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Effects of tacrine (THA) on spatial reference memory and cholinergic enzymes in specific rat brain regions
Life Sciences, Vol. 58, No. 1, pp. 47-54, 19% Copyright 0 1995 Elswier Science Inc. Printed in the USA. All rights reserved 0024-3205/% $15.00 t .oO
0024-3205(95)022.54-6
EFFECTS OF TACRINE (THA) ON SPATIAL REFERENCE MEMORY CHOLINERGIC ENZYMES IN SPECIFIC RAT BRAIN REGIONS
AND
Joy J. Jackson and Magdi R.I. Soliman Florida A&M University, College of Pharmacy and Pharmaceutical Florida 32307. U.S.A.
Sciences, Tallahassee,
(Received in final form October 18, 1995)
Summary Cognitive function of rats treated with saline (control), THA (8 mg/kg, i.p.), scopolamine (5 mg/kg, i.p.). or a combination of THA (8 mg/kg) and scopolamine (5 mg/kg) was tested in the Morris water maze. The latency to find the platform in the water maze was used to evaluate performance. THA did not Scopolamine significantly alter the latency period as compared to control rats. resulted in a highly significant (~~0.01) increase in latency period (183% increase) as compared to saline treated controls. However, when THA was concurrently administered with scopolamine, it was able to completely reverse the performance decrement induced by scopolamine. Immediately following spatial reference memory testing, animals were sacrificed by decapitation one hour post injection. Brains were immediately removed and the cortex, hippocampus, hypothalamus, and pituitary were dissected and their choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were determined spectrophotometrically. THA administration resulted in a significant increase in ChAT activity in the cortex (23% increase). However, when THA was concurrently administered with scopolamine, a significant increase in ChAT activity was observed in cortex (77% increase), hippocampus (32% increase), hypothalamus (97% increase), and pituitary (92.5% increase). THA administration resulted in a significant decrease in AChE activity (p
Corresponding author: Magdi R.I. Soliman, Ph.D., College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Fax: 904-561-2673
Tallahassee,
Florida 32307, USA. Phone: 904-599-3933;
48
THA, Spatial Reference
Memory & Cholinergic
Enzymes
Vol. 58, No. 1, 1996
One of the most devastating diseases involved in impairment of recent memory is Alzheimer’s Disease (AD). Recent efforts have focused on identifying neurotransmitters which may play a specific role in AD. It has been speculated for some time that studying changes in the brain bioavailability of neurotransmitters such as acetylcholine (ACh), dopamine, and serotonin may lead to pharmacological interventions capable of halting the progression of AD (1). To date, ACh is the most frequently studied neurotransmitter and seems to hold the most promising role in the treatment of AD (1,2). Studies of brain acetylcholine availability were triggered by the observation that patients taking medication with high anticholinergic properties often exhibited memory problems (1). This clinical observation, along with neuropathological studies documenting deterioration in the nucleus basalis of Meynert, the primary site of ACh synthesis, led to an acetylcholine hypothesis of AD (1,3,4). Deficits in cholinergic functioning have been most closely linked to the cognitive and behavioral symptoms of AD (5,6,7,8,9,10). The levels of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are markedly reduced in the brain of patients with this disease. The most dramatic reduction in the activity of these enzymes was found in the cerebral cortex, hippocampus, and correlated with areas which contained a great density of neurofibrillary tangles (6,10,11,12,13,14,15). Two lines of evidence support a relationship between cholinergic deficits and cognitive impairment in AD. First, there is a correlation between post-mortem and biopsy markers of cholinergic activity and the severity of dementia (16). Secondly, there are similarities between the cognitive impairment observed after administration of the muscarinic antagonist scopolamine to normal elderly volunteers and patients suffering from AD (16). Effective treatments for AD are urgently needed. A number of cholinergic compounds have been used to treat this disease in particular cholinesterase inhibitors and cholinergic agonists or precursors (e.g. physostigmine, bethanechol, and lecithin). Improvement in memory and cognition in such patients was limited (11). The most promising improvement of memory has been reported with 9-Amino-1,2,3,4-tetrahydroaminoacridine also known as tacrine (11,12). Tacrine (Cognex, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI) is a potent, centrally active, reversible acetylcholinesterase inhibitor that has been used alone or in combination with lecithin to treat patients with AD (9). Although tacrine has shown promising results, there is still controversy surrounding the use of tacrine in AD. Brain slices and synaptosomes prepared from brain have been used to study the effects of tacrine on neurotransmitter receptors and ion channels (11). Tacrine does not replace cholinergic neurons and does not treat the underlying pathology of the disease (17). Tacrine has been shown to increase the concentration of ACh by inhibiting acetylcholinesterase (3,6,13,17). Other studies have shown, however, that tacrine does not increase the levels of ACh (11). Cognitive benefits have been seen in some patients using tacrine, while other studies have not demonstrated positive effects (9,ll). Therefore, the purpose of this investigation was to evaluate the effects of tacrine on spatial reference memory and to elucidate its effects on cholinergic enzymes, in specific rat brain regions.
