Cholecystokinin
Protects
against Kiminobu
Basal
Sugaya,
Cholinergic
Forebrain
Masanori
Takahashi
Neurons
Lesion and Kazuhiko
Kubota
Research Institute for Biosciences, Science Universityof Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan Received
December
2, 1991
Accepted
February
29, 1992
ABSTRACT Alzheimer's Disease (AD) patients have a severe degeneration of cholinergic neurons in their cerebral cortices. Basal forebrain (BF)-lesioned rat is used as a model animal of a cholinergic defi cit in the cerebral cortex. Cholinergic markers were decreased in the cerebral cortex of BF-lesioned rats. Intracerebroventricular continuous infusion of cholecystokinin octapeptide (CCK8) following BF lesion obviously preserved these cholinergic markers. These results suggest that CCK8 prevents the de generation of cholinergic neurons in the cerebral cortex following BF lesion. Keywords:
Alzheimer's
disease,
Cholecystokinin,
The cholinergic deficit seen in Alzheimer's Disease (AD) is associated with cell loss in the nucleus basalis of Meynert (nbM) (1). In mammalian brain, some cell groups found in the basal forebrain (BF) are particular ly given interest as an analogue of nbM (2). The large neurons of the BF provide the major extrinsic cho linergic innervation of the cerebral cortex (CC). This origin form cell group known as the nucleus basalis magnocellularis (NBM) is diffusely localized in the re gion of the ventromedial portion of the globus pallidus, the substaintia innominata and the preoptic magnocellu lar nucleus. Lesions of the rat NBM provide an animal model of cholinergic dysfunction in the CC of patients with AD, and it causes a dramatic decrease in the cho line acetyltransferase (ChAT) activity, acetylcholin esterase (AChE) activity, high affinity choline uptake (3) and acetylcholine (ACh) release (4) in the CC. Similar deficits are observed in AD brain (1, 5). Learn ing and memory impairments, characteristic of AD, are also produced in NBM-lesioned animals (6). Cholecysto kinin octapeptide (CCK8) and it's binding site are widely distributed in the brain (7). Centrally adminis tered CCK8 increased 2-deoxy glucose (2DG) uptake (8) and modulated dopamine (DA) release in the nu cleus accumbens (9) and ACh release from the cerebral cortex in vivo (10). These evidences suggest that CCK8 can play a role of neurotransmitter or neuromodulator in the central nervous system. Most of all, it can mod ulate the memory processing (11). In these studies, we determine the effect of infusions
Basal
forebrain
lesion
of CCK8 into the ventricle on the post-lesion functional changes of cholinergic neurons by examining ChAT activity and ACh release level in continuous micro punches of the cortex. Male Sprague-Dawley rats (Sankyo Lab. Co., Tokyo), weighing 250-280 g at the start of the experiments, were used. They were housed in cages maintained at 22 ± 2 °C with a 12/12 hr light dark cycle. The rats had free access to laboratory chow (CE2, Nihon Clea Co.) and water throughout the ex periments. Each rat was anesthetized with sodium pen tobarbital (50 mg/kg, i.p.) and mounted in a stereotaxic apparatus (David copf). Unilateral lesions of the NBM were performed via injection of ibotenic acid (Sigma) (5 ,ug/1 ,ul, in a vehicle of sterile filtered 200 mM phos phate buffer, pH 7.4). Ibotenic acid was delivered with a graduated microliter glass pipette. NBM was stereo taxically localized using the following coordinates: AP = -2 .3 mm from the bregma, ML = ± 3.7 mm, 7.5 mm below the dura. The toxin was delivered over 10 min and the micropipette was left in place for another 5 min following injection, and the vehicle was injected contralaterally. After that, the rat was stereotaxically implanted unilaterally into the ventricle with a perma nent stainless steel L-shaped microcannula. The coor dinates used for cannula plantation were AP = 0.8 mm from the bregma, ML = ± 1.5 mm, 3.6 mm below the dura. The cannula was then anchored to the skull with acrylic cement. The mini osmotic pump (alzet MODEL 2002) filled with CCK8 (Peptide Institute, Inc.; 10 ng/12,ul) or vehicle was connected to the cannula with
