Neuropeptides (1991) 19,77-90 0 Longman Group UK Ltd 1991
The Effects of Intraperitoneally Administered Phencyclidine on the Central Nervous System: Behavioral and’Neurochemical Studies Q. YANG*,t,
T. MOROJI”,
Y. TAKAMATSU”,
Y. HAGINO”
and M. OKUWA*,S
*Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156 Japan; tDepartment of Psychiatry, West China University of Medical Science, Chengdu, Sichuan, China; *New Product Development Department, Shionogi & Company Ltd, Osaka, Japan (Reprint requests to TM)
Abstract-The effects of intraperitoneally (IP) injected phencyclidine (phencyclohexyl piperidine; PCP) on the metabolism of dopamine (DA) and cholecystokinin-like immunoreactivity (CCK-LI) in the rat brain were investigated in connection with PCP-induced behavioral changes. The predominant behavior change elicited by 2.5 mg/kg PCP was locomotion, while with higher doses (5 and 10 mg/kg) sniffing