Materials and Methods Animals: Male Sprague-Dawley rats weighing 280-400 g were used in this experiment. Animals were housed in groups of 5 and kept under 12 hour light/dark cycle (fluorescent light,
Vol. 58, No. 1, 1996
THA, Spatial Reference Memory & Cholinerglc Enzymes
49
30-40 lux) and constant temperature (23 “C) and humidity (50-60 Sr,). Water and food (Purina Lab Chow) were provided ad libitum. Drug Treatment: After a training period of 5 days, animals were injected i.p. at 11:OO hr with either the muscarinic antagonist, scopolamine hydrobromide (5 mg/kg), the anticholinesterase agent, 9-Amino- 1,2,3,4-tetrahydroaminoacridine (Tacrine, 8 mg/kg) or a combination of scopolamine (5 mg/kg) and tacrine (8 mg/kg). Control rats in each group were injected i.p. with normal saline. All drugs were injected one hour before behavioral testing. The dose of tacrine (8 mg/kg) has been shown to be an effective dose by previous investigators (11). No overt cholinergic effects such as tremors or salivation were noted in the tacrine treated animals. Exoerimental Procedures: A. Snatial Reference Memorv: Spatial reference memory was tested using the Morris water maze (18). The water maze consisted of a stainless steel tub with a diameter of 46 inches, and a height of 23 inches. A stainless steel platform (2 inches below the water surface) with an area of 12.25 square inches and a height of 11 inches was used as the escape platform. Powdered milk (5 g/L) was then added to the water to make it opaque. The tub was then divided into four quadrants and the escape platform was placed in the lower right quadrant (quadrant 4) of the tub. Each day of testing consisted of 2 swims in the water maze. During the first swim, the rat was placed in quadrant 1. The amount of time the rat required to find the submerged platform, up to 80 seconds was recorded. After a 20 second rest period, the rat was then placed in a random quadrant (excluding the quadrant with the platform) and the amount of time required to find the escape platform was recorded again. The training period lasted for 5 days prior to treatment for each group and the rats were tested again one hour post injection. The mean latency periods for each animal was calculated. B. Cholinernic Enzvmes: Immediately following spatial reference testing, the animals were decapitated and the brains were removed and frozen at -7O’C. The hypothalamus, hippocampus, cortex, and pituitary were dissected. Choline acetyltransferase (ChAT) activity was determined spectrophotometrically using the method of Chad and Wolfgram (1972). Briefly, tissue was homogenized (1% w/v) in ice cold 0.5M sodium phosphate buffer (pH 7.0). In each test tube, 20 pL of 0.5 M phosphate buffer, 1 M acetyl Co A, 1 M choline chloride, 3 n&l sodium chloride, 1.1 mM ethylenediamine tetraacetic acid (EDTA), 6.5mM dithioerythritol (DTE), and 0.76 mM neostigmine bromide were added along with 40 PL of creatine (0.5 M) and boiled distilled water. The mixture was pre-incubated at 37’C for 5 minutes and 200 pL of tissue homogenate was added. For the blanks, 200 pL of boiled homogenate was added. The mixture was incubated at 37°C for 20 minutes, boiled at 100°C for 2 minutes, and 800 pL of sodium arsenite (2.5 mM) was added. Test tubes were centrifuged for 5 minutes and 1 mL of suspension was collected. CDithiopyrimidne (PDS, 10 pL, 1 n&l) was added. The tubes were allowed to stand for 15 minutes and were read spectrophotometrically at 342 nm. Acetylcholinesterase (AChE) activity was determined by the method of Ellman (1961). Tissue was homogenized in cold buffer with Triton X-100. In a test tube, 2.6 mL of buffer without Triton, 0.4 mL of homogenate, and 100 pL of dithiobisnitrobenzoic acid (DTNB) were added. The test tube was mixed and used to zero the machine. The substrate (20 pL) was added and mixed. Initial absorbance and absorbance after 4 minutes was read at 412 nm.
