四氢孕酮和地西泮对暴露于架高十字迷宫小鼠的作用
作者：于榕 鲁映青 盛美萍 史念慈 姚明辉
单位：上海医科大学基础医学院药理教研室，上海 200032
关键词：四氢孕酮；地西泮；焦虑；抗焦虑；十字迷宫；自发活动
中国临床药理学与治疗学000102
目的 比较孕酮的还原性代谢产物四氢孕酮和地西泮抗焦虑作用。方法 C57小鼠腹腔注射四氢孕酮、地西泮或赋形剂，20 min后观察在架高十字迷宫试验中的表现，测定其自发活动。结果 腹腔注射四氢孕酮0.1 mg.kg-1，明显缩短小鼠进入十字迷宫开臂的潜伏期[从(31.30±8.39)s减少到(8.80±6.00)s，P<0.001]，并显著增加进入十字迷宫开臂的次数(从1.20± 0.42增到4.80±1.75,P<0.001)及在开臂的滞留时间占总时间的百分比(从7.13%增加到32.50%,P<0.001)。而腹腔注射地西泮0.25 mg.kg-1的抗焦虑作用弱于四氢孕酮。自发活动实验显示，0.5 mg.kg-1地西泮明显减少小鼠自发活动(P<0.01)，而0.1 mg.kg-1地西泮和0.25 mg.kg-1的四氢孕酮均不影响小鼠自发活动。结论 试验结果提示神经甾体和地西泮对小鼠行为有不同的作用。四氢孕酮具选择性抗焦虑作用而不影响自发活动，可望成为地西泮抗焦虑的代用品。
, http://www.100md.com
Comparison of anxiolytic effects of allopregnanolone and diazepam on the elevated plus-maze in mice
YU Rong LU Ying-Qing SHENG Mei-Ping SHI Nian-Ci YAO Ming-Hui
(Department of Pharmacology,Shanghai Medical University,Shanghai 200032)
Aim To compare the anxiolytic effects of reduced metabolite of progesterone and benzodiazepine.Methods The effects of allopregnanolone and diazepam on spontaneous locomotor activity and on exploration in the elevated plus-maze were studied in C57 mice 20 min after vehicle or drug intraperitoneal administration.Results Allopregnanolone (0.1 mg.kg-1,ip) elicited marked anxiolytic effects in terms of significantly reducing the latency to enter the open arm from (31.30±8.39)s to (8.80±6.00)s,(P<0.001),and significantly increasing both the number of open arm entries from 1.20 ± 0.42 to 4.80 ±1.75,(P<0.001) and the proportion of total time spent on the open arm from 7.13% to 32.50%,(P<0.001).Meanwhile,the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect comparing to that of the allopregnanolone.Analysis of spontaneous locomotor activity showed while 0.5 mg.kg-1 of diazepam decreased the locomotor activity (P<0.01),neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam affect the locomotor activity score.Conclusion Together,these results provide evidence for differential behavioral actions of the neurosteroids and benzodiazepines.Since the allopregnanolone produce a selective anxiolytic effect without affecting the spontaneous locomotor activity,the allopregnanolone may be a better alternative for diazepam in treating anxiety.
, 百拇医药
Key words allopregnanolone;diazepam;anxiety;elevated plus-maze;anxiolytic;spontaneous locomotor activity
Neurosteroids are endogenous metabolites of certain steroid hormones that rapidly alter the excitability of neurons by direct actions on ligand- gated ion channels [1].The gamma-aminobutyric acid (GABA A) receptor Cl-channel complex is a major target for the action of neuroactive steroids in the central nervous system,and it is now well recognized that certain neurosteroids can potentiate GABA-mediated inhibitory response[2]. GABA A receptors are known to contain allosteric modulatory sites for therapeutically useful drugs such as benzodiazepines (BZD) and barbiturates.It is now well recognized that certain neurosteroids, in particular the endogenous allopreg-nanolone,can produce a powerful enhancement of GABA A receptor responses in vitro and is suggested to have anxiolytic activity like benzodiazepines [3].Considering the fact that the subunit requirement varies for various anxiolytics to elicit at least some of their pharmacological effects via the GABA A receptor,and that benzodiazepines and neuroactive steroids have their distinctive receptors on the GABA A receptor complex,we therefore hypothesized that neuroactive steroids have anxiolytic activity and their anxiolytic profile might be different from that of benzodiazepines.Consequently,in the present study,we explored the anxiolytic activity of allopregnanolone and compare the potential differences between the anxiolytic action of allopregnanolone and diazepam.
