0361~9230/91 $3.00 + .OO
Brain ResearchBuNerin,Vol. 26, pp. 8174320.Q PergamonPress plc,1991.F’rintedin the U.S.A.
RAPID CO~~U~ICA~IO~
Protection Against Hypoxia-Induced Passive Avoidance Deficits: Interactions Between DuP 996 and Ketanserin VICTOR J. DENOBLE,’ KIM1 F. DENOBLE AND KAREN R. SPENCER The ~uP~n? Merck P~r~ceut~c~l Cornpony E40014430, P.O. Box 80400, Wilmington, DE 19880-0400 Received 27 December 1990 DENOBLE, V, J., K. F. DENOBLE AND K. R. SPENCER. Protection against hypoxia-induced passive nvoidance deficits: lnteractions between DuP 9% and ketanserin. BRAIN RES BULL 26(5) 817420, 1991. -DuP 996 and ketanserin have previously been shown to protect against experimentally induced passive avoidance (PA) deficits. In the present experiment the potential interaction between DuP 996 and ketanserin on hypoxia-induced amnesia was evaluated. Exposure to hypoxia (6.5% oxygen) produced a reliable deficit in PA retention which was attenuated by posthypoxia treatment with DuP 996 (0.0141 mg!kg SC). Similar effects were found with ketanserin at 1.0 and 3.0 mg/kg SC. ~oa~nis~tion of ketanserin, at a dose that did not protect against hypoxia (0.3 mg& SC), and DuP 9% (at doses of 0.005, 0.1, 0.03, 0.1, 0.3 and 1.0 mglkg SC) revealed a potentiation of both previously inactive doses of DuP 996 (e.g., 0.005, 0.3, and 1.0 mglkg SC) and an increase in the protective effect of previously active doses of DuP 996 (0.01, 0.03, 0.1 msflcg SC). These results suggest that combined administration of DuP 996, a neurotransmitter release enhancer, with ketanserin, a serotonin (5HT) antagonist, may provide a useful treatment for dementia. Neurotransmitter release enhancer DuP 996 Drug interactions
Ketanserin
Hypoxia
Rats
Amnesia
Dementia
5HT, selective (e.g., pirenperone, ketanserin), can enhancememory of a previously learned inhibitory response in mice (2,3) and can reverse h~xia-induced PA retention deficits in rats (11). Further, using a visual recognition task in monkeys, SHT antagonists were effective in preventing a performance deficit induced by exposure to hypoxia and also increased the accuracy of responding in monkeys who had a low level of accuracy. These studies support the notion that SHT antagonists administered after avoidance training or after an ex~ment~ly induced memory deficit (exposure to hypoxia) can enhance retrieval in mice (3), protect rats from experimentally induced amnesia (1 l), and enhance performance in monkeys (6). The interaction between the serotonergic nervous system and other neurotransmitter systems is well established (3). Therefore, the present experiment evaluated the effects of combined ad~nis~ation of a 5HT antagonist with the neurotransmitter release enhancer DuP 996 on hypoxia-induced PA deficits.
COGNITIVE deficits resulting from neuropathological brain changes or normal aging are most likely due to alterations in
multiple ne~o~~s~~r systems. Cholinergic, nora~nergic, dopaminergic, and peptidergic ne~o~~s~~er systems have all been implicated in the mediation of learning and memory decline associated with aging (7,9). A majority of research has focused on the cholinergic nervous system with particular reference to cognitive deficits resulting from Alzheimer’s disease (AD). In that regard a novel compound, 3,3-bis(~p~n~nylme~yl)-l-pheny~ndolin-Z-one (DuP 996) has been shown to enhance stimulated release of acetylcholine (ACh), dopamine (DA), and serotonin (5HT) from rat cortical, hippocampal and striatal slices in vitro and increase release of ACh from the cortex of awake freely moving rats in viva without changes in ace~Ichol~es~~e (AChF?) activity (8), In addition, previous studies have shown that DuP 9% protects against hypoxia-induced amnesia of a PA response in rats at a dose range of O.Ol0.1 mg/kg SC (5). A recent series of reports has shown that a number of .%IT receptor antagonists, both nonselective (e.g., metergoline), and
Animals
METHOD
Male Sprague-Dawley rats (Charles River Breeding Labo-
‘Requestsfor reprints should be addressed to Victor J. DeNoble, DuPont Merck Pharmaceutical Company, Experimental Station, &$00/4430, P.0. Box 80400, Wilmington, DE 198804400.