a polyethylene tube, which was then placed sub cutaneously on the back of the rat. The mini pump assured constant delivery of CCK8 (10 ng/day) for two weeks. Animals were sacrificed by decapitation at two weeks after NBM lesion. The brains were rapidly taken out and sliced into 400-,um sections with a Mcllwain tis sue chopper (Brinkman). Each of ten sections ranging from +2.0 mm bregma to -2.0 mm bregma was dis sected free. Three micropunches (1.6-mm diameter) were taken from each hemisphere of the cerebral cortex (CC-1 to CC-5, Fig. 1). Sixty punches were taken in total, and 30 of them were frozen on dry ice for later determination of ChAT activity, while the remaining 30 punches were used for measurement of ACh release. ACh release was determined in micropunches from every other one of the serial cortical slices. The micro punches were preloaded with [3H]-choline (0.1,uM; 80 Ci/mmol) for 20 min in Krebs Ringer bicarbonate buff er of the following composition: 118 mM NaCl, 4.70 mM KC1, 1.50 mM MgSO4, 1.15 mM KH2PO4, 1.25 mM CaCl2, 25 mM NaHCO3, 11.1 mM glucose, bub bled with 95%02 and 5% CO2 at 37°C. The punches (3 punches/chamber) were then transferred into a su perfusion chamber system (Brandel) and continuously superfused with oxygenated Krebs buffer containing 10
,uM hemicholinium-3, at a flow rate of 1 ml/min. After the experiment, the micropunches were solubilized by Soluene-350 (Packard). The radioactivities in the super fusate and solubilized tissue were measured by a liquid scintillation counter. For the measurement of ChAT activity, each of three tissue punches was homogenized using an ultrasonicator (30% power, Microtip, 6 sec) in 100,ul of 50 mM Tris-HC1 buffer (PH 7.4). The homog enate was resuspended in 1/5 volume of 2.5% Triton-X 100 and the same volume of 10 mM EDTA. Eight microliters of sample suspension and 20 ,ul of a sub strate mixture consisting of 50 mM sodium phosphate buffer, 0.2 mM [3H]-Acetyl CoA (NEN, 1 pCi/mmol), 20 mM EDTA, 300 mM NaCl, 8 mM choline chloride and 0.1 mM physostigmine salicylate were mixed in a scintillation mini vial. Following a 15 min incubation at 37°C, the vials were placed in ice, and the reaction was stopped by adding 1 ml of ice-cold 10 mM sodium phos phate buffer. Then 0.4 ml of acetonitrile containing 2 mg Kalignost was added. Synthesized [3H]-ACh was extracted in 2 ml toluene scintillation cocktail (4 g/l PPO and 0.1 g/1 POPOP) and was measured by a scin tillation counter. The protein concentration of each ali quot was measured by a Bio-Rad protein assay kit. The total amount of [3H]-choline uptake by each mi
Fig. 1. Dissection of the rat brain and ACh release from the micropunches of the CC (sham-operated hemisphere). Fol lowing a washing equilibration period of 30 min, 2.5-min fractions were collected in scintillation vials. The first 6 fractions were collected at rest, and then the punches were exposed to Krebs with 40 mM potassium for 5 min (horizontal line). This outflow of tritium has been shown to be a good measure of [3H]-AChrelease. The percent fractional release of transmitter over each 2.5-min period was calculated.