THA, Spatial Reference Memory & Cholinergic Enzymes
50
Vol. 58, No. 1, 1996
Statistical Analysis: Results were analyzed using the unpaired t-test and two-way analysis of variance. Comparison of means of the different groups was conducted using the Bonferroni multiple comparison test. The level of significance was set at p < 0.05.
Results Snatial Reference Memory Spatial reference memory of rats treated with saline (control), scopolamine, tacrine, or a combination of tacrine and scopolamine was tested in the Morris water maze. The latency to find the platform in the water maze was used to evaluate performance. Tacrine, administered alone did not significantly alter the latency period as compared to control rats. Scopolamine treatment resulted in a highly significant (p
0
CON
m
SC0
m
THA
m
S + T
**
GROUP
Fig. 1 Effect of scopolamine (SCO), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on spatial reference memory in rats (n = lo/group). **: ~~0.01 compared to the control (CON) group
ChAT Activity Tacrine administration resulted in a significant increase (p
Vol. 58, No. 1, 1996
THA, Spatial Reference Memory & Cholinergic Enzymes
m ,o
2
a
SC0
mS+T
THA
7.50
E 8
200
s
175
-
150
E
125
> F
100
Y s
7= SO
E
25
**
l*
225
0
HYP
HIP
PIT
COR GROUP
Fig. 2 Effect of scopolamine (SCO), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on ChAT activity in the hypothalamus (HYP), hippocampus (HIP), cortex (COR), and pituitary (PIT). Control ChAT activity values in HYP, HIP, COR and PIT were 0.55 + 0.01, 0.57 f 0.02, 0.60 f 0.06 and 0.53 f 0.05 nanomoles CoASH formed/min/gm tissue repectively. *: ~~0.05 compared to the controls; **: p
0
CON
m
SC0
m
THA
m
S + T
10 9 8 7 6 l **
5 43 2 1 oHYP
PIT
HIP Group
Fig. 3 Effect of scopolamine (X0), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on AChE activity in the hypothalamus (HYP), hippocampus (HIP), cortex (COR), and pituitary (PIT). *. ~~0.05 compared to the controls (CON); **: pcO.001 compared to the controls; ***: p
51
52
THA, Spatial Reference
Memory & Cholinergic
Enzymes
Vol. 58, No. 1, 1996
AChE Activity Administration of tacrine alone resulted in a significant decrease (p
Discussion
Several drugs that enhance cholinergic activity have been investigated as potential therapeutic agents in the treatment of AD. Tacrine is a potent, centrally active, reversible AChE inhibitor that prevents the degradation of endogenously released ACh (19). There is, however, some question about the efficacy of tacrine. There have been a number of studies reporting facilitation of learning in rodents after treatment with tacrine. It was shown that treatment of old mice with tacrine enhanced retention of a learned response (20). However, it was shown that THA-treated rats were not significantly better in acquisition of the Morris water maze compared to control rats (6). Studies in AD patients using tacrine alone has demonstrated modest efficacy (21). It has been shown, in trials using tacrine in combination with