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Effects of antagonists at the NMDA receptor complex in two models of anxiety
European Neuropsychopharmacology, 4 (1994) 503-512
503
© 1994 Elsevier Science B.V. All rights reserved 0924-977X/94/$07.00 NEUPSY 00202
Effects of antagonists at the N M D A receptor complex in two models of anxiety A d a m Plaznik a'b'', W o j c i e c h Palejko a, Maciej N a z a r a a n d M a r i a Jessa b aDepartment of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, AI. Sobieskiego 1/9 and bDepartment of Experimental and Clinical Pharmacology, Medical Academy, Krakowskie Przedmieseie 26/28, Warsaw, Poland (Received 14 December 1993) (Revision received 12 April 1994) (Accepted 4 June 1994)
Key words: N M D A receptor complex antagonist; Diazepam; Open field test; Vogel conflict test; (Rat) Summary The effects of an antagonist at the strychnine insensitive glycine site (5,7-dichlorokynurenic acid, i.c.v.), and of noncompetitive (MK-801, i.p.) and competitive (CGP 37849, i.p.; CGP 39551, i.p.; AP-7, i.c.v.) N M D A antagonists were compared with diazepam (i.p.) in two animal models of anxiety (the open field exploratory behavior of non-habituated rats, and the Vogel conflict test). All drugs when applied in appropriate doses increased punished drinking in the Vogel test, without producing any significant changes in free drinking and the stimulus threshold at their lowest anticonflict doses. The effective doses were as follows: diazepam 1.5 and 2.5 mg/kg; MK-801 0.005 and 0.01 mg/kg; CGP 39551 5.0 and 20.0 mg/kg; CGP 37849 1.0 and 2.5 mg/kg; 5,7-dichlorokynurenic acid 5.0 lag (i.c.v); AP-7 0.5 lag (i.c.v.). In the open field diazepam (0.05 mg/kg), MK-801 (0.1 mg/kg), CGP 37849 (0.01, 0.1, 1.0 mg/kg), and AP-7 2.5 lag (i.c.v.) significantly increased exploratory activity in the central sectors of the open field (anti-neophobic reaction), without changing motor activity of the rat. MK-801 at the highest tested dose of 0.2 mg/kg significantly stimulated animal locomotor activity. CGP 37849 in the largest dose examined (10 mg/kg) significantly depressed the motor behavior of rats. Overall, it appeared that different NMDA antagonists showed an anxiolytic-like profile, similar to that of the benzodiazepine diazepam. Among different NMDA receptor complex antagonists studied, CGP 37849 was characterized by the largest distinction between the doses showing an anxiolytic-like action in the open field test, and changing rat motor behavior.
Introduction There is evidence for an anxiolytic-like action of NMDA receptor antagonists in mice and rats. Anti-anxiety-like properties of N M D A antagonists have been demonstrated in a range of rodent models, including social interaction, elevated plusmaze, separation-induced vocalization, and con"Corresponding author. Fax: 48 2 642 5375.
SSDI 0 9 2 4 - 9 7 7 X ( 9 4 ) 0 0 0 8 1 - L
flict procedures (cf. Wiley and Balster, 1992). However, this effect usually coincides with locomotor stimulation which may make animals more responsive in the absence of any specific change in emotionality. Anticonflict effects of NMDA antagonists were not obtained in primates (Mansbach et al., 1991; Rupniak et al., 1993). Moreover, noncompetitive NMDA antagonists do not appear to offer advantages over existing anxiolytics because of their potency to induce motor impairment and ataxia (Koek and Colpaert, 1990; Willets
504 et al. 1990). In some tests N M D A antagonists were about 100 times less potent than diazepam in antagonizing the suppressive effects of punishment, or neophobic reactions in mice and rats (Stephens et al., 1986; Sanger and Jackson, 1989). However, there are still some reasons to believe that this rapidly developing class of drugs may offer some advantages over the benzodiazepines in the treatment of anxiety disorders. Recently, significant progress has been made in developing potent, competitive N M D A receptor antagonists with good penetration into the brain after peripheral administration (Schmutz et al., 1990). Interestingly, these drugs are devoid of locomotor stimulatory properties (Bubser et al., 1992; Maj et al., 1992). Alternative approaches reducing NMDA-mediated neurotransmission, such as interaction with the modulatory glycine site, may also eliminate some of the undesirable effects. For example, in contrast to MK-801 (( + )-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate) the competitive N M D A receptor antagonist CGP 39551 (the carboxy-ethyl ester of CGP 37849: DL-(E)-2-amino4-methyl-5-phosphono-3-pentenoic acid) did not increase DA metabolism in subcortical structures (Bubser et al., 1992), and the glycine antagonists 7chlorokynurenic acid and HA-966 (1-hydroxy-3aminopyrrolidone-2) selectively blocked N M D A induced convulsions without producing phencyclidine-like behavioral effects (Koek and Colpaert, 1990). It is noteworthy that significant increases in punished response were found after the combined administration of MK-801 and diazepam, thus suggesting an enhancement of the anticonflict potency of benzodiazepines by N M D A antagonists (Kuribara et al., 1990). Further, a competitive N M D A receptor antagonist, AP-7 (DL-2-amino-7phosphonoheptanoic acid), was reported to produce discriminative stimulus similar to diazepam (Bennett and Amrick, 1986). To clarify further the issue of an involvement of NMDA-related mechanisms in emotional control, in the present paper we have compared the effects of an antagonist at the strychnine insensitive glycine sites, and of noncompetitive and competitive N M D A receptor antagonists with diazepam, in two animal models of anxiety (Stefanski et al., 1992). In the light of the above reviewed findings, the main objective of the present study was to reevaluate the effects of channel blockers, competitive antagonists and negative allosteric modulators acting at the glycine site, in the conflict and ethologically oriented tests.
Methods
Animals Male Wistar rats (200_+20 g), bought from a breeder (license of the Ministry of Agriculture), were used in the study. The animals were housed in standard laboratory conditions, under a 12-h light/ dark 12h cycle (lights on at 6 a.m.), in a temperature controlled (21+2°C), ventilated colony, with free access to food and tap water. The rats were kept four per cage (30 x 30 x 20 cm) or individually after i.c.v, cannula implantation. Each experimental and control group consisted of 6-8 animals. Rats used in the Vogel conflict test, shock threshold or baseline drinking experiments had the access to water limited to 60 min within 24 h. Vogel's conflict test Apparatus consisted of four plastic boxes ( 3 0 x 3 0 x 6 0 cm), with a grid floor made of stainless steel rods. A water drinking tube was mounted on the wall of a cage, an electric shock generator was connected with the grid floor and the wire embedded in glass drinking tube. During the first 2 days of the 4-day training, animals were deprived of water 23 h daily. The last 2 days consisted of a pretest when animals with a sustained 23/24-h deprivation cycle were placed in the test apparatus with no electric shocks delivered, for 15 min. Subsequently, the rats were allowed to drink water for 45 min in their home cages. After 4 days of training, drinking levels for all animals were usually stabilized (animals drinking less than 5 ml of water during a 15-min session were not taken into consideration). The rats were randomly divided into two control groups (both receiving solvent), and experimental groups receiving drug injections. During the experimental procedure animals were put into the apparatus and electric shocks were delivered in cycles of 5 s with 4-s intervals. Shock current was set at 0.4 mA. The amount of water consumed during 15 min of test session was recorded and taken as a measure of conflict behavior. All experiments were performed between 12 a.m. and 6 p.m. The time between testing and drug administration was as follows: diazepam and MK801 30 min, CGP 37849 and CGP 39551 60 min, AP-7 and 5,7-dichlorokynurenic acid (i.c.v.) 5 min. In order to estimate the putative influence of drugs on shock threshold and baseline drinking, the following control experiments were performed.