cropunch and [3H]-ACh basal release were not changed after NBM lesion (data not shown). There were no sig nificant differences in potassium-evoked [3H]-ACh re lease between each section (CC-1 to CC-5, Fig. 1). Potassium-evoked [3H]-ACh release was significantly depleted (about 60% of depletion) at two weeks follow ing lesion (Fig. 2a). ChAT activity was also depleted (about 40% of depletion) at that time (Fig. 2b). Infu sions of CCK8 (10 ng/animal/day) into the ventricle significantly preserved K+ evoked [3H]-ACh release (CC-1 and CC-2) even after the lesion, and this pres ervation effect was weak in the rostral part of the CC (CC-3 to CC-5, Fig. 2a). The ChAT activity in the frontal part of the brain (CC-1 and CC-2) after the CCK8 treatment was significantly preserved as a control level, and this preservation effect tended to be weaker in the more rostral part of the CC (Fig. 2b). Since there were no significant differences in the basal ACh release and [3H]-choline uptake, many cholinergic
nerve cells in the CC may not have died during this period. The decreases of ACh release and ChAT activ ity following the NBM lesion indicate that there is a functional change of the cholinergic nerve cells in the CC. There are three possibilities for the CCK8 action sites in this study. The first is that CCK8 directly acts on the CC, because a high density of CCK8 binding sites was found in the CC, and CCK8 increased the ACh concentration in rat CC (9) and the 2DG uptake in mouse CC (7). The second is that CCK8 directly acts on the NBM neuron and prevents the cell death from the ibotenic lesion because CCK8 can block some effects of kainic acid in hippocampal slices (12). The last is that CCK8 acts on another neural system which mediates the CCK8 effect. In either case, CCK8 pre serves the cholinergic input from the NBM or CCK8 activates the cholinergic system in the CC. These activ ities might cause the increase of ACh transmission and the cerebral blood flow in CC (13) and preserve the CC
Fig. 2. Effect of CCK8 infusions on the decrease of K+ evoked ACh release from CC micropunches (a) and ChAT activ ity determined in the homogenate of CC micropunches (b) 2 weeks after NBM lesion. The values given are mean % ACh basal release ± S.E.M. (a) or mean ChAT activity ± S.E.M. Percent ACh basal release was calculated as the total % in crease from the base line at the peak. ChAT activity was expressed as nmol/mg protein/min. Rl, sham-operated and 10 ng/animal/day (i.c.v.) CCK8-treated (control); •, lesioned (untreated); J, lesioned and 10 ng/animal/day (i.c.v.) CCK8 treated (CCK8). Student's t-tests were used for comparisons; n = 6 to 8 animals per group; * * (P < 0.01), * (P < 0.05), significant difference between the untreated group and the CCK8-treated group.
cholinergic system against destruction. While ChAT activity was significantly preserved, ACh release was not significantly preserved in CC-3 to CC-5. This differ ence can be explained by a difference of the distribu tion of CCK8 receptor or CCK8 receptor subtypes. This phenomenon and the mechanism of the preserva tion effect of CCK8 require further investigation. In AD therapy, many trials have been done for the purpose of increasing the cholinergic transmissions by giving an AChE inhibitor or synthetic cholinergic ago nist. None of these trials resulted in any dramatic changes in the memory impairment of AD patients like we have seen in Parkinsonism by levodopa therapy. This may be partly because the memory function is more complex than the coordinate function of moving and also because the cholinergic system is mostly de stroyed in AD brain and there is no ability to respond to cholinomimetic drugs. Fortunately, the CCK system still survives even in a severe stage of AD (14); there fore, CCK8 might have some effect in this stage of AD brain and enhance the cholinergic transmission. In some cases of AD, the clinician uses a major tranquiliz er to control the DA system. CCK8 also modulates the DA system and works in schizophrenia (15). Even if the animal model is not a real AD, it mimics the de struction of the cholinergic system in the CC. CCK8 prevents the destruction of the cholinergic system in this model. These results indicate that CCK8 has poten tial as an antidimentia drug for AD. Acknowledgments These experiments were partially supported by a grant from the Science University of Tokyo. We thank Vikki Harmon for proof reading this manuscript and Yuichi Matsumoto for technical assist ance during the use of the superfusion system (Brandel).