lecithin in AD patients, that the treatment did not result in any convincing clinically significant effects on cognition (1,2,15,22). On the other hand, in other studies with AD patients, tacrine was shown to Tacrine given in conjunction with produce modest cognitive benefit (l&9,19,22,23). scopolamine has also been shown to reverse the scopolamine induced deficit in passive avoidance in rats (23). Our results are in agreement with the reports that tacrine alone does not improve memory, but reverses the memory deficit induced by scopolamine administration. In AD, large and consistent reduction in the activities of ChAT and AChE in both the hippocampus and cortex have been reported and these decreases appear to correlate with the degree of cognitive impairment (14). In the hippocampus and cortex of normal aged humans and rodents, the activities of ChAT and AChE have been reported to decrease in association with a corresponding cognitive decline (14). Tacrine has been found to inhibit the depolarization release of ACh in neostriatal tissue and to a lesser extent in the neocortex (11). Tacrine also caused a slight, but non-significant inhibition of ACh from hippocampal and cortical slices (24). The decrease in release caused by tacrine was considered by these authors to be due to stimulation of presynaptic receptors by ACh which accumulated in the synaptic cleft after AChE inhibition. Intramuscular doses of tacrine in the rat inhibited brain cholinesterase after the fist dose (20 mg/kg) and ACh levels increased by 70% (25). A second dose (15 mg/kg) was administered 6 hours later, when cholinesterase levels had partially recovered and ACh levels had returned to normal. Although brain cholinesterase was depressed by the second dose of tacrine, to a level comparable with that seen after the first dose, levels of ACh did not increase. Actually levels of ACh were 25% below control values. In our study, a significant decrease in AChE was seen in the cortex and hippocampus which agrees with previous studies that have shown decreases in these brain regions. We also observed a significant decrease in AChE in the hypothalamus. An interesting observation in our study is the increase of ChAT activity in the cortex following the administration of tacrine. In addition, when tacrine was concurrently administered with scopolamine, a significant increase was seen in the cortex, hippocampus, hypothalamus, and pituitary. The exact mechanism of the tacrine-induced increase in ChAT activity is not known and requires further investigation.
Vol. 58, No. 1, 1996
‘WA, Spatial Reference Memory & Cholinergic Enzymes
However, the increase in ChAT activity produced by tacrine, may in part explain its ability to reverse the scopolamine induced decrease in spatial reference memory and may play a role in its beneficial effect in improving cognitive ability. Acknowledgment This study was supported by NIH grants GM081 11 and RR03020
References 1. 2. 3. 4. 5. 6. 7.
8. 9. 10.
