G P C were given together within 2 min after acquisition and the retention tests were performed either 3 h (fig. 2A) or 24 h (fig. 2B) later.
3.2. Effects o f a - G P C on [ 3 H ] A C h release
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Fig. 2. Effect of post-training administration of a-GPC on SCOl lamine-induced memory disruption of the passive avoidance sponse. Immediately after acquisition of the passive avoidance sponse rats received i.p. scopolamine (1 mg/kg) or i.p. vehicle p.o. a-GPC (100-600 mg/kg) or p.o. vehicle. Rats were tested retention of the behavioural response 3 h (A) or 24 h (B) later. B represent the means+_S.E.M, of three independent experiments 10 rats in each group. ** P < 0.001 vs. controls; * P < 0.001 scopolamine. ( [] ) Controls; ( [] ) scopolamine; ( [] ) scopolamine + GPC 100 mg/kg; ([]) scopolamine + a-GPC 300 mg/kg; ([]) scol lamine + a-GPC 600 mg/kg.
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4. Discussion
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T h e basal f o r e b r a i n cholinergic system plays a maj~ role in m e m o r y f u n c t i o n in b o t h h u m a n s a n d expe~ m e n t a l a n i m a l s (for a review see: Bartus et al., 198~ a n d it has b e e n suggested that cholinergic impairme: may be responsible for the m e m o r y d i s t u r b a n c e assoc ated with old age in r o d e n t s (Lippa et al., 1980; Strot et al., 1980). In line with those, anticholinergic dru~
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Fig. 3. Effects of a-GPC on potassium-stimulated [3H]ACh release in rat hippocampal slices. (A) five groups of five rats each were treated with either saline or different doses (50-600 mg/kg p.o.) of a-GPC and killed by decapitation 3 h later. Hippocampal slices were preloaded with [3H]choline and 30 mM potassium-stimulated [3H]ACh release was determined as described in Materials and methods. (B) rats were treated with saline or a-GPC (300 mg/kg p.o.) and killed by decapitation at different times (2-8 h) after drug administration. Hippocampal slices were preloaded with [3H]choline and both basal and 30 mM potassium-stimulated [3HIACh release were measured as described in Materials and methods. Points are the means_+S.E.M. of three independent experiments run in triplicate. * P < 0.001 vs. controls (©) Controls; (o) a-GPC,
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