4
5
6
7
8
9
10
11
12
13
REFERENCES
14 1
2
3
Etienne, P., Robitaille, Y., Wood, P., Gauthier, S., Nair, N.P.V. and Quirion, R.: Nucleus basalis neuronal loss, neu ritic plaques and choline acetyltransferase activity in advanced Alzheimer's Disease. Neuroscience 19, 1279-1291 (1986) Wenk, G. and Olton, D.S.: Basal forebrain cholinergic neurons and Alzheimer's Disease. In Animal Models of De mentia, Edited by Coyle, J.T., p. 81-101, Alan R. Liss, New York (1987) Arendash, G.W., Millard, W.J., Dunn, A. and Meyer, E.M.:
15
Longterm neuropathological and neurochemical effects of nucleus basalis lesions in the rat. Science 238, 952-956 (1987) Meyer, E.M., Arendash, G.W., Judkins, J.H., Ying, L., Wade, C. and Kern, W.R.: Effects of nucleus basalis lesion on the muscarinic and nicotinic modulation of [3H]-acetyl choline release. The rat cerebral cortex. J. Neurochem. 49, 1758 1762(1987) . Nilsson, L., Nordberg, A., Hardy, J., Wester, P. and Winblad, B.: Physostigminerestores [3H]-acetylcholineefflux from Alzheimer brain slices to normal level. J. Neural Transm. 67, 275-285 (1986) Salamone, J.D., Beart, P.M., Alpert, J.E. and Iverson, S.D.: Impairment in T-maze reinforced alternation performance fol lowing nucleus basalis magnocellularis lesions in rats. Behav. Brain. Res. 13, 63-70 (1984) Morency, M.A., Qirion, R., Nair, N.P.V. and Mishra, R.K.: Localization of cholecystokinin binding sites in canine brain using quantitative autoradiography. Prog. Neuropsychophar macol. Biol. Psychiatry 15, 291-296 (1991) Sugaya, K. and Kubota, K.: Autoradiographic demonstration of the antagonism of anthramycin and diazepam against cholecystokinin in the mouse brain using the [14C]-2-deoxy glucose method. Japan. J. Pharmacol. 48, 1-6 (1988) Marshall, F.H., Barnes, S., Woodruff, G.N. and Hunter, J.C.: Cholecystokinin modulates the release of dopamine from the anterior and posterior nucleus accumbens by two different mechanisms.J. Neurochem. 56, 917-922 (1991) Magnani, M., Mantovani, P. and Pepeu, G.: Effect of cholecystokinin octapeptide and ceruletide on release of acetylcholine from cerebral cortex of the rat in vivo. Neuropharmacology23, 1305-1309 (1984) Flood, J.F., Smith, G.E. and Moreley, J.E.: Modulation memory processing by cholecystokinin: Dependence on the vagus nerve. Science 236, 832-833 (1987) Aitken, P.G., Jaffe, D.B. and Nadler, J.V.: Cholecystokinin blocks some effects of kainic acid in CA3 region of hippo campal slices. Peptides 12, 127-129 (1991) Bisesold, D., Inanami, 0., Sato, A. and Sato, Y.: Stimula tion of the nucleus basalis of Meynert increases cerebral cor tical blood flow in rats. Neurosci. Lett. 98, 39-44 (1989) Rossor, M.N., Rehfeld, J.F., Emson, P.C., Mountjoy, C.Q., Roth, M. and Iversen, L.L.: Normal cortical concentration of cholecystokinin-like immunoreactivity with reduced choline acetyltransferase activity in senile dementia of Alzheimer type. Life Sci. 29, 405-410 (1981) Nair, N.P., Bloom, D.M., Nestoros, J.N. and Schwartz, G.: Therapeutic efficacy of cholecystokinin in neurolepitic-resist ant schizophrenic subjects. Psychopharmacol. Bull. 19, 134 137 (1983)
Protects
against Kiminobu
Basal
Sugaya,
Cholinergic
Forebrain
Masanori
Takahashi
Neurons
Lesion and Kazuhiko
Kubota
Research Institute for Biosciences, Science Universityof Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan Received
December
2, 1991
Accepted
February
29, 1992