11. 12. 13. 14. 15.
16.
17.
N.L. KELTNER, JAMA 271(13) 985991 (1994). N. MALTBY, G.A. BROE, H. CREASEY, A.F. JORM, H. CHRISTENSEN, and W.S. BROOKS, BMJ 308 879-883 (1994). C.CANTI, E. MARTI, J. MARSAL, and C. SOLSONA, Bri J Pharmacol112 19- 22 (1994). A. SAHGAL, A.B. KEITH, S. LLOYD, J.M. KERWIN, E.K. PERRY and J.A. EDWARDSON, Pharmacol Biochem Behav 37 597-605 (1990). P.A. NEWHOUSE, T. SUNDERLAND,P.N. TARIOT, C.L. BLUMHARDT, H. WEINGARTNER, and A. MELLOW, Psychopharmacology % 171-175 (1988). A. ADEM, A.K. MOHAMMED, and B. WINBLAD, Neural Trans 2 113-128 (1990). K.L. DAVIS, L.J.THAL,E.R. GAMZU,C.S. DAV1SR.F. WOOLSON, S.1. GRACON, D.A. DRACHMAN, L.S. SCHEIDER, P.J. WHITEHOUSE, T.M. HOOVER, J.C. MORRIS, C.H. KAWAS, D.S. KNOPMAN, N.L. EARL, V. KUMAR, and R.S. DOODY, New Engl J Med 327 1253-1259 (1992). S.A. EAGGER, R. LEVY, and B.J. SAHAKIAN, Lancet 337 989-992 (1991). M. FARLOW, S.I. GRACON, L.A. HERSHEY, K.W. LWEIS, C.H. SADOWSKY, and J. DOLAN-URENO, JAMA 268 2523-2529 (1992). A. FISHER, R. BRANDEIS, I. KARTON, Z. PITTEL, D. GURWITZ, R. HARING, M. SAPIR, A. LEVY, and E. HELDMAN, J Pharmacol Exp Ther 257 392403 (1991). S.E. FREEMAN, and R.M. DAWSON, Neurobiology 36 257-277 (1991). 1. NORDGREN, B. KARLIN, and M. KIMLAND, Pharmacol Tox x236-240 (1992). T. NABESHIMA, E. MARUYAMA, A. KATOH, and T. KAMEYAMA, Jpn J Pharmacol57311-319(1991). P. RIEKKINEN, JR., J. SIRVIO, and P. RIEKKINEN, Pharmacol Biochem Behav 36 237-241 (1990). S. GAUTHIER, R. BOUCHARD, A. LAMONTAGNE, P. BAILEY, J.H. BERGMAN, Y. TESFAYE, M. SAINT-MARTIN, Y. BACHER, L. CARRIER, R. CHARBONNEAU, A.M. CLARFIELD, B. COLLIER, D. DASTOOR, L. GAUTHIER, M. GERMAIN, C. KISSEL, M. KERIEGER, S. KUSHNER, H. MASSON, J. MORIN, V. NAIR, L. NEIRNCK, and S. SUISSA, N Engl J Med 322 1272-1276 (1990). K.P. EBMEIER, R. HUNTER, S.M. CURRAN, N.J. DOUGAL, C.L. MURRAY, D.J. WYPER, J. PATTERSON, M.T. HANSON, K. SIEGFRIED, and G.M. GODWIN, Psychopharmacology _IQ8 103-109 (1992). M.A. WINKER, JAMA 27-I 985991 (1994).
53
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18. 19. 20. 21. 22. 23. 24. 25.
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R.G.M. MORRIS, J. Neurosci. Meth. 11 47- 60 (1984) M.J. KNAPP, D.S. KNOPMAN, P.R. SOLOMON, W.W. PENDLEBURY, C.S. DAVIS, and S.I. GRACON, JAMA 271985-991 (1994). J.F FLOOD and A. CHERKIN, J Gerontol43 B54-56 (1988). W.K. SUMMERS, J.O. VISSELMAN, G.M. MARSH, and K. CANDELORA, Biol Psycho1 16 145- 153 (198 1). K.M. PERRYMAN and L.J. FI’ITEN, Life Sciences 53 479-486 (1990). P. CHOPIN and M. BRILEY, Psychopharmacology 106 26-10 (1992). P.E. POTTER, S. NITTA, I. CHAUDHRY, I. LALEHARI, P. GOLDINER, and F.F. FOOLDES, Neurochem Int 14 433-438 (1989). S. HALLACK and E. GIACOBINI, Neuropharmacology 28 199-206 (1989).
0024-3205(95)022.54-6
EFFECTS OF TACRINE (THA) ON SPATIAL REFERENCE MEMORY CHOLINERGIC ENZYMES IN SPECIFIC RAT BRAIN REGIONS
AND
Joy J. Jackson and Magdi R.I. Soliman Florida A&M University, College of Pharmacy and Pharmaceutical Florida 32307. U.S.A.