505 DIAZEPAM
Central entries
- OPEN FIELD TEST Activity 30
200
O o 20
100 10
C
.05
DIAZEPAM
T i m e in c e n t r a l s e c t o r
2O
.05
C
.1 mg/kg
.1 mg/kg
- VOGEL TEST
o
ml
s
0
_L
O O
10
C
.05
I
.1 mg/kg
o
•
[:;sk
C
S
1.5
2.5
mg/kg
Fig. 1. The influence of diazepam on rat behavior in the open field test and on punished water intake in the Vogel conflict test. Data are shown as mean 4-SEM. Ordinate: number of photobeam interruptions (activity); number of entries into the central part of the arena (central entries); time (s) spent in the central sector of the open field (time in central sector); the amount of water intake (ml) (Vogel test). C, control rats; S, shocked rats (Vogel test). The number of rats in each experimental group was 8. O, differs from control rats (open field) and from shocked rats (Vogel test); O, differs from the control non-shocked group. ©, P < 0 . 0 5 ; © O , 0 0 , P<0.01.
Baseline drinking test The animals were prepared in the same manner as in the Vogel test, except that during the experimental procedure no shocks were delivered. Water amounts consumed spontaneously after active drug dose and vehicle administration were recorded and compared.~ Shock threshold test (flinch-jump test) The rats were deprived of water prior to the test as in the Vogel test. The experiment was performed in the Vogel test cages and shocks were delivered to the grid floor in 0.75-s pulses (Evans, 1961). After 3 min of habituation shock titration was continued upward or downward in a stepwise manner (0.05 mA, 0.05-0.85 mA range) depending upon responsiveness of the animal. The time interval between shocks was 15 s. The time between drug administration and testing was the same as in the Vogel test.
Open field test The open field apparatus used in this experiment consisted of two round arenas (80 cm diameter), each equipped symmetrically with three photocells. Testing was performed in a soundproof chamber under dim light and continuous white noise (65 dB) without previous habituation. General activity (number of photobeam interruptions) was scored for 10 min. Animals were also observed by an experimenter via closed-circuit television. The number of entries into the central part of the open field was recorded (this parameter was defined as a movement of the animal from the wall to the central area of the open field over a distance of approx. 15 cm). The time spent in the central sector (area defined as a centrally situated 35 cm diameter circle) was also recorded as a third parameter. The time between drug administration and testing was the same as in the Vogel test.
506 MK-801
- OPEN
FIELD TEST
C e n t r a l entries
Activity 300
20 o
200
o 10
100
,005
.01
.05
.1
n
0
.2mg/kg
C
C
.005
.01
MK-801
T i m e in c e n t r a l s e c t o r
.05
1
2mg/kg
C
- VOGEL
TEST
o 7
40 s
ml
o o
6
0
5
30
4 20
3 2
10
1 0
0 C
.005 .01
.05
1
C
C
.2mg/kg
S
n
,001
"In
n C
S
,005 .01
,05
mg/kg
Fig. 2. The influence of MK-801 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats was 7 8. For other explanations see Fig. 1.
Intracerebroventricular injections
Drugs
Rats were anesthetized with ketamine and positioned in a stereotaxic apparatus (Stoelting & Co., USA) with the incisor bar set in horizontal plane. A 10 mm long stainless steel guide cannula (0.7 mm external diameter, 0.5 mm internal diameter) was placed 1.5 mm laterally to the sagittal suture (right side), 1.2 mm posteriorly to bregma and 3.5 mm below the dura in the lateral ventricle (Pellegrino et al., 1967). The guide cannula was fixed to the skull with jewellery screws and dental acrylic cement. Seven days following implantation, the animals were subjected to behavioral tests. I.c.v. drug injections were delivered with a 10-pl Hamilton microsyringe connected to the 12.5 mm long stainless steel injector (0.3 mm external diameter) via polyethylene tubing. Drug solutions were administered 5 rain prior to testing in a volume of 5 gl over 60 s. The injection needle remained in place for an additional 30-60 s before it was removed and the stylet replaced.
Diazepam (Polfa, Poland) was suspended in 1% Tween 80. (+)-MK-801 hydrogen maleate (( + )-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-iminemaleate) (Research Biochemicals Inc., USA), CGP 37849 (DL-(E)-2-amino-4-methyl-5phosphono-3-pentenoic acid) and its carboxyethyl ester CGP 39551 (Ciba-Geigy, Switzerland), as well as AP-7 (DL-2 amino-7-phosphonoheptanoic acid, Tocris Neuramin, UK), were dissolved in distilled water. 5,7-Dichlorokynurenic acid (Tocris Neuramin, UK) was dissolved in dilute aqueous NaOH with pH adjusted to 6~7. Drug solutions were prepared immediately before administration (i.p., 2 ml/kg or 5 ~1 for intracerebral injections).
Histological analysis All animals were killed after the final testing day. The brains of operated rats were removed, stored in 5% formaldehyde solution and verified histologically. The frozen tissue was dissected in the slices and the place of injection examined with a magnifying glass. Only those animals with verified
507 CGP 37849 - OPEN FIELD TEST
Central entries
Activity 200
20
100
10
O
O
h
0
.005
.01
.1
C
1
C
5 10mg/k 9
.005
,01
O
In .1
1
C
CGP 37849 - VOGEL TEST
T i m e in c e n t r a l s e c t o r
60 s
6
50
5
40
4
o O
ml
©
3
30
o 20
2
O
1
10 0
"" C
0
.005
,01
.1
C
5 10mg/kg
5 10mg/kg
S
,3
n 1
2.5 mg/kg
Fig. 3. The influence o f C G P 37849 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The n u m b e r o f rats in each experimental group was 8. F o r other explanations see Fig. 1.
i.c.v, injection sites were included in the study.
Statistical analysis The data are shown as means +_SEM. Statistical analysis was performed using one-way ANOVA, followed by Student's t-test for independent samples. The confidence limit of P<0.05 was considered as statistically significant.