, 百拇医药
1 Materials and methods
1.1 Animals Male C57 mice (18～22 g)(№02-229) were obtained from Shanghai Medical University animal department.Animals were allowed to acclimatize with free access to food and water for a 48 h period before testing.
1.2 Drugs Allopregnanolone was purchased from Sigma Chemical Co.(St Louis,MO.USA).Stock solution of allopregnanolone was prepared in dimethyl sulfoxide(SP JinShan Chemical Plant,Lot № 790917) and further dilution was made using 0.9% saline.Diazepam injection (2 ml:10 mg) was obtained from Shanghai SunRise Pharmaceutical Co.Ltd.( Lot № 990201.All drug solutions were administered ip in a volume equaling 1% of the animal's body weight.
, 百拇医药
1.3 Elevated plus-maze test The plus-maze apparatus was made of Plexiglas and consisted of two open arms 30.5 cm long with a 5 cm wide runway and two closed arms 30.5 cm long with 5 cm wide runway enclosed by clear Plexiglas walls 15 cm high.The arms extended from a central platform,and the runways of both arms were made of black Plexiglas.The apparatus was mounted on a standing pole 38.5 cm above the floor.The apparatus was similar to that described by Lister[4].Behavioral evaluations were carried out in a quiet,temperature controlled room(23 ℃).Animals were brought in the room 4 h prior to experimentation.During the test period,the mouse was placed in the center of the plus-maze facing an open arm.During the 5 min test,the delay of entry as well as the number of entries and time spent in the open arms were scored by direct observation.A mouse was taken to have entered an arm when all four legs were on the arm (outside the start position).Diazepam,allopregnanolone or vehicle was administered 20 min prior to placement of individual mice in the test apparatus.
, http://www.100md.com
1.4 Locomotor activity test The effects of neurosteroids on motor activity were evaluated with an SMUP automated animal activity scoring system (Department of Physiology,Shanghai Medical University).It was modified from the Jaggle cage method.Briefly,the locomotor activity of the caged animal was transformed into electrical signal which was input into a the computer via a SMUP-PC bio-signal process system.The recorded signal was then integrated for two consecutive 15 s as a locomotor activity score.Each animals were caged 20 min after administration of diazepam,allopregnanolone or vehicle.The recording started 2 min after caging for conditioning.After each test the cage was carefully cleaned.The motor activity was expressed in terms of mean score per animal.
, http://www.100md.com
1.5 Data analysis 10 mice were tested for each group.Data are presented as the ±s.Data were subjected to one-way analysis of variance (ANOVA),the criterion for significance being P <0.05.
2 Results
2.1 The elevated plus-maze test Allopregnanolone elicit significant anxiolytic activity as shown by either the decrease in the latency of entry or the increase in the proportion of time spent on the open arms.Of the 300 second test period,vehicle-treated mice spent an average of (31.10 ±8.39) s in the closed arms before venturing into the open arms.The latency to enter declined significantly to a value of (8.80±6.00) s (P<0.001) following allopregnanolone administration (0.1 mg.kg-1,ip).Allopregnanolone also significantly increased both the number of open arm entries from 1.20±0.42 to 4.80±1.75,(P<0.001) and the proportion of total time spent on the open arm from 7.13% to 32.50%,( P<0.001).Meanwhile,the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect comparing to that of the allopregnanolone.Diazepam reduced the latency of entry to (23.20 ±18.48) s without significance,but it significantly increased both the number of open arm entries to 3.00±1.41 (P<0.001) and the proportion of time spent in the open arms of the maze to (21.90 ±14.82)% (P<0.01).