Sciences, Tallahassee,
(Received in final form October 18, 1995)
Summary Cognitive function of rats treated with saline (control), THA (8 mg/kg, i.p.), scopolamine (5 mg/kg, i.p.). or a combination of THA (8 mg/kg) and scopolamine (5 mg/kg) was tested in the Morris water maze. The latency to find the platform in the water maze was used to evaluate performance. THA did not Scopolamine significantly alter the latency period as compared to control rats. resulted in a highly significant (~~0.01) increase in latency period (183% increase) as compared to saline treated controls. However, when THA was concurrently administered with scopolamine, it was able to completely reverse the performance decrement induced by scopolamine. Immediately following spatial reference memory testing, animals were sacrificed by decapitation one hour post injection. Brains were immediately removed and the cortex, hippocampus, hypothalamus, and pituitary were dissected and their choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were determined spectrophotometrically. THA administration resulted in a significant increase in ChAT activity in the cortex (23% increase). However, when THA was concurrently administered with scopolamine, a significant increase in ChAT activity was observed in cortex (77% increase), hippocampus (32% increase), hypothalamus (97% increase), and pituitary (92.5% increase). THA administration resulted in a significant decrease in AChE activity (p
Corresponding author: Magdi R.I. Soliman, Ph.D., College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Fax: 904-561-2673
Tallahassee,
Florida 32307, USA. Phone: 904-599-3933;
48
THA, Spatial Reference
Memory & Cholinergic
Enzymes
Vol. 58, No. 1, 1996
One of the most devastating diseases involved in impairment of recent memory is Alzheimer’s Disease (AD). Recent efforts have focused on identifying neurotransmitters which may play a specific role in AD. It has been speculated for some time that studying changes in the brain bioavailability of neurotransmitters such as acetylcholine (ACh), dopamine, and serotonin may lead to pharmacological interventions capable of halting the progression of AD (1). To date, ACh is the most frequently studied neurotransmitter and seems to hold the most promising role in the treatment of AD (1,2). Studies of brain acetylcholine availability were triggered by the observation that patients taking medication with high anticholinergic properties often exhibited memory problems (1). This clinical observation, along with neuropathological studies documenting deterioration in the nucleus basalis of Meynert, the primary site of ACh synthesis, led to an acetylcholine hypothesis of AD (1,3,4). Deficits in cholinergic functioning have been most closely linked to the cognitive and behavioral symptoms of AD (5,6,7,8,9,10). The levels of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are markedly reduced in the brain of patients with this disease. The most dramatic reduction in the activity of these enzymes was found in the cerebral cortex, hippocampus, and correlated with areas which contained a great density of neurofibrillary tangles (6,10,11,12,13,14,15). Two lines of evidence support a relationship between cholinergic deficits and cognitive impairment in AD. First, there is a correlation between post-mortem and biopsy markers of cholinergic activity and the severity of dementia (16). Secondly, there are similarities between the cognitive impairment observed after administration of the muscarinic antagonist scopolamine to normal elderly volunteers and patients suffering from AD (16). Effective treatments for AD are urgently needed. A number of cholinergic compounds have been used to treat this disease in particular cholinesterase inhibitors and cholinergic agonists or precursors (e.g. physostigmine, bethanechol, and lecithin). Improvement in memory and cognition in such patients was limited (11). The most promising improvement of memory has been reported with 9-Amino-1,2,3,4-tetrahydroaminoacridine also known as tacrine (11,12). Tacrine (Cognex, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI) is a potent, centrally active, reversible acetylcholinesterase inhibitor that has been used alone or in combination with lecithin to treat patients with AD (9). Although tacrine has shown promising results, there is still controversy surrounding the use of tacrine in AD. Brain slices and synaptosomes prepared from brain have been used to study the effects of tacrine on neurotransmitter receptors and ion channels (11). Tacrine does not replace cholinergic neurons and does not treat the underlying pathology of the disease (17). Tacrine has been shown to increase the concentration of ACh by inhibiting acetylcholinesterase (3,6,13,17). Other studies have shown, however, that tacrine does not increase the levels of ACh (11). Cognitive benefits have been seen in some patients using tacrine, while other studies have not demonstrated positive effects (9,ll). Therefore, the purpose of this investigation was to evaluate the effects of tacrine on spatial reference memory and to elucidate its effects on cholinergic enzymes, in specific rat brain regions.