Results
Open field data Diazepam in a dose of 0.05 mg/kg, but not 0.1 mg/kg, significantly increased the number of entries into the central part of the open field (t=3.06, df 14, P<0.01), as well as time spent in the central sector of this arena (t=3.79, df 14, P<0.01) (Fig. 1). MK-801 at a dose of 0.1 mg/kg stimulated rat exploratory activity in the central sectors of the open field (entries, t=2.71, df 16, P<0.05; time, t=2.32, df 16, P<0.05) (Fig. 2); however, these rats tended also to have higher
levels of locomotor activity. MK-801 injected in a dose of 0.2 mg/kg significantly increased the animals' motility (t=5.0, df 14, P<0.01). CGP 37849 enhanced selectively in a limited range of doses (0.01, 0.1 and 1.0 mg/kg) rat behavior in the central parts of the open field (first experiment, time, F = 4.89, df 3,27, P < 0.01; second experiment, entries, F=3.54, df 3,28, P<0.05) (Fig. 3). At the largest dose examined (10 mg/kg) CGP 37849 significantly depressed motor behavior (t = 2.3, df 14, P<0.05) (Fig. 3). CGP 39551 administered in a wide dose range from 0.1 to 5.0 mg/kg did not affect rat behavior in the open field (motor activity, second experiment, F =0.66, df 3,28, P>0.05; entries, second experiment, F=1.58, df 3,28, P>0.05) (Fig. 4). 5,7-Dichlorokynurenic acid applied i.c.v. (0.5-5.0 ~tg) changed neither rat motor (F= 1.71, df 4,25, P>0.05) nor exploratory activity (entries, F = 1.67, df4,25, P>0.05) (Fig. 5), in a significant way. After the dose of 5 ~tg of 5,7dichlorokynurenic acid rats tended to have lower levels of locomotor activity. AP-7 administered centrally at a dose of 2.5 lag significantly increased
508 Central entries
CGP 39551 - OPEN FIELD TEST Activity 200
20
100
10
C
,1
C
.5
C
2,5 5 mg/kg
T i m e in c e n t r a l s e c t o r
.1
C
.5
CGP 39551 - VOGEL
20
7
2.5 5mg/kg
TEST
m[ O
O
6 5 4
10
3 2 1
C
.1
.5
2.5
5mg/kg
0
C
S
1
5
20 mg/kg
Fig. 4. The influence o f C G P 39551 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The n u m b e r o f rats in each experimental group was 8. F o r other explanations see Fig. 1.
exploratory behavior of rats in the central part of the open field (entries, t =2.9, df 19, P<0.01; time, t=2.5, df 19, P<0.05) (Fig. 6). At a dose of 5.0 lag of AP-7, significant ataxia prevented accurate measurement of open field behavior, with all animals remaining in one place during the whole observation period.
Vogel's conflict test In the Vogel conflict test all examined drugs in some doses significantly increased the punished consumption of water (Figs. 1-6). The clear-cut effect appeared after administration of diazepam at 1.5 and 2.5 mg/kg (F= 4.25, df 3,28, P<0.05) (Fig. 1). MK-801 was active at doses of 0.005 and 0.01, but not 0.001 and 0.05 mg/kg (second experiment, F = 11.09, df 4,33, P<0.01) (Fig. 2). CGP 37849 had also disinhibitory effect in the Vogel conflict test (1.0 and 2.5 mg/kg) (F=4.81, df 4,33, P<0.01) (Fig. 3). CGP 39551 prevented the shock-induced suppression of drinking at 5 and 20 mg/kg only (F = 5.68, df 4,34, P<0.01) (Fig. 4). Both intraventricularly administered drugs, 5,7-dichlorokynure-
nic acid (F=6.89, df 4,29, P<0.01) and AP-7 (F=3.23, df 4,29, P<0.05), caused a marked increase in punished drinking after the doses of 5.0 lag and 0.5 lag, respectively (Figs. 5 and 6). Probably because of seasonal (the experiment was performed over several months) and betweengroup variabilities, the control groups differed in the amount of water consumption, but not in their reactivity to the shock (Fig. 1-6). In the control experiments all compounds failed to produce any significant effects on free (unpunished) drinking and the stimulus threshold at their lowest anti-conflict doses (Table 1).
Discussion
Diazepam significantly increased punished consumption of water in the Vogel test, and disinhibited rat reaction to a novel environment in the open field test. These effects of the benzodiazepine occurred independently of changes in motor activity as well as spontaneous water intake and
509 5,7-DICHLOROKYNURENIC ACID OPEN FIELD TEST - Activity
Central entries
200
1
100
C
.5
1.25
2.5
5,ug i.c.v.
C
T i m e in c e n t r a l s e c t o r
.5
.25
2.5
5aJg i.c.v.
5,7-DIC HLOROKYNU RENIC ACID VOGEL TEST
40
6
ml
o
5 30
.1_ 4
20
3 2
10 1 0 C
0 .5
1.25
2.5
5/.Jg i.c.v.
C
S
n
2.5
5
10 ~ug i.e.v.
Fig. 5. The influence of 5,7-dichlorokynurenic acid on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats in each experimental group was 6-8. For other explanations see Fig. 1.
pain threshold. The open field test appeared to be more sensitive to the benzodiazepine derivative, and helped to reveal the anxiolytic-like potency of diazepam, in doses as small as ~g/kg. Recently, anxiolytic-like effects of chlordiazepoxide and diazepam, given in a similar dose range in the open field test, were reported by us and other authors (Bruhwyler, 1990; Stefanski et al., 1992). In the present study we examined again the action of diazepam in the most potent and consistent doses obtained from our previous experiments (Stefanski et al., 1992). The examined N M D A receptor antagonists, irrespective of their site of action at the N M D A receptor complex, significantly affected rat emotional behavior. The results obtained in the Vogel test seem to be more consistent, since in contrast to the open field test all five N M D A antagonists selectively increased punished consumption of water. This finding confirms the data of other authors on the action of N M D A antagonists in conflict paradigms (Bennett and Amrick, 1986; Stephens et al., 1986; Kuribara et al., 1990;
McMillan et al., 1991; Xie and Commissaris, 1992; Wiley et al., 1992). MK-801, a noncompetitive N M D A receptor antagonist, disinhibited punished behavior in the Vogel test in a dose as small as 0.005 mg/kg. The drug administered in a dose of 0.03 mg/kg also increased conflict responding in the Cook and Davidson conditioned conflict paradigm (Corbett and Dunn, 1993). A slightly larger dose of 0.05 mg/kg of MK-801 was reported to induce hyperactivity, hyper-reactivity, reductions in rearing behavior and deficits in tongue extension (Hargreaves and Cain, 1992). The weaker effects of MK-801 observed in the present experiment in the open field may be explained by the omission in the experimental procedure of habituation to the test conditions. Habituation should have served to decrease rat spontaneous locomotion, thus revealing drug-induced stimulation of rat motility. Higher doses of MK-801 are usually necessary to increase rodent locomotion and rearing (Liljequist et al., 1991; Bubser et al., 1992). Accordingly, in the present experiment a potent enhancement of rat motor activity could be
510 Central entnes
AP-7 - OPEN FIELD TEST Activity 200
20
o o 100
lO
C
,5
2.5
C
5,ug i.c.
.5
T i m e in c e n t r a l s e c t o r
2.5
5 # g i.c.v.