ABSTRACT Alzheimer's Disease (AD) patients have a severe degeneration of cholinergic neurons in their cerebral cortices. Basal forebrain (BF)-lesioned rat is used as a model animal of a cholinergic defi cit in the cerebral cortex. Cholinergic markers were decreased in the cerebral cortex of BF-lesioned rats. Intracerebroventricular continuous infusion of cholecystokinin octapeptide (CCK8) following BF lesion obviously preserved these cholinergic markers. These results suggest that CCK8 prevents the de generation of cholinergic neurons in the cerebral cortex following BF lesion. Keywords:
Alzheimer's
disease,
Cholecystokinin,
The cholinergic deficit seen in Alzheimer's Disease (AD) is associated with cell loss in the nucleus basalis of Meynert (nbM) (1). In mammalian brain, some cell groups found in the basal forebrain (BF) are particular ly given interest as an analogue of nbM (2). The large neurons of the BF provide the major extrinsic cho linergic innervation of the cerebral cortex (CC). This origin form cell group known as the nucleus basalis magnocellularis (NBM) is diffusely localized in the re gion of the ventromedial portion of the globus pallidus, the substaintia innominata and the preoptic magnocellu lar nucleus. Lesions of the rat NBM provide an animal model of cholinergic dysfunction in the CC of patients with AD, and it causes a dramatic decrease in the cho line acetyltransferase (ChAT) activity, acetylcholin esterase (AChE) activity, high affinity choline uptake (3) and acetylcholine (ACh) release (4) in the CC. Similar deficits are observed in AD brain (1, 5). Learn ing and memory impairments, characteristic of AD, are also produced in NBM-lesioned animals (6). Cholecysto kinin octapeptide (CCK8) and it's binding site are widely distributed in the brain (7). Centrally adminis tered CCK8 increased 2-deoxy glucose (2DG) uptake (8) and modulated dopamine (DA) release in the nu cleus accumbens (9) and ACh release from the cerebral cortex in vivo (10). These evidences suggest that CCK8 can play a role of neurotransmitter or neuromodulator in the central nervous system. Most of all, it can mod ulate the memory processing (11). In these studies, we determine the effect of infusions
Basal
forebrain
lesion
of CCK8 into the ventricle on the post-lesion functional changes of cholinergic neurons by examining ChAT activity and ACh release level in continuous micro punches of the cortex. Male Sprague-Dawley rats (Sankyo Lab. Co., Tokyo), weighing 250-280 g at the start of the experiments, were used. They were housed in cages maintained at 22 ± 2 °C with a 12/12 hr light dark cycle. The rats had free access to laboratory chow (CE2, Nihon Clea Co.) and water throughout the ex periments. Each rat was anesthetized with sodium pen tobarbital (50 mg/kg, i.p.) and mounted in a stereotaxic apparatus (David copf). Unilateral lesions of the NBM were performed via injection of ibotenic acid (Sigma) (5 ,ug/1 ,ul, in a vehicle of sterile filtered 200 mM phos phate buffer, pH 7.4). Ibotenic acid was delivered with a graduated microliter glass pipette. NBM was stereo taxically localized using the following coordinates: AP = -2 .3 mm from the bregma, ML = ± 3.7 mm, 7.5 mm below the dura. The toxin was delivered over 10 min and the micropipette was left in place for another 5 min following injection, and the vehicle was injected contralaterally. After that, the rat was stereotaxically implanted unilaterally into the ventricle with a perma nent stainless steel L-shaped microcannula. The coor dinates used for cannula plantation were AP = 0.8 mm from the bregma, ML = ± 1.5 mm, 3.6 mm below the dura. The cannula was then anchored to the skull with acrylic cement. The mini osmotic pump (alzet MODEL 2002) filled with CCK8 (Peptide Institute, Inc.; 10 ng/12,ul) or vehicle was connected to the cannula with