817
XIX
D~NOBLE.
ratories, Kingston, NY) weighing 200-225 g were used. The animals were housed four per cage (26.0 W x 45.0 L x 20.0 H cm) with free access to food and water. They were maintained on a 12-hour light/dark cycle (lights on from 0600 to 1800 h) at a room temperature of 22 rt: 1°C with a relative humidity of 5Ok 10%. In each experiment rats were used once.
DENOBL~
AND
TABLE 1 HYPOXIA-IND~ICED
DISK~PTI~N
OF PASSIVE AVOIDANCE
Oxygen
21 % 10
Passive Avoidance
Training
PA training began by placing the rat into the clear compartment of the two-compartment PA box. Following a 10-s delay, the slide door was raised providing access to the dark compartment. Once the rat moved completely into the dark compartment, the slide door was lowered and after a 10-s delay, a 1S-mA inescapable shock was applied to the grid floor for 3 s. This was followed by an additional 10-s period at the end of which the rat received another 3-s shock (1.5 rnA). The rats were immediately removed from the dark comp~ent after receiving the second shock, injected with vehicle or test compound, and returned to their home cage. Rats not entering the dark compartment within 90 s were removed from the study. Of the animals tested, less than 1% were removed from the study for not entering the dark compartment within the allotted time (90 s) during acquisition training. A retention test was given 4 h later. It proceeded in the same manner as the training session except no shock was applied to the grid floor when the rats entered the dark compartment. During the retention test, the rats were provided access to the dark compartment for 300 s. To determine if the compounds increased entry latencies by altering the rats physical ability to enter the dark chamber during the retention test, the highest effective dose of each compound
0
RETENTIOh
Median Retention: Latency (S)
Concentration
Apparatus
The experimental sessions were conducted in a two-compartment PA box. One compartment, made of clear plastic with a perforated clear plastic floor, measured 21 (L) x 24.5 (H) x 17 (W) cm and was illuminated with a m-watt incandescent light bulb placed 36 cm above the floor of the PA box. The other comparmient. made of black plastic, measured 30.5 (L) x 20.3 (H) x 21.5 (W) cm with a floor made of 4-mm stainless steel rods spaced 1.2 cm apart. A Coulboum Instruments Grid Floor Shocker was connected to the steel rods providing a scrambled footshock, The two compartments were separated by a solenoid-operated slide door (Lafayette Inst~ment Co., Lafayette, IN). A Coulboum Instruments Electronic Counter, activated by the opening or closing of the slide door, recorded acquisition and retention latencies. These latencies were defined as the time, in seconds, it took an animal to enter (all four paws on the grid floor) the dark compartment. For memos dis~ption, rats were exposed to an hypoxic environment for 30 min immediately prior to PA training. The hypoxia chamber was constructed of clear plastic, measured 32.5 (L) x 22.5 (H) x 23 (W) cm, and was continuously perfused with a gas mixture of oxygen and nitrogen. The flow rate was adjusted such that the gas turnover in the chamber was 15 liters per min. To determine the effects of different oxygen concentrations on PA retention, the rats were exposed to gas mixtures containing different percentages of oxygen (21%, lo%, 9%, 8%. 7%, or 6.5%) supplemented with nitrogen for 30 min prior to PA training. Oxygen concentrations were continuously monitored in the hypoxia chamber with an oxygen sensor (Sensitron, Inc., Reading, PA).