Materials and Methods Animals: Male Sprague-Dawley rats weighing 280-400 g were used in this experiment. Animals were housed in groups of 5 and kept under 12 hour light/dark cycle (fluorescent light,
Vol. 58, No. 1, 1996
THA, Spatial Reference Memory & Cholinerglc Enzymes
49
30-40 lux) and constant temperature (23 “C) and humidity (50-60 Sr,). Water and food (Purina Lab Chow) were provided ad libitum. Drug Treatment: After a training period of 5 days, animals were injected i.p. at 11:OO hr with either the muscarinic antagonist, scopolamine hydrobromide (5 mg/kg), the anticholinesterase agent, 9-Amino- 1,2,3,4-tetrahydroaminoacridine (Tacrine, 8 mg/kg) or a combination of scopolamine (5 mg/kg) and tacrine (8 mg/kg). Control rats in each group were injected i.p. with normal saline. All drugs were injected one hour before behavioral testing. The dose of tacrine (8 mg/kg) has been shown to be an effective dose by previous investigators (11). No overt cholinergic effects such as tremors or salivation were noted in the tacrine treated animals. Exoerimental Procedures: A. Snatial Reference Memorv: Spatial reference memory was tested using the Morris water maze (18). The water maze consisted of a stainless steel tub with a diameter of 46 inches, and a height of 23 inches. A stainless steel platform (2 inches below the water surface) with an area of 12.25 square inches and a height of 11 inches was used as the escape platform. Powdered milk (5 g/L) was then added to the water to make it opaque. The tub was then divided into four quadrants and the escape platform was placed in the lower right quadrant (quadrant 4) of the tub. Each day of testing consisted of 2 swims in the water maze. During the first swim, the rat was placed in quadrant 1. The amount of time the rat required to find the submerged platform, up to 80 seconds was recorded. After a 20 second rest period, the rat was then placed in a random quadrant (excluding the quadrant with the platform) and the amount of time required to find the escape platform was recorded again. The training period lasted for 5 days prior to treatment for each group and the rats were tested again one hour post injection. The mean latency periods for each animal was calculated. B. Cholinernic Enzvmes: Immediately following spatial reference testing, the animals were decapitated and the brains were removed and frozen at -7O’C. The hypothalamus, hippocampus, cortex, and pituitary were dissected. Choline acetyltransferase (ChAT) activity was determined spectrophotometrically using the method of Chad and Wolfgram (1972). Briefly, tissue was homogenized (1% w/v) in ice cold 0.5M sodium phosphate buffer (pH 7.0). In each test tube, 20 pL of 0.5 M phosphate buffer, 1 M acetyl Co A, 1 M choline chloride, 3 n&l sodium chloride, 1.1 mM ethylenediamine tetraacetic acid (EDTA), 6.5mM dithioerythritol (DTE), and 0.76 mM neostigmine bromide were added along with 40 PL of creatine (0.5 M) and boiled distilled water. The mixture was pre-incubated at 37’C for 5 minutes and 200 pL of tissue homogenate was added. For the blanks, 200 pL of boiled homogenate was added. The mixture was incubated at 37°C for 20 minutes, boiled at 100°C for 2 minutes, and 800 pL of sodium arsenite (2.5 mM) was added. Test tubes were centrifuged for 5 minutes and 1 mL of suspension was collected. CDithiopyrimidne (PDS, 10 pL, 1 n&l) was added. The tubes were allowed to stand for 15 minutes and were read spectrophotometrically at 342 nm. Acetylcholinesterase (AChE) activity was determined by the method of Ellman (1961). Tissue was homogenized in cold buffer with Triton X-100. In a test tube, 2.6 mL of buffer without Triton, 0.4 mL of homogenate, and 100 pL of dithiobisnitrobenzoic acid (DTNB) were added. The test tube was mixed and used to zero the machine. The substrate (20 pL) was added and mixed. Initial absorbance and absorbance after 4 minutes was read at 412 nm.
THA, Spatial Reference Memory & Cholinergic Enzymes
50
Vol. 58, No. 1, 1996
Statistical Analysis: Results were analyzed using the unpaired t-test and two-way analysis of variance. Comparison of means of the different groups was conducted using the Bonferroni multiple comparison test. The level of significance was set at p < 0.05.