a polyethylene tube, which was then placed sub cutaneously on the back of the rat. The mini pump assured constant delivery of CCK8 (10 ng/day) for two weeks. Animals were sacrificed by decapitation at two weeks after NBM lesion. The brains were rapidly taken out and sliced into 400-,um sections with a Mcllwain tis sue chopper (Brinkman). Each of ten sections ranging from +2.0 mm bregma to -2.0 mm bregma was dis sected free. Three micropunches (1.6-mm diameter) were taken from each hemisphere of the cerebral cortex (CC-1 to CC-5, Fig. 1). Sixty punches were taken in total, and 30 of them were frozen on dry ice for later determination of ChAT activity, while the remaining 30 punches were used for measurement of ACh release. ACh release was determined in micropunches from every other one of the serial cortical slices. The micro punches were preloaded with [3H]-choline (0.1,uM; 80 Ci/mmol) for 20 min in Krebs Ringer bicarbonate buff er of the following composition: 118 mM NaCl, 4.70 mM KC1, 1.50 mM MgSO4, 1.15 mM KH2PO4, 1.25 mM CaCl2, 25 mM NaHCO3, 11.1 mM glucose, bub bled with 95%02 and 5% CO2 at 37°C. The punches (3 punches/chamber) were then transferred into a su perfusion chamber system (Brandel) and continuously superfused with oxygenated Krebs buffer containing 10
,uM hemicholinium-3, at a flow rate of 1 ml/min. After the experiment, the micropunches were solubilized by Soluene-350 (Packard). The radioactivities in the super fusate and solubilized tissue were measured by a liquid scintillation counter. For the measurement of ChAT activity, each of three tissue punches was homogenized using an ultrasonicator (30% power, Microtip, 6 sec) in 100,ul of 50 mM Tris-HC1 buffer (PH 7.4). The homog enate was resuspended in 1/5 volume of 2.5% Triton-X 100 and the same volume of 10 mM EDTA. Eight microliters of sample suspension and 20 ,ul of a sub strate mixture consisting of 50 mM sodium phosphate buffer, 0.2 mM [3H]-Acetyl CoA (NEN, 1 pCi/mmol), 20 mM EDTA, 300 mM NaCl, 8 mM choline chloride and 0.1 mM physostigmine salicylate were mixed in a scintillation mini vial. Following a 15 min incubation at 37°C, the vials were placed in ice, and the reaction was stopped by adding 1 ml of ice-cold 10 mM sodium phos phate buffer. Then 0.4 ml of acetonitrile containing 2 mg Kalignost was added. Synthesized [3H]-ACh was extracted in 2 ml toluene scintillation cocktail (4 g/l PPO and 0.1 g/1 POPOP) and was measured by a scin tillation counter. The protein concentration of each ali quot was measured by a Bio-Rad protein assay kit. The total amount of [3H]-choline uptake by each mi
Fig. 1. Dissection of the rat brain and ACh release from the micropunches of the CC (sham-operated hemisphere). Fol lowing a washing equilibration period of 30 min, 2.5-min fractions were collected in scintillation vials. The first 6 fractions were collected at rest, and then the punches were exposed to Krebs with 40 mM potassium for 5 min (horizontal line). This outflow of tritium has been shown to be a good measure of [3H]-AChrelease. The percent fractional release of transmitter over each 2.5-min period was calculated.