SPENCER
3oti
c/r c/c
300
300
x4
17fl*
7%
124” 39-i
6.5 o/c
Significantly different from 21% oxygen *p
Brain ResearchBuNerin,Vol. 26, pp. 8174320.Q PergamonPress plc,1991.F’rintedin the U.S.A.
RAPID CO~~U~ICA~IO~
Protection Against Hypoxia-Induced Passive Avoidance Deficits: Interactions Between DuP 996 and Ketanserin VICTOR J. DENOBLE,’ KIM1 F. DENOBLE AND KAREN R. SPENCER The ~uP~n? Merck P~r~ceut~c~l Cornpony E40014430, P.O. Box 80400, Wilmington, DE 19880-0400 Received 27 December 1990 DENOBLE, V, J., K. F. DENOBLE AND K. R. SPENCER. Protection against hypoxia-induced passive nvoidance deficits: lnteractions between DuP 9% and ketanserin. BRAIN RES BULL 26(5) 817420, 1991. -DuP 996 and ketanserin have previously been shown to protect against experimentally induced passive avoidance (PA) deficits. In the present experiment the potential interaction between DuP 996 and ketanserin on hypoxia-induced amnesia was evaluated. Exposure to hypoxia (6.5% oxygen) produced a reliable deficit in PA retention which was attenuated by posthypoxia treatment with DuP 996 (0.0141 mg!kg SC). Similar effects were found with ketanserin at 1.0 and 3.0 mg/kg SC. ~oa~nis~tion of ketanserin, at a dose that did not protect against hypoxia (0.3 mg& SC), and DuP 9% (at doses of 0.005, 0.1, 0.03, 0.1, 0.3 and 1.0 mglkg SC) revealed a potentiation of both previously inactive doses of DuP 996 (e.g., 0.005, 0.3, and 1.0 mglkg SC) and an increase in the protective effect of previously active doses of DuP 996 (0.01, 0.03, 0.1 msflcg SC). These results suggest that combined administration of DuP 996, a neurotransmitter release enhancer, with ketanserin, a serotonin (5HT) antagonist, may provide a useful treatment for dementia. Neurotransmitter release enhancer DuP 996 Drug interactions
Ketanserin
Hypoxia
Rats
Amnesia
Dementia
5HT, selective (e.g., pirenperone, ketanserin), can enhancememory of a previously learned inhibitory response in mice (2,3) and can reverse h~xia-induced PA retention deficits in rats (11). Further, using a visual recognition task in monkeys, SHT antagonists were effective in preventing a performance deficit induced by exposure to hypoxia and also increased the accuracy of responding in monkeys who had a low level of accuracy. These studies support the notion that SHT antagonists administered after avoidance training or after an ex~ment~ly induced memory deficit (exposure to hypoxia) can enhance retrieval in mice (3), protect rats from experimentally induced amnesia (1 l), and enhance performance in monkeys (6). The interaction between the serotonergic nervous system and other neurotransmitter systems is well established (3). Therefore, the present experiment evaluated the effects of combined ad~nis~ation of a 5HT antagonist with the neurotransmitter release enhancer DuP 996 on hypoxia-induced PA deficits.
COGNITIVE deficits resulting from neuropathological brain changes or normal aging are most likely due to alterations in
multiple ne~o~~s~~r systems. Cholinergic, nora~nergic, dopaminergic, and peptidergic ne~o~~s~~er systems have all been implicated in the mediation of learning and memory decline associated with aging (7,9). A majority of research has focused on the cholinergic nervous system with particular reference to cognitive deficits resulting from Alzheimer’s disease (AD). In that regard a novel compound, 3,3-bis(~p~n~nylme~yl)-l-pheny~ndolin-Z-one (DuP 996) has been shown to enhance stimulated release of acetylcholine (ACh), dopamine (DA), and serotonin (5HT) from rat cortical, hippocampal and striatal slices in vitro and increase release of ACh from the cortex of awake freely moving rats in viva without changes in ace~Ichol~es~~e (AChF?) activity (8), In addition, previous studies have shown that DuP 9% protects against hypoxia-induced amnesia of a PA response in rats at a dose range of O.Ol0.1 mg/kg SC (5). A recent series of reports has shown that a number of .%IT receptor antagonists, both nonselective (e.g., metergoline), and
Animals
METHOD
Male Sprague-Dawley rats (Charles River Breeding Labo-
‘Requestsfor reprints should be addressed to Victor J. DeNoble, DuPont Merck Pharmaceutical Company, Experimental Station, &$00/4430, P.0. Box 80400, Wilmington, DE 198804400.