Results Snatial Reference Memory Spatial reference memory of rats treated with saline (control), scopolamine, tacrine, or a combination of tacrine and scopolamine was tested in the Morris water maze. The latency to find the platform in the water maze was used to evaluate performance. Tacrine, administered alone did not significantly alter the latency period as compared to control rats. Scopolamine treatment resulted in a highly significant (p
0
CON
m
SC0
m
THA
m
S + T
**
GROUP
Fig. 1 Effect of scopolamine (SCO), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on spatial reference memory in rats (n = lo/group). **: ~~0.01 compared to the control (CON) group
ChAT Activity Tacrine administration resulted in a significant increase (p
Vol. 58, No. 1, 1996
THA, Spatial Reference Memory & Cholinergic Enzymes
m ,o
2
a
SC0
mS+T
THA
7.50
E 8
200
s
175
-
150
E
125
> F
100
Y s
7= SO
E
25
**
l*
225
0
HYP
HIP
PIT
COR GROUP
Fig. 2 Effect of scopolamine (SCO), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on ChAT activity in the hypothalamus (HYP), hippocampus (HIP), cortex (COR), and pituitary (PIT). Control ChAT activity values in HYP, HIP, COR and PIT were 0.55 + 0.01, 0.57 f 0.02, 0.60 f 0.06 and 0.53 f 0.05 nanomoles CoASH formed/min/gm tissue repectively. *: ~~0.05 compared to the controls; **: p
0
CON
m
SC0
m
THA
m
S + T
10 9 8 7 6 l **
5 43 2 1 oHYP
PIT
HIP Group
Fig. 3 Effect of scopolamine (X0), tacrine (THA), and a combination of scopolamine and tacrine (S + T) on AChE activity in the hypothalamus (HYP), hippocampus (HIP), cortex (COR), and pituitary (PIT). *. ~~0.05 compared to the controls (CON); **: pcO.001 compared to the controls; ***: p
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AChE Activity Administration of tacrine alone resulted in a significant decrease (p
Discussion
Several drugs that enhance cholinergic activity have been investigated as potential therapeutic agents in the treatment of AD. Tacrine is a potent, centrally active, reversible AChE inhibitor that prevents the degradation of endogenously released ACh (19). There is, however, some question about the efficacy of tacrine. There have been a number of studies reporting facilitation of learning in rodents after treatment with tacrine. It was shown that treatment of old mice with tacrine enhanced retention of a learned response (20). However, it was shown that THA-treated rats were not significantly better in acquisition of the Morris water maze compared to control rats (6). Studies in AD patients using tacrine alone has demonstrated modest efficacy (21). It has been shown, in trials using tacrine in combination with lecithin in AD patients, that the treatment did not result in any convincing clinically significant effects on cognition (1,2,15,22). On the other hand, in other studies with AD patients, tacrine was shown to Tacrine given in conjunction with produce modest cognitive benefit (l&9,19,22,23). scopolamine has also been shown to reverse the scopolamine induced deficit in passive avoidance in rats (23). Our results are in agreement with the reports that tacrine alone does not improve memory, but reverses the memory deficit induced by scopolamine administration. In AD, large and consistent reduction in the activities of ChAT and AChE in both the hippocampus and cortex have been reported and these decreases appear to correlate with the degree of cognitive impairment (14). In the hippocampus and cortex of normal aged humans and rodents, the activities of ChAT and AChE have been reported to decrease in association with a corresponding cognitive decline (14). Tacrine has been found to inhibit the depolarization release of ACh in neostriatal tissue and to a lesser extent in the neocortex (11). Tacrine also caused a slight, but non-significant inhibition of ACh from hippocampal and cortical slices (24). The decrease in release caused by tacrine was considered by these authors to be due to stimulation of presynaptic receptors by ACh which accumulated in the synaptic cleft after AChE inhibition. Intramuscular doses of tacrine in the rat inhibited brain cholinesterase after the fist dose (20 mg/kg) and ACh levels increased by 70% (25). A second dose (15 mg/kg) was administered 6 hours later, when cholinesterase levels had partially recovered and ACh levels had returned to normal. Although brain cholinesterase was depressed by the second dose of tacrine, to a level comparable with that seen after the first dose, levels of ACh did not increase. Actually levels of ACh were 25% below control values. In our study, a significant decrease in AChE was seen in the cortex and hippocampus which agrees with previous studies that have shown decreases in these brain regions. We also observed a significant decrease in AChE in the hypothalamus. An interesting observation in our study is the increase of ChAT activity in the cortex following the administration of tacrine. In addition, when tacrine was concurrently administered with scopolamine, a significant increase was seen in the cortex, hippocampus, hypothalamus, and pituitary. The exact mechanism of the tacrine-induced increase in ChAT activity is not known and requires further investigation.
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However, the increase in ChAT activity produced by tacrine, may in part explain its ability to reverse the scopolamine induced decrease in spatial reference memory and may play a role in its beneficial effect in improving cognitive ability. Acknowledgment This study was supported by NIH grants GM081 11 and RR03020
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