AP-7 - VOGEL TEST
90
8
80
7
70
6
60
ml
O
_L
4
40
3
30
"
i
S
.1
•
5
50
5y
•
2
20 ,o
1
o
0
c
,5
2.5
C
5 ~g i.c.v.
.5
2.5 ,ug i.c.v.
Fig. 6. The influence of AP-7 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats in each experimental group was 7 8. For other explanations see Fig. 1.
observed after the highest dose of 0.2 mg/kg. This coincided with a significant stimulation of rat behavior in the central parts of the open field. This interpretation (motor-related) of MK-801 open field data is compatible with other results obtained
with noncompetitive N M D A antagonists (Wedzony et al., 1993). Interestingly, CGP 37849 yielded selective and potent anxiolytic-like effects both in the open field and in the Vogel test, in a dose range similar to that of diazepam. After the highest dose
TABLE l THE I N F L U E N C E O F D I A Z E P A M , C G P 37849, C G P 39551, MK-801, AP-7 A N D 5 , 7 - D I C H L O R O K Y N U R E N I C ACID ON BASELINE D R I N K I N G A N D SHOCK T H R E S H O L D IN A N T I C O N F L I C T DOSES, ACTIVE IN THE V O G E L TEST. Drug
Dose
n
Spontaneous drinking (ml) (mean _+SEM)
n
Pain-flinch (mA) (mean 4- SEM)
Control/Tween Diazepam
1.5 mg/kg
5 5
5.98 4- 0.90 7.40 4- 0.20
7 7
0.37 4- 0. | 0 0.36 _+0.02
Control/H20 C G P 37849 C G P 39551 MK-801
1.0 mg/kg 5.0 mg/kg 0.005 mg/kg
9 7 7 7
5.83 _+0.45 6.23 + 0.60 7.34_+ 0.80 6.36 + 1.40
8 8 8 8
0.38 _+0.02 0.43 + 0.10 0.38 _+0.10 0.36 _+0.03
Control/H20 AP-7 5,7-Dichlorokynurenic acid
0.5 lag i.c.v./5 lal 5.0 lag i.c.v./5 lal
5 8 7
6.07 4- 1.10 6.25 _+0.60 5.20_+ 1.00
7 6 7
0.40_+ 0.02 0.43 _+0.02 0.44_+0.01
The data are shown as means_+ SEM. n, number of rats.
511 of 10.0 mg/kg of CGP 37849 a significant reduction in rat motor behavior was found, confirming other authors' data (Maj et al., 1992). The action of CGP 37849 was characterized by the largest difference between the dose changing rat motor activity and exploratory behavior in the central part of the open field (1000-fold). The same coefficients calculated for AP-7 and MK-801 were much smaller (2-fold). CGP 37849 and CGP 39551 are potent competitive N M D A receptor antagonists with oral activity (Schmutz et al., 1990). In contrast to MK-801, the carboxy-ethyl ester of CGP 37849, CGP 39551, did not increase the dopaminergic activity of the basal ganglia and even decreased the DOPAC/DA ratio in the frontal cortex (Bubser et al., 1992). In agreement with our findings, CGP 39551 was reported to differ from MK-801 and CGP 37849 both behaviorally and biochemically in that its effects were much less intense (Maj et al., 1992; L6scher et al., 1993). It has been proposed that the differences between competitive and noncompetitive N M D A receptor antagonists are due to the existence in the brain of binding sites for noncompetitive N M D A antagonists, that are not coupled to the N M D A binding site (Rao et al., 1990). However, these differences may not be important since local infusions of competitive N M D A receptor antagonists were reported to stimulate behavior, and after higher doses to induce ataxia (Schmidt, 1986; Tiedtke et al., 1990). Accordingly, intracerebroventricular (this report) or intrahippocampal (data in preparation) administration of the competitive N M D A antagonist AP-7 induced ataxia and potent locomotor stimulation, respectively. The effects of centrally injected 5,7-dichlorokynurenic acid, an antagonist at a glycine regulatory site on the N M D A receptor, were limited, observed after the highest dose tested and concerned rat behavior in the Vogel test only. Previously, it was found that strychnine insensitive glycine antagonists are active in non-conflict procedures with rodents (Trulas et al., 1989; Winslow et al., 1990; Kehne et al., 1991; Corbett and Dunn, 1993), but inactive in a conflict procedure with pigeons (Koek and Colpaert, 1991). Moreover, 1-amino cyclopropane carboxylic acid, a ligand at strychnine insensitive glycine receptors, was significantly less efficacious than chlordiazepoxide in an elevated plus-maze (Trulas et al., 1989). Also in the Cook and Davidson conditioned conflict paradigm the disinhibition of shock-induced suppression of conflict responding by 5,7-dichlorokynurenic acid
was not as robust as that observed with diazepam (Corbett and Dunn, 1993). Overall, these data indicate a weak anxiolytic-like profile of action of glycine antagonists. However, the fact that rats administered 5,7-dichlorokynurenic acid failed to generalize to a MK-801 discriminative stimulus cue indicates that antagonists at the strychnine insensitive glycine site could be devoid of psychotomimetic effects in man (Corbett and Dunn, 1993). The results in this study show that in two animal models of anxiety the behavioral profile of MK801, CGP 37849 and AP-7 appeared to be similar to that of the benzodiazepine. However, the motor effects of CGP 37849 and MK-801 and the atactic influence of AP-7 observed at the higher dose level indicate that these drugs may be less advantageous than benzodiazepines in the treatment of anxiety disorder. Though the N M D A receptor system contributes somewhat to the control of emotional processes, the effect seems to be less direct and consistent than was previously thought.
Acknowledgement The paper was supported by grant No. 17 from the Institute of Psychiatry and Neurology, Warsaw, Poland.