817
XIX
D~NOBLE.
ratories, Kingston, NY) weighing 200-225 g were used. The animals were housed four per cage (26.0 W x 45.0 L x 20.0 H cm) with free access to food and water. They were maintained on a 12-hour light/dark cycle (lights on from 0600 to 1800 h) at a room temperature of 22 rt: 1°C with a relative humidity of 5Ok 10%. In each experiment rats were used once.
DENOBL~
AND
TABLE 1 HYPOXIA-IND~ICED
DISK~PTI~N
OF PASSIVE AVOIDANCE
Oxygen
21 % 10
Passive Avoidance
Training
PA training began by placing the rat into the clear compartment of the two-compartment PA box. Following a 10-s delay, the slide door was raised providing access to the dark compartment. Once the rat moved completely into the dark compartment, the slide door was lowered and after a 10-s delay, a 1S-mA inescapable shock was applied to the grid floor for 3 s. This was followed by an additional 10-s period at the end of which the rat received another 3-s shock (1.5 rnA). The rats were immediately removed from the dark comp~ent after receiving the second shock, injected with vehicle or test compound, and returned to their home cage. Rats not entering the dark compartment within 90 s were removed from the study. Of the animals tested, less than 1% were removed from the study for not entering the dark compartment within the allotted time (90 s) during acquisition training. A retention test was given 4 h later. It proceeded in the same manner as the training session except no shock was applied to the grid floor when the rats entered the dark compartment. During the retention test, the rats were provided access to the dark compartment for 300 s. To determine if the compounds increased entry latencies by altering the rats physical ability to enter the dark chamber during the retention test, the highest effective dose of each compound
0
RETENTIOh
Median Retention: Latency (S)
Concentration
Apparatus
The experimental sessions were conducted in a two-compartment PA box. One compartment, made of clear plastic with a perforated clear plastic floor, measured 21 (L) x 24.5 (H) x 17 (W) cm and was illuminated with a m-watt incandescent light bulb placed 36 cm above the floor of the PA box. The other comparmient. made of black plastic, measured 30.5 (L) x 20.3 (H) x 21.5 (W) cm with a floor made of 4-mm stainless steel rods spaced 1.2 cm apart. A Coulboum Instruments Grid Floor Shocker was connected to the steel rods providing a scrambled footshock, The two compartments were separated by a solenoid-operated slide door (Lafayette Inst~ment Co., Lafayette, IN). A Coulboum Instruments Electronic Counter, activated by the opening or closing of the slide door, recorded acquisition and retention latencies. These latencies were defined as the time, in seconds, it took an animal to enter (all four paws on the grid floor) the dark compartment. For memos dis~ption, rats were exposed to an hypoxic environment for 30 min immediately prior to PA training. The hypoxia chamber was constructed of clear plastic, measured 32.5 (L) x 22.5 (H) x 23 (W) cm, and was continuously perfused with a gas mixture of oxygen and nitrogen. The flow rate was adjusted such that the gas turnover in the chamber was 15 liters per min. To determine the effects of different oxygen concentrations on PA retention, the rats were exposed to gas mixtures containing different percentages of oxygen (21%, lo%, 9%, 8%. 7%, or 6.5%) supplemented with nitrogen for 30 min prior to PA training. Oxygen concentrations were continuously monitored in the hypoxia chamber with an oxygen sensor (Sensitron, Inc., Reading, PA).
SPENCER
3oti
c/r c/c
300
300
x4
17fl*
7%
124” 39-i
6.5 o/c
Significantly different from 21% oxygen *p