cropunch and [3H]-ACh basal release were not changed after NBM lesion (data not shown). There were no sig nificant differences in potassium-evoked [3H]-ACh re lease between each section (CC-1 to CC-5, Fig. 1). Potassium-evoked [3H]-ACh release was significantly depleted (about 60% of depletion) at two weeks follow ing lesion (Fig. 2a). ChAT activity was also depleted (about 40% of depletion) at that time (Fig. 2b). Infu sions of CCK8 (10 ng/animal/day) into the ventricle significantly preserved K+ evoked [3H]-ACh release (CC-1 and CC-2) even after the lesion, and this pres ervation effect was weak in the rostral part of the CC (CC-3 to CC-5, Fig. 2a). The ChAT activity in the frontal part of the brain (CC-1 and CC-2) after the CCK8 treatment was significantly preserved as a control level, and this preservation effect tended to be weaker in the more rostral part of the CC (Fig. 2b). Since there were no significant differences in the basal ACh release and [3H]-choline uptake, many cholinergic
nerve cells in the CC may not have died during this period. The decreases of ACh release and ChAT activ ity following the NBM lesion indicate that there is a functional change of the cholinergic nerve cells in the CC. There are three possibilities for the CCK8 action sites in this study. The first is that CCK8 directly acts on the CC, because a high density of CCK8 binding sites was found in the CC, and CCK8 increased the ACh concentration in rat CC (9) and the 2DG uptake in mouse CC (7). The second is that CCK8 directly acts on the NBM neuron and prevents the cell death from the ibotenic lesion because CCK8 can block some effects of kainic acid in hippocampal slices (12). The last is that CCK8 acts on another neural system which mediates the CCK8 effect. In either case, CCK8 pre serves the cholinergic input from the NBM or CCK8 activates the cholinergic system in the CC. These activ ities might cause the increase of ACh transmission and the cerebral blood flow in CC (13) and preserve the CC
Fig. 2. Effect of CCK8 infusions on the decrease of K+ evoked ACh release from CC micropunches (a) and ChAT activ ity determined in the homogenate of CC micropunches (b) 2 weeks after NBM lesion. The values given are mean % ACh basal release ± S.E.M. (a) or mean ChAT activity ± S.E.M. Percent ACh basal release was calculated as the total % in crease from the base line at the peak. ChAT activity was expressed as nmol/mg protein/min. Rl, sham-operated and 10 ng/animal/day (i.c.v.) CCK8-treated (control); •, lesioned (untreated); J, lesioned and 10 ng/animal/day (i.c.v.) CCK8 treated (CCK8). Student's t-tests were used for comparisons; n = 6 to 8 animals per group; * * (P < 0.01), * (P < 0.05), significant difference between the untreated group and the CCK8-treated group.
cholinergic system against destruction. While ChAT activity was significantly preserved, ACh release was not significantly preserved in CC-3 to CC-5. This differ ence can be explained by a difference of the distribu tion of CCK8 receptor or CCK8 receptor subtypes. This phenomenon and the mechanism of the preserva tion effect of CCK8 require further investigation. In AD therapy, many trials have been done for the purpose of increasing the cholinergic transmissions by giving an AChE inhibitor or synthetic cholinergic ago nist. None of these trials resulted in any dramatic changes in the memory impairment of AD patients like we have seen in Parkinsonism by levodopa therapy. This may be partly because the memory function is more complex than the coordinate function of moving and also because the cholinergic system is mostly de stroyed in AD brain and there is no ability to respond to cholinomimetic drugs. Fortunately, the CCK system still survives even in a severe stage of AD (14); there fore, CCK8 might have some effect in this stage of AD brain and enhance the cholinergic transmission. In some cases of AD, the clinician uses a major tranquiliz er to control the DA system. CCK8 also modulates the DA system and works in schizophrenia (15). Even if the animal model is not a real AD, it mimics the de struction of the cholinergic system in the CC. CCK8 prevents the destruction of the cholinergic system in this model. These results indicate that CCK8 has poten tial as an antidimentia drug for AD. Acknowledgments These experiments were partially supported by a grant from the Science University of Tokyo. We thank Vikki Harmon for proof reading this manuscript and Yuichi Matsumoto for technical assist ance during the use of the superfusion system (Brandel).