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512 ultrasonic vocalizations. Eur. J. Pharmacol. 193, 283 292. Koek, W. and Colpaert, F.C. (1990) Selective blockade of NMDA-induced convulsions by NMDA antagonists and putative glycine antagonists: relationship with phencyclidine-like behavioural effects. J. Pharmacol. Exp. Ther. 252, 349 357. Koek, W. and Colpaert, F.C. (1991) Use of conflict procedure in pigeons to characterize anxiolytic drug activity: evaluation of NMDA antagonists. Life Sci. 49, PL37~PL42. Kuribara, H., Fujiwara, S., Yasuda, H. and Tadokoro, H. (1990) The anticonflict effect of MK-801, and NMDA antagonist: investigation by punishment procedure in mice. Jpn. J. Pharmacol. 54, 249 252. Liljequist, S., Ossowska, K., Grabowska-Anden, M. and Anden, N.E. (1991) Effect of NMDA receptor antagonist, MK-801, on locomotor activity and on the metabolism of dopamine in various brain areas of mice. Eur. J. Pharmacol. 195, 55-61. L6scher, W., Annies, R. and H6nack, D. (1993) Comparison of competitive and uncompetitive NMDA receptor antagonists with regard to monoaminergic neuronal activity and behavioural effects in rats. Eur. J. Pharmacol. 242, 263 274. Maj, J., Rogoz, Z., Skuza, G. and Sowinska, H. (1992) The effect of CGP 37849 and CGP 39551 competitive NMDA receptor antagonists in the forced swimming test. Pol. J. Pharmacol. Pharm. 44, 337 346. Mansbach, R.S., Willetts, J., Jortani, S.A. and Balster, R.L. (1991) NMDA lack of antipunishment effect in squirrel monkeys. Pharmacol. Biochem. Behav. 39, 977 981. McMillan, D.E., Hardwick, W.C., DeCosta, B.R. and Rice, K.C. (1991) Effects of drugs that bind to PCP and Sigma receptors on punished responding. J. Pharmacol. Exp. Ther. 258, 1015 1018. Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J. (1967) A Stereotaxic Atlas of the Rat Brain. Plenum Press, New York. Rao, T.S., Kim, H.S., Lehmann, J., Martin, L.L. and Wood, P.L. (1990) Selective activation of dopaminergic pathways in the mesocortex by compounds that act at the phencyclidine (PCP) binding site: tentative evidence for PCP recognition sites not coupled to NMDA receptors. Neuropharmacology 29, 255 231. Rupniak, N.M.J., Boyce, S., Tye, S., Cook, G. and Iversen, S.D. (1993) Anxiolytic-like and antinociceptive effects of MK-801 accompanied by sedation and ataxia in primates. Pharmacol. Biochem. Behav. 44, 153 156. Sanger, D.J. and Jackson, A. (1989) Effects of phencyclidine and other NMDA antagonists on the schedule controlled behaviour of rats. J. Pharmacol. Exp. Ther. 248, 1215 1221.
Schmidt, W.J. (1986) Intrastriatal injections of DL-2-amino-5phosphonovaleric acid (AP-5) induces sniffing stereotype that is antagonized by haloperidol and clozapine. Psychopharmacology 90, 123 128. Schmutz, M., Portet, Ch., Jeker, A.~ Klebs, K.~ Vassout, A., Allgeier, H., Heckendorn, R., Fagg, G.E., Olpe, H.R. and Riezen, H. (1990) The competitive NMDA receptor antagonists CGP 37849 and CGP 39551 are potent, orally active anticonvulsants in rodents. N.S. Arch. Pharmacol. 342, 61 66. Stefanski, R., Palejko, W., Kostowski, W. and Plaznik, A. (1992) The comparison of benzodiazepine derivatives and serotonergic agonists and antagonists in two animal models of anxiety. Neuropharmacology 12, 1251 1258. Stephens, D.N., Meldrum, B.S., Weidmann, R., Schneider, C. and Grutzner, M. (1986) Does the excitatory amino acid receptor antagonist 2-APH exhibit anxiolytic activity'? Psychopharmacology 90, 166-169. Tiedtke, P.I., Bischoff, C. and Schmidt, W.J. (1990) MK-801induced stereotypy and its antagonism by neuroleptic drugs. J. Neural Transm. (Gen. Sect.) 81, 173 178. Trulas, R., Jackson, B. and Skolnick, Ph. (1989) Anxiolytic properties of l-aminocyclopropanecarboxylicacid, a ligand at strychnine-insensitive glycine receptors. Pharmacol. Biochem. Behav. 34, 313 316. Wedzony, K., Golembiowska, K. and Klimek, V. (1993) MK801-induced symptoms of sensitization the lack of correlation with the extracellular concentration of dopamine in the rat. Brain Res. 333 336. Wiley, J.L. and Balster, R.L. (1992) Preclinical evaluation of N-methyl-D-aspartate antagonists for antianxiety effects: a review. In: Kamenka, J.M. and Domino, E.F. (Eds.), Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, MI, pp. 801 815. Wiley, J.L., Porter, J.H., Compton, A.D. and Balster, R.L. (1992) Antipunishment effects of acute and repeated administration of phencyclidine and NPC 12626 in rats. Life Sci. 50, 1519 1528. Willetts, J., Balster, R.L. and Leander, J.D. (1990) The behavioural pharmacology of NMDA receptor antagonists. Trends Pharmacol. Sci. I 1,423 428. Winslow, J.T., Insel, T.R., Trullas, R. and Skolnick, Ph. (1990) Rat pup isolation calls are reduced by functional antagonists of the NMDA receptor complex. Eur. J. Pharmacol. 190, 11 21. Xie, Z. and Commissaris, L. (1992) Anxiolytic-like effects of the noncompetitive NMDA antagonist MK-801. Pharmacol. Biochem. Behav. 43, 471 477.
503
© 1994 Elsevier Science B.V. All rights reserved 0924-977X/94/$07.00 NEUPSY 00202
Effects of antagonists at the N M D A receptor complex in two models of anxiety A d a m Plaznik a'b'', W o j c i e c h Palejko a, Maciej N a z a r a a n d M a r i a Jessa b aDepartment of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, AI. Sobieskiego 1/9 and bDepartment of Experimental and Clinical Pharmacology, Medical Academy, Krakowskie Przedmieseie 26/28, Warsaw, Poland (Received 14 December 1993) (Revision received 12 April 1994) (Accepted 4 June 1994)
Key words: N M D A receptor complex antagonist; Diazepam; Open field test; Vogel conflict test; (Rat) Summary The effects of an antagonist at the strychnine insensitive glycine site (5,7-dichlorokynurenic acid, i.c.v.), and of noncompetitive (MK-801, i.p.) and competitive (CGP 37849, i.p.; CGP 39551, i.p.; AP-7, i.c.v.) N M D A antagonists were compared with diazepam (i.p.) in two animal models of anxiety (the open field exploratory behavior of non-habituated rats, and the Vogel conflict test). All drugs when applied in appropriate doses increased punished drinking in the Vogel test, without producing any significant changes in free drinking and the stimulus threshold at their lowest anticonflict doses. The effective doses were as follows: diazepam 1.5 and 2.5 mg/kg; MK-801 0.005 and 0.01 mg/kg; CGP 39551 5.0 and 20.0 mg/kg; CGP 37849 1.0 and 2.5 mg/kg; 5,7-dichlorokynurenic acid 5.0 lag (i.c.v); AP-7 0.5 lag (i.c.v.). In the open field diazepam (0.05 mg/kg), MK-801 (0.1 mg/kg), CGP 37849 (0.01, 0.1, 1.0 mg/kg), and AP-7 2.5 lag (i.c.v.) significantly increased exploratory activity in the central sectors of the open field (anti-neophobic reaction), without changing motor activity of the rat. MK-801 at the highest tested dose of 0.2 mg/kg significantly stimulated animal locomotor activity. CGP 37849 in the largest dose examined (10 mg/kg) significantly depressed the motor behavior of rats. Overall, it appeared that different NMDA antagonists showed an anxiolytic-like profile, similar to that of the benzodiazepine diazepam. Among different NMDA receptor complex antagonists studied, CGP 37849 was characterized by the largest distinction between the doses showing an anxiolytic-like action in the open field test, and changing rat motor behavior.