, 百拇医药
2.2 Analysis of the spontaneous locomotor activity One of the major concerns regarding measurement of potential anxiolytic actions of drugs is whether changes in activity represent anxiolytic actions or effects on locomotor activity.Therefore,the neurosteroid effect on locomotor activity was measured.Analysis of locomotor activity revealed that neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam depressed locomotor activity scores;while 0.5 mg.kg-1 of diazepam significantly decreased the score from 9027±3128 to 5068±1607 (P<0.01,Tab 1,Fig 1).These results,therefore,have demonstrated that ip injection of allopregnanolone resulted in an anxiolytic activity without affecting the spontaneous locomotor activity.
, http://www.100md.com
Tab 1 Comparison of the effects of allopregnanolone and diazepam on the spontaneous locomotor activities in C57 mice(
±s,n=10 ) Group
Locomotor activity score
control
9027±3128
allopregnanolong/0.1mg.kg-1
10110±2879
, http://www.100md.com
diazwpam/0.25mg.kg-1
9418±1380
diazepam/0.5mg.kg-1
5068±1607**
compared with control group **P<0.01
Fig 1 A typical tracing of the spontaneous locomotor activity in C57 mice
, 百拇医药
A:control;B:allopregnanolone 0.1 mg.kg-1;C:diazepam 0.25 mg.kg-1;D:diazepam 0.5 mg.kg-1
3 Discussion
Several line of recent investigation has shown that neurosteroid allopregnanolone allosterically modulate GABA through a unique binding site on the GABA A receptor Cl- channel complex.This binding site has been found to be distinctly different from that of the benzodiazepines or the barbiturates site,respectively.In particular,the 3 a-hydroxy ring A-reduced metabolite of progesterone,allopregnanolone produced a potent enhancement of GABA A receptor responses in vitro.Since benzodiazepines are believed to produce anxiolytic activity via the binding to the GABA A receptor complex,it is reasonable to speculate that allopregnanolone has a similar anxiolytic activity like benzodiazepines.However,benzodiazepines and neurosteroids have their distinctive receptors on the GABA A receptor complex,we therefore compared the anxiolytic activity allopregnanolone and that of diazepam.We found that allopregnanolone produced marked anxiolytic effect at dose of 0.1 mg.kg-1 on the elevated plus-maze test in C57 mice,while the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect.
, 百拇医药
The change in the elevated plus-maze could simply reflect changes in locomotor activity.Therefore,we were concerned that drug actions on locomotion could influence several of the elevated plus-maze measures which may confound interpretation of drug effects on the elevated plus-maze test of anxiety.To prevent this ambiguity,we have also examined the effects of neurosteroid and benzodiazepine on the spontaneous locomotor activity.In the present study,we found that neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam affect the spontaneous locomotor activity,while 0.5 mg.kg-1 of diazepam significantly reduced the locomotor activity score.Therefore,this finding indicate that the allopregnanolone-induced changes in the elevated plus-maze do not arise because of changes in locomotor activity.
, 百拇医药
In conclusion,the present study demonstrated the differential behavioral actions of neurosteroid in the elevated plus-maze test of anxiety.This selective anxiolytic effect of allopregnanolone suggested that the allopregnanolone may be a better alternative for diazepam in treating the anxiety.