4
5
6
7
8
9
10
11
12
13
REFERENCES
14 1
2
3
Etienne, P., Robitaille, Y., Wood, P., Gauthier, S., Nair, N.P.V. and Quirion, R.: Nucleus basalis neuronal loss, neu ritic plaques and choline acetyltransferase activity in advanced Alzheimer's Disease. Neuroscience 19, 1279-1291 (1986) Wenk, G. and Olton, D.S.: Basal forebrain cholinergic neurons and Alzheimer's Disease. In Animal Models of De mentia, Edited by Coyle, J.T., p. 81-101, Alan R. Liss, New York (1987) Arendash, G.W., Millard, W.J., Dunn, A. and Meyer, E.M.:
15
Longterm neuropathological and neurochemical effects of nucleus basalis lesions in the rat. Science 238, 952-956 (1987) Meyer, E.M., Arendash, G.W., Judkins, J.H., Ying, L., Wade, C. and Kern, W.R.: Effects of nucleus basalis lesion on the muscarinic and nicotinic modulation of [3H]-acetyl choline release. The rat cerebral cortex. J. Neurochem. 49, 1758 1762(1987) . Nilsson, L., Nordberg, A., Hardy, J., Wester, P. and Winblad, B.: Physostigminerestores [3H]-acetylcholineefflux from Alzheimer brain slices to normal level. J. Neural Transm. 67, 275-285 (1986) Salamone, J.D., Beart, P.M., Alpert, J.E. and Iverson, S.D.: Impairment in T-maze reinforced alternation performance fol lowing nucleus basalis magnocellularis lesions in rats. Behav. Brain. Res. 13, 63-70 (1984) Morency, M.A., Qirion, R., Nair, N.P.V. and Mishra, R.K.: Localization of cholecystokinin binding sites in canine brain using quantitative autoradiography. Prog. Neuropsychophar macol. Biol. Psychiatry 15, 291-296 (1991) Sugaya, K. and Kubota, K.: Autoradiographic demonstration of the antagonism of anthramycin and diazepam against cholecystokinin in the mouse brain using the [14C]-2-deoxy glucose method. Japan. J. Pharmacol. 48, 1-6 (1988) Marshall, F.H., Barnes, S., Woodruff, G.N. and Hunter, J.C.: Cholecystokinin modulates the release of dopamine from the anterior and posterior nucleus accumbens by two different mechanisms.J. Neurochem. 56, 917-922 (1991) Magnani, M., Mantovani, P. and Pepeu, G.: Effect of cholecystokinin octapeptide and ceruletide on release of acetylcholine from cerebral cortex of the rat in vivo. Neuropharmacology23, 1305-1309 (1984) Flood, J.F., Smith, G.E. and Moreley, J.E.: Modulation memory processing by cholecystokinin: Dependence on the vagus nerve. Science 236, 832-833 (1987) Aitken, P.G., Jaffe, D.B. and Nadler, J.V.: Cholecystokinin blocks some effects of kainic acid in CA3 region of hippo campal slices. Peptides 12, 127-129 (1991) Bisesold, D., Inanami, 0., Sato, A. and Sato, Y.: Stimula tion of the nucleus basalis of Meynert increases cerebral cor tical blood flow in rats. Neurosci. Lett. 98, 39-44 (1989) Rossor, M.N., Rehfeld, J.F., Emson, P.C., Mountjoy, C.Q., Roth, M. and Iversen, L.L.: Normal cortical concentration of cholecystokinin-like immunoreactivity with reduced choline acetyltransferase activity in senile dementia of Alzheimer type. Life Sci. 29, 405-410 (1981) Nair, N.P., Bloom, D.M., Nestoros, J.N. and Schwartz, G.: Therapeutic efficacy of cholecystokinin in neurolepitic-resist ant schizophrenic subjects. Psychopharmacol. Bull. 19, 134 137 (1983)