Introduction There is evidence for an anxiolytic-like action of NMDA receptor antagonists in mice and rats. Anti-anxiety-like properties of N M D A antagonists have been demonstrated in a range of rodent models, including social interaction, elevated plusmaze, separation-induced vocalization, and con"Corresponding author. Fax: 48 2 642 5375.
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flict procedures (cf. Wiley and Balster, 1992). However, this effect usually coincides with locomotor stimulation which may make animals more responsive in the absence of any specific change in emotionality. Anticonflict effects of NMDA antagonists were not obtained in primates (Mansbach et al., 1991; Rupniak et al., 1993). Moreover, noncompetitive NMDA antagonists do not appear to offer advantages over existing anxiolytics because of their potency to induce motor impairment and ataxia (Koek and Colpaert, 1990; Willets
504 et al. 1990). In some tests N M D A antagonists were about 100 times less potent than diazepam in antagonizing the suppressive effects of punishment, or neophobic reactions in mice and rats (Stephens et al., 1986; Sanger and Jackson, 1989). However, there are still some reasons to believe that this rapidly developing class of drugs may offer some advantages over the benzodiazepines in the treatment of anxiety disorders. Recently, significant progress has been made in developing potent, competitive N M D A receptor antagonists with good penetration into the brain after peripheral administration (Schmutz et al., 1990). Interestingly, these drugs are devoid of locomotor stimulatory properties (Bubser et al., 1992; Maj et al., 1992). Alternative approaches reducing NMDA-mediated neurotransmission, such as interaction with the modulatory glycine site, may also eliminate some of the undesirable effects. For example, in contrast to MK-801 (( + )-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate) the competitive N M D A receptor antagonist CGP 39551 (the carboxy-ethyl ester of CGP 37849: DL-(E)-2-amino4-methyl-5-phosphono-3-pentenoic acid) did not increase DA metabolism in subcortical structures (Bubser et al., 1992), and the glycine antagonists 7chlorokynurenic acid and HA-966 (1-hydroxy-3aminopyrrolidone-2) selectively blocked N M D A induced convulsions without producing phencyclidine-like behavioral effects (Koek and Colpaert, 1990). It is noteworthy that significant increases in punished response were found after the combined administration of MK-801 and diazepam, thus suggesting an enhancement of the anticonflict potency of benzodiazepines by N M D A antagonists (Kuribara et al., 1990). Further, a competitive N M D A receptor antagonist, AP-7 (DL-2-amino-7phosphonoheptanoic acid), was reported to produce discriminative stimulus similar to diazepam (Bennett and Amrick, 1986). To clarify further the issue of an involvement of NMDA-related mechanisms in emotional control, in the present paper we have compared the effects of an antagonist at the strychnine insensitive glycine sites, and of noncompetitive and competitive N M D A receptor antagonists with diazepam, in two animal models of anxiety (Stefanski et al., 1992). In the light of the above reviewed findings, the main objective of the present study was to reevaluate the effects of channel blockers, competitive antagonists and negative allosteric modulators acting at the glycine site, in the conflict and ethologically oriented tests.
Methods
Animals Male Wistar rats (200_+20 g), bought from a breeder (license of the Ministry of Agriculture), were used in the study. The animals were housed in standard laboratory conditions, under a 12-h light/ dark 12h cycle (lights on at 6 a.m.), in a temperature controlled (21+2°C), ventilated colony, with free access to food and tap water. The rats were kept four per cage (30 x 30 x 20 cm) or individually after i.c.v, cannula implantation. Each experimental and control group consisted of 6-8 animals. Rats used in the Vogel conflict test, shock threshold or baseline drinking experiments had the access to water limited to 60 min within 24 h. Vogel's conflict test Apparatus consisted of four plastic boxes ( 3 0 x 3 0 x 6 0 cm), with a grid floor made of stainless steel rods. A water drinking tube was mounted on the wall of a cage, an electric shock generator was connected with the grid floor and the wire embedded in glass drinking tube. During the first 2 days of the 4-day training, animals were deprived of water 23 h daily. The last 2 days consisted of a pretest when animals with a sustained 23/24-h deprivation cycle were placed in the test apparatus with no electric shocks delivered, for 15 min. Subsequently, the rats were allowed to drink water for 45 min in their home cages. After 4 days of training, drinking levels for all animals were usually stabilized (animals drinking less than 5 ml of water during a 15-min session were not taken into consideration). The rats were randomly divided into two control groups (both receiving solvent), and experimental groups receiving drug injections. During the experimental procedure animals were put into the apparatus and electric shocks were delivered in cycles of 5 s with 4-s intervals. Shock current was set at 0.4 mA. The amount of water consumed during 15 min of test session was recorded and taken as a measure of conflict behavior. All experiments were performed between 12 a.m. and 6 p.m. The time between testing and drug administration was as follows: diazepam and MK801 30 min, CGP 37849 and CGP 39551 60 min, AP-7 and 5,7-dichlorokynurenic acid (i.c.v.) 5 min. In order to estimate the putative influence of drugs on shock threshold and baseline drinking, the following control experiments were performed.
505 DIAZEPAM
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Fig. 1. The influence of diazepam on rat behavior in the open field test and on punished water intake in the Vogel conflict test. Data are shown as mean 4-SEM. Ordinate: number of photobeam interruptions (activity); number of entries into the central part of the arena (central entries); time (s) spent in the central sector of the open field (time in central sector); the amount of water intake (ml) (Vogel test). C, control rats; S, shocked rats (Vogel test). The number of rats in each experimental group was 8. O, differs from control rats (open field) and from shocked rats (Vogel test); O, differs from the control non-shocked group. ©, P < 0 . 0 5 ; © O , 0 0 , P<0.01.
Baseline drinking test The animals were prepared in the same manner as in the Vogel test, except that during the experimental procedure no shocks were delivered. Water amounts consumed spontaneously after active drug dose and vehicle administration were recorded and compared.~ Shock threshold test (flinch-jump test) The rats were deprived of water prior to the test as in the Vogel test. The experiment was performed in the Vogel test cages and shocks were delivered to the grid floor in 0.75-s pulses (Evans, 1961). After 3 min of habituation shock titration was continued upward or downward in a stepwise manner (0.05 mA, 0.05-0.85 mA range) depending upon responsiveness of the animal. The time interval between shocks was 15 s. The time between drug administration and testing was the same as in the Vogel test.
Open field test The open field apparatus used in this experiment consisted of two round arenas (80 cm diameter), each equipped symmetrically with three photocells. Testing was performed in a soundproof chamber under dim light and continuous white noise (65 dB) without previous habituation. General activity (number of photobeam interruptions) was scored for 10 min. Animals were also observed by an experimenter via closed-circuit television. The number of entries into the central part of the open field was recorded (this parameter was defined as a movement of the animal from the wall to the central area of the open field over a distance of approx. 15 cm). The time spent in the central sector (area defined as a centrally situated 35 cm diameter circle) was also recorded as a third parameter. The time between drug administration and testing was the same as in the Vogel test.