YU Rong,male,35 years old,Ph.D,associate professor,neuropharmacology
References
1，Majewska MD.Neurosteroids: endogenous bimodal modulators of GABA A receptor.Mechanism of action and physiological significance.Prog Neurobiol,1992;38:379
, http://www.100md.com
2，Lambert JJ,Belelli D,Hill-Venning C ,et al.Neu-rosteroids and GABAA receptor function.Trends Pharmacol,1990;188:403
3，Yu R,Ticku MK.Chronic neurosteroid treatment produces functional hetrologous uncoupling at the GABAA-benzodiazepine receptor complex in mam-malian cortical neurons. Mol Pharmacol,1995;47:603
4，Lister RG.The use of plus-maze to measure anxiety in the mouse.Psychopharmacology,1987;92:180
received：2000-02-10, 百拇医药
单位：上海医科大学基础医学院药理教研室，上海 200032
关键词：四氢孕酮；地西泮；焦虑；抗焦虑；十字迷宫；自发活动
中国临床药理学与治疗学000102
目的 比较孕酮的还原性代谢产物四氢孕酮和地西泮抗焦虑作用。方法 C57小鼠腹腔注射四氢孕酮、地西泮或赋形剂，20 min后观察在架高十字迷宫试验中的表现，测定其自发活动。结果 腹腔注射四氢孕酮0.1 mg.kg-1，明显缩短小鼠进入十字迷宫开臂的潜伏期[从(31.30±8.39)s减少到(8.80±6.00)s，P<0.001]，并显著增加进入十字迷宫开臂的次数(从1.20± 0.42增到4.80±1.75,P<0.001)及在开臂的滞留时间占总时间的百分比(从7.13%增加到32.50%,P<0.001)。而腹腔注射地西泮0.25 mg.kg-1的抗焦虑作用弱于四氢孕酮。自发活动实验显示，0.5 mg.kg-1地西泮明显减少小鼠自发活动(P<0.01)，而0.1 mg.kg-1地西泮和0.25 mg.kg-1的四氢孕酮均不影响小鼠自发活动。结论 试验结果提示神经甾体和地西泮对小鼠行为有不同的作用。四氢孕酮具选择性抗焦虑作用而不影响自发活动，可望成为地西泮抗焦虑的代用品。
, http://www.100md.com
Comparison of anxiolytic effects of allopregnanolone and diazepam on the elevated plus-maze in mice
YU Rong LU Ying-Qing SHENG Mei-Ping SHI Nian-Ci YAO Ming-Hui
(Department of Pharmacology,Shanghai Medical University,Shanghai 200032)
Aim To compare the anxiolytic effects of reduced metabolite of progesterone and benzodiazepine.Methods The effects of allopregnanolone and diazepam on spontaneous locomotor activity and on exploration in the elevated plus-maze were studied in C57 mice 20 min after vehicle or drug intraperitoneal administration.Results Allopregnanolone (0.1 mg.kg-1,ip) elicited marked anxiolytic effects in terms of significantly reducing the latency to enter the open arm from (31.30±8.39)s to (8.80±6.00)s,(P<0.001),and significantly increasing both the number of open arm entries from 1.20 ± 0.42 to 4.80 ±1.75,(P<0.001) and the proportion of total time spent on the open arm from 7.13% to 32.50%,(P<0.001).Meanwhile,the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect comparing to that of the allopregnanolone.Analysis of spontaneous locomotor activity showed while 0.5 mg.kg-1 of diazepam decreased the locomotor activity (P<0.01),neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam affect the locomotor activity score.Conclusion Together,these results provide evidence for differential behavioral actions of the neurosteroids and benzodiazepines.Since the allopregnanolone produce a selective anxiolytic effect without affecting the spontaneous locomotor activity,the allopregnanolone may be a better alternative for diazepam in treating anxiety.
, 百拇医药
Key words allopregnanolone;diazepam;anxiety;elevated plus-maze;anxiolytic;spontaneous locomotor activity
Neurosteroids are endogenous metabolites of certain steroid hormones that rapidly alter the excitability of neurons by direct actions on ligand- gated ion channels [1].The gamma-aminobutyric acid (GABA A) receptor Cl-channel complex is a major target for the action of neuroactive steroids in the central nervous system,and it is now well recognized that certain neurosteroids can potentiate GABA-mediated inhibitory response[2]. GABA A receptors are known to contain allosteric modulatory sites for therapeutically useful drugs such as benzodiazepines (BZD) and barbiturates.It is now well recognized that certain neurosteroids, in particular the endogenous allopreg-nanolone,can produce a powerful enhancement of GABA A receptor responses in vitro and is suggested to have anxiolytic activity like benzodiazepines [3].Considering the fact that the subunit requirement varies for various anxiolytics to elicit at least some of their pharmacological effects via the GABA A receptor,and that benzodiazepines and neuroactive steroids have their distinctive receptors on the GABA A receptor complex,we therefore hypothesized that neuroactive steroids have anxiolytic activity and their anxiolytic profile might be different from that of benzodiazepines.Consequently,in the present study,we explored the anxiolytic activity of allopregnanolone and compare the potential differences between the anxiolytic action of allopregnanolone and diazepam.