506 MK-801
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Fig. 2. The influence of MK-801 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats was 7 8. For other explanations see Fig. 1.
Intracerebroventricular injections
Drugs
Rats were anesthetized with ketamine and positioned in a stereotaxic apparatus (Stoelting & Co., USA) with the incisor bar set in horizontal plane. A 10 mm long stainless steel guide cannula (0.7 mm external diameter, 0.5 mm internal diameter) was placed 1.5 mm laterally to the sagittal suture (right side), 1.2 mm posteriorly to bregma and 3.5 mm below the dura in the lateral ventricle (Pellegrino et al., 1967). The guide cannula was fixed to the skull with jewellery screws and dental acrylic cement. Seven days following implantation, the animals were subjected to behavioral tests. I.c.v. drug injections were delivered with a 10-pl Hamilton microsyringe connected to the 12.5 mm long stainless steel injector (0.3 mm external diameter) via polyethylene tubing. Drug solutions were administered 5 rain prior to testing in a volume of 5 gl over 60 s. The injection needle remained in place for an additional 30-60 s before it was removed and the stylet replaced.
Diazepam (Polfa, Poland) was suspended in 1% Tween 80. (+)-MK-801 hydrogen maleate (( + )-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-iminemaleate) (Research Biochemicals Inc., USA), CGP 37849 (DL-(E)-2-amino-4-methyl-5phosphono-3-pentenoic acid) and its carboxyethyl ester CGP 39551 (Ciba-Geigy, Switzerland), as well as AP-7 (DL-2 amino-7-phosphonoheptanoic acid, Tocris Neuramin, UK), were dissolved in distilled water. 5,7-Dichlorokynurenic acid (Tocris Neuramin, UK) was dissolved in dilute aqueous NaOH with pH adjusted to 6~7. Drug solutions were prepared immediately before administration (i.p., 2 ml/kg or 5 ~1 for intracerebral injections).
Histological analysis All animals were killed after the final testing day. The brains of operated rats were removed, stored in 5% formaldehyde solution and verified histologically. The frozen tissue was dissected in the slices and the place of injection examined with a magnifying glass. Only those animals with verified
507 CGP 37849 - OPEN FIELD TEST
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i.c.v, injection sites were included in the study.
Statistical analysis The data are shown as means +_SEM. Statistical analysis was performed using one-way ANOVA, followed by Student's t-test for independent samples. The confidence limit of P<0.05 was considered as statistically significant.
Results
Open field data Diazepam in a dose of 0.05 mg/kg, but not 0.1 mg/kg, significantly increased the number of entries into the central part of the open field (t=3.06, df 14, P<0.01), as well as time spent in the central sector of this arena (t=3.79, df 14, P<0.01) (Fig. 1). MK-801 at a dose of 0.1 mg/kg stimulated rat exploratory activity in the central sectors of the open field (entries, t=2.71, df 16, P<0.05; time, t=2.32, df 16, P<0.05) (Fig. 2); however, these rats tended also to have higher
levels of locomotor activity. MK-801 injected in a dose of 0.2 mg/kg significantly increased the animals' motility (t=5.0, df 14, P<0.01). CGP 37849 enhanced selectively in a limited range of doses (0.01, 0.1 and 1.0 mg/kg) rat behavior in the central parts of the open field (first experiment, time, F = 4.89, df 3,27, P < 0.01; second experiment, entries, F=3.54, df 3,28, P<0.05) (Fig. 3). At the largest dose examined (10 mg/kg) CGP 37849 significantly depressed motor behavior (t = 2.3, df 14, P<0.05) (Fig. 3). CGP 39551 administered in a wide dose range from 0.1 to 5.0 mg/kg did not affect rat behavior in the open field (motor activity, second experiment, F =0.66, df 3,28, P>0.05; entries, second experiment, F=1.58, df 3,28, P>0.05) (Fig. 4). 5,7-Dichlorokynurenic acid applied i.c.v. (0.5-5.0 ~tg) changed neither rat motor (F= 1.71, df 4,25, P>0.05) nor exploratory activity (entries, F = 1.67, df4,25, P>0.05) (Fig. 5), in a significant way. After the dose of 5 ~tg of 5,7dichlorokynurenic acid rats tended to have lower levels of locomotor activity. AP-7 administered centrally at a dose of 2.5 lag significantly increased
508 Central entries
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exploratory behavior of rats in the central part of the open field (entries, t =2.9, df 19, P<0.01; time, t=2.5, df 19, P<0.05) (Fig. 6). At a dose of 5.0 lag of AP-7, significant ataxia prevented accurate measurement of open field behavior, with all animals remaining in one place during the whole observation period.
Vogel's conflict test In the Vogel conflict test all examined drugs in some doses significantly increased the punished consumption of water (Figs. 1-6). The clear-cut effect appeared after administration of diazepam at 1.5 and 2.5 mg/kg (F= 4.25, df 3,28, P<0.05) (Fig. 1). MK-801 was active at doses of 0.005 and 0.01, but not 0.001 and 0.05 mg/kg (second experiment, F = 11.09, df 4,33, P<0.01) (Fig. 2). CGP 37849 had also disinhibitory effect in the Vogel conflict test (1.0 and 2.5 mg/kg) (F=4.81, df 4,33, P<0.01) (Fig. 3). CGP 39551 prevented the shock-induced suppression of drinking at 5 and 20 mg/kg only (F = 5.68, df 4,34, P<0.01) (Fig. 4). Both intraventricularly administered drugs, 5,7-dichlorokynure-
nic acid (F=6.89, df 4,29, P<0.01) and AP-7 (F=3.23, df 4,29, P<0.05), caused a marked increase in punished drinking after the doses of 5.0 lag and 0.5 lag, respectively (Figs. 5 and 6). Probably because of seasonal (the experiment was performed over several months) and betweengroup variabilities, the control groups differed in the amount of water consumption, but not in their reactivity to the shock (Fig. 1-6). In the control experiments all compounds failed to produce any significant effects on free (unpunished) drinking and the stimulus threshold at their lowest anti-conflict doses (Table 1).