, 百拇医药
1 Materials and methods
1.1 Animals Male C57 mice (18～22 g)(№02-229) were obtained from Shanghai Medical University animal department.Animals were allowed to acclimatize with free access to food and water for a 48 h period before testing.
1.2 Drugs Allopregnanolone was purchased from Sigma Chemical Co.(St Louis,MO.USA).Stock solution of allopregnanolone was prepared in dimethyl sulfoxide(SP JinShan Chemical Plant,Lot № 790917) and further dilution was made using 0.9% saline.Diazepam injection (2 ml:10 mg) was obtained from Shanghai SunRise Pharmaceutical Co.Ltd.( Lot № 990201.All drug solutions were administered ip in a volume equaling 1% of the animal's body weight.
, 百拇医药
1.3 Elevated plus-maze test The plus-maze apparatus was made of Plexiglas and consisted of two open arms 30.5 cm long with a 5 cm wide runway and two closed arms 30.5 cm long with 5 cm wide runway enclosed by clear Plexiglas walls 15 cm high.The arms extended from a central platform,and the runways of both arms were made of black Plexiglas.The apparatus was mounted on a standing pole 38.5 cm above the floor.The apparatus was similar to that described by Lister[4].Behavioral evaluations were carried out in a quiet,temperature controlled room(23 ℃).Animals were brought in the room 4 h prior to experimentation.During the test period,the mouse was placed in the center of the plus-maze facing an open arm.During the 5 min test,the delay of entry as well as the number of entries and time spent in the open arms were scored by direct observation.A mouse was taken to have entered an arm when all four legs were on the arm (outside the start position).Diazepam,allopregnanolone or vehicle was administered 20 min prior to placement of individual mice in the test apparatus.
, http://www.100md.com
1.4 Locomotor activity test The effects of neurosteroids on motor activity were evaluated with an SMUP automated animal activity scoring system (Department of Physiology,Shanghai Medical University).It was modified from the Jaggle cage method.Briefly,the locomotor activity of the caged animal was transformed into electrical signal which was input into a the computer via a SMUP-PC bio-signal process system.The recorded signal was then integrated for two consecutive 15 s as a locomotor activity score.Each animals were caged 20 min after administration of diazepam,allopregnanolone or vehicle.The recording started 2 min after caging for conditioning.After each test the cage was carefully cleaned.The motor activity was expressed in terms of mean score per animal.
, http://www.100md.com
1.5 Data analysis 10 mice were tested for each group.Data are presented as the ±s.Data were subjected to one-way analysis of variance (ANOVA),the criterion for significance being P <0.05.
2 Results
2.1 The elevated plus-maze test Allopregnanolone elicit significant anxiolytic activity as shown by either the decrease in the latency of entry or the increase in the proportion of time spent on the open arms.Of the 300 second test period,vehicle-treated mice spent an average of (31.10 ±8.39) s in the closed arms before venturing into the open arms.The latency to enter declined significantly to a value of (8.80±6.00) s (P<0.001) following allopregnanolone administration (0.1 mg.kg-1,ip).Allopregnanolone also significantly increased both the number of open arm entries from 1.20±0.42 to 4.80±1.75,(P<0.001) and the proportion of total time spent on the open arm from 7.13% to 32.50%,( P<0.001).Meanwhile,the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect comparing to that of the allopregnanolone.Diazepam reduced the latency of entry to (23.20 ±18.48) s without significance,but it significantly increased both the number of open arm entries to 3.00±1.41 (P<0.001) and the proportion of time spent in the open arms of the maze to (21.90 ±14.82)% (P<0.01).