Discussion
Diazepam significantly increased punished consumption of water in the Vogel test, and disinhibited rat reaction to a novel environment in the open field test. These effects of the benzodiazepine occurred independently of changes in motor activity as well as spontaneous water intake and
509 5,7-DICHLOROKYNURENIC ACID OPEN FIELD TEST - Activity
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Fig. 5. The influence of 5,7-dichlorokynurenic acid on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats in each experimental group was 6-8. For other explanations see Fig. 1.
pain threshold. The open field test appeared to be more sensitive to the benzodiazepine derivative, and helped to reveal the anxiolytic-like potency of diazepam, in doses as small as ~g/kg. Recently, anxiolytic-like effects of chlordiazepoxide and diazepam, given in a similar dose range in the open field test, were reported by us and other authors (Bruhwyler, 1990; Stefanski et al., 1992). In the present study we examined again the action of diazepam in the most potent and consistent doses obtained from our previous experiments (Stefanski et al., 1992). The examined N M D A receptor antagonists, irrespective of their site of action at the N M D A receptor complex, significantly affected rat emotional behavior. The results obtained in the Vogel test seem to be more consistent, since in contrast to the open field test all five N M D A antagonists selectively increased punished consumption of water. This finding confirms the data of other authors on the action of N M D A antagonists in conflict paradigms (Bennett and Amrick, 1986; Stephens et al., 1986; Kuribara et al., 1990;
McMillan et al., 1991; Xie and Commissaris, 1992; Wiley et al., 1992). MK-801, a noncompetitive N M D A receptor antagonist, disinhibited punished behavior in the Vogel test in a dose as small as 0.005 mg/kg. The drug administered in a dose of 0.03 mg/kg also increased conflict responding in the Cook and Davidson conditioned conflict paradigm (Corbett and Dunn, 1993). A slightly larger dose of 0.05 mg/kg of MK-801 was reported to induce hyperactivity, hyper-reactivity, reductions in rearing behavior and deficits in tongue extension (Hargreaves and Cain, 1992). The weaker effects of MK-801 observed in the present experiment in the open field may be explained by the omission in the experimental procedure of habituation to the test conditions. Habituation should have served to decrease rat spontaneous locomotion, thus revealing drug-induced stimulation of rat motility. Higher doses of MK-801 are usually necessary to increase rodent locomotion and rearing (Liljequist et al., 1991; Bubser et al., 1992). Accordingly, in the present experiment a potent enhancement of rat motor activity could be
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5 ~g i.c.v.
.5
2.5 ,ug i.c.v.
Fig. 6. The influence of AP-7 on rat behavior in the open field test and on punished water intake in the Vogel conflict test. The number of rats in each experimental group was 7 8. For other explanations see Fig. 1.
observed after the highest dose of 0.2 mg/kg. This coincided with a significant stimulation of rat behavior in the central parts of the open field. This interpretation (motor-related) of MK-801 open field data is compatible with other results obtained
with noncompetitive N M D A antagonists (Wedzony et al., 1993). Interestingly, CGP 37849 yielded selective and potent anxiolytic-like effects both in the open field and in the Vogel test, in a dose range similar to that of diazepam. After the highest dose
TABLE l THE I N F L U E N C E O F D I A Z E P A M , C G P 37849, C G P 39551, MK-801, AP-7 A N D 5 , 7 - D I C H L O R O K Y N U R E N I C ACID ON BASELINE D R I N K I N G A N D SHOCK T H R E S H O L D IN A N T I C O N F L I C T DOSES, ACTIVE IN THE V O G E L TEST. Drug
Dose
n
Spontaneous drinking (ml) (mean _+SEM)
n
Pain-flinch (mA) (mean 4- SEM)
Control/Tween Diazepam
1.5 mg/kg
5 5
5.98 4- 0.90 7.40 4- 0.20
7 7
0.37 4- 0. | 0 0.36 _+0.02
Control/H20 C G P 37849 C G P 39551 MK-801
1.0 mg/kg 5.0 mg/kg 0.005 mg/kg
9 7 7 7
5.83 _+0.45 6.23 + 0.60 7.34_+ 0.80 6.36 + 1.40
8 8 8 8
0.38 _+0.02 0.43 + 0.10 0.38 _+0.10 0.36 _+0.03
Control/H20 AP-7 5,7-Dichlorokynurenic acid
0.5 lag i.c.v./5 lal 5.0 lag i.c.v./5 lal
5 8 7
6.07 4- 1.10 6.25 _+0.60 5.20_+ 1.00
7 6 7
0.40_+ 0.02 0.43 _+0.02 0.44_+0.01
The data are shown as means_+ SEM. n, number of rats.
511 of 10.0 mg/kg of CGP 37849 a significant reduction in rat motor behavior was found, confirming other authors' data (Maj et al., 1992). The action of CGP 37849 was characterized by the largest difference between the dose changing rat motor activity and exploratory behavior in the central part of the open field (1000-fold). The same coefficients calculated for AP-7 and MK-801 were much smaller (2-fold). CGP 37849 and CGP 39551 are potent competitive N M D A receptor antagonists with oral activity (Schmutz et al., 1990). In contrast to MK-801, the carboxy-ethyl ester of CGP 37849, CGP 39551, did not increase the dopaminergic activity of the basal ganglia and even decreased the DOPAC/DA ratio in the frontal cortex (Bubser et al., 1992). In agreement with our findings, CGP 39551 was reported to differ from MK-801 and CGP 37849 both behaviorally and biochemically in that its effects were much less intense (Maj et al., 1992; L6scher et al., 1993). It has been proposed that the differences between competitive and noncompetitive N M D A receptor antagonists are due to the existence in the brain of binding sites for noncompetitive N M D A antagonists, that are not coupled to the N M D A binding site (Rao et al., 1990). However, these differences may not be important since local infusions of competitive N M D A receptor antagonists were reported to stimulate behavior, and after higher doses to induce ataxia (Schmidt, 1986; Tiedtke et al., 1990). Accordingly, intracerebroventricular (this report) or intrahippocampal (data in preparation) administration of the competitive N M D A antagonist AP-7 induced ataxia and potent locomotor stimulation, respectively. The effects of centrally injected 5,7-dichlorokynurenic acid, an antagonist at a glycine regulatory site on the N M D A receptor, were limited, observed after the highest dose tested and concerned rat behavior in the Vogel test only. Previously, it was found that strychnine insensitive glycine antagonists are active in non-conflict procedures with rodents (Trulas et al., 1989; Winslow et al., 1990; Kehne et al., 1991; Corbett and Dunn, 1993), but inactive in a conflict procedure with pigeons (Koek and Colpaert, 1991). Moreover, 1-amino cyclopropane carboxylic acid, a ligand at strychnine insensitive glycine receptors, was significantly less efficacious than chlordiazepoxide in an elevated plus-maze (Trulas et al., 1989). Also in the Cook and Davidson conditioned conflict paradigm the disinhibition of shock-induced suppression of conflict responding by 5,7-dichlorokynurenic acid
was not as robust as that observed with diazepam (Corbett and Dunn, 1993). Overall, these data indicate a weak anxiolytic-like profile of action of glycine antagonists. However, the fact that rats administered 5,7-dichlorokynurenic acid failed to generalize to a MK-801 discriminative stimulus cue indicates that antagonists at the strychnine insensitive glycine site could be devoid of psychotomimetic effects in man (Corbett and Dunn, 1993). The results in this study show that in two animal models of anxiety the behavioral profile of MK801, CGP 37849 and AP-7 appeared to be similar to that of the benzodiazepine. However, the motor effects of CGP 37849 and MK-801 and the atactic influence of AP-7 observed at the higher dose level indicate that these drugs may be less advantageous than benzodiazepines in the treatment of anxiety disorder. Though the N M D A receptor system contributes somewhat to the control of emotional processes, the effect seems to be less direct and consistent than was previously thought.
Acknowledgement The paper was supported by grant No. 17 from the Institute of Psychiatry and Neurology, Warsaw, Poland.
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