, 百拇医药
2.2 Analysis of the spontaneous locomotor activity One of the major concerns regarding measurement of potential anxiolytic actions of drugs is whether changes in activity represent anxiolytic actions or effects on locomotor activity.Therefore,the neurosteroid effect on locomotor activity was measured.Analysis of locomotor activity revealed that neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam depressed locomotor activity scores;while 0.5 mg.kg-1 of diazepam significantly decreased the score from 9027±3128 to 5068±1607 (P<0.01,Tab 1,Fig 1).These results,therefore,have demonstrated that ip injection of allopregnanolone resulted in an anxiolytic activity without affecting the spontaneous locomotor activity.
, http://www.100md.com
Tab 1 Comparison of the effects of allopregnanolone and diazepam on the spontaneous locomotor activities in C57 mice(
±s,n=10 ) GroupLocomotor activity score
control
9027±3128
allopregnanolong/0.1mg.kg-1
10110±2879
, http://www.100md.com
diazwpam/0.25mg.kg-1
9418±1380
diazepam/0.5mg.kg-1
5068±1607**
compared with control group **P<0.01
Fig 1 A typical tracing of the spontaneous locomotor activity in C57 mice
, 百拇医药
A:control;B:allopregnanolone 0.1 mg.kg-1;C:diazepam 0.25 mg.kg-1;D:diazepam 0.5 mg.kg-1
3 Discussion
Several line of recent investigation has shown that neurosteroid allopregnanolone allosterically modulate GABA through a unique binding site on the GABA A receptor Cl- channel complex.This binding site has been found to be distinctly different from that of the benzodiazepines or the barbiturates site,respectively.In particular,the 3 a-hydroxy ring A-reduced metabolite of progesterone,allopregnanolone produced a potent enhancement of GABA A receptor responses in vitro.Since benzodiazepines are believed to produce anxiolytic activity via the binding to the GABA A receptor complex,it is reasonable to speculate that allopregnanolone has a similar anxiolytic activity like benzodiazepines.However,benzodiazepines and neurosteroids have their distinctive receptors on the GABA A receptor complex,we therefore compared the anxiolytic activity allopregnanolone and that of diazepam.We found that allopregnanolone produced marked anxiolytic effect at dose of 0.1 mg.kg-1 on the elevated plus-maze test in C57 mice,while the diazepam (0.25 mg.kg-1) produced a lower anxiolytic effect.
, 百拇医药
The change in the elevated plus-maze could simply reflect changes in locomotor activity.Therefore,we were concerned that drug actions on locomotion could influence several of the elevated plus-maze measures which may confound interpretation of drug effects on the elevated plus-maze test of anxiety.To prevent this ambiguity,we have also examined the effects of neurosteroid and benzodiazepine on the spontaneous locomotor activity.In the present study,we found that neither 0.1 mg.kg-1 of allopregnanolone nor 0.25 mg.kg-1 of diazepam affect the spontaneous locomotor activity,while 0.5 mg.kg-1 of diazepam significantly reduced the locomotor activity score.Therefore,this finding indicate that the allopregnanolone-induced changes in the elevated plus-maze do not arise because of changes in locomotor activity.
, 百拇医药
In conclusion,the present study demonstrated the differential behavioral actions of neurosteroid in the elevated plus-maze test of anxiety.This selective anxiolytic effect of allopregnanolone suggested that the allopregnanolone may be a better alternative for diazepam in treating the anxiety.
YU Rong,male,35 years old,Ph.D,associate professor,neuropharmacology
References
1，Majewska MD.Neurosteroids: endogenous bimodal modulators of GABA A receptor.Mechanism of action and physiological significance.Prog Neurobiol,1992;38:379
, http://www.100md.com
2，Lambert JJ,Belelli D,Hill-Venning C ,et al.Neu-rosteroids and GABAA receptor function.Trends Pharmacol,1990;188:403
3，Yu R,Ticku MK.Chronic neurosteroid treatment produces functional hetrologous uncoupling at the GABAA-benzodiazepine receptor complex in mam-malian cortical neurons. Mol Pharmacol,1995;47:603
4，Lister RG.The use of plus-maze to measure anxiety in the mouse.Psychopharmacology,1987;92:180
received：2000-02-10, 百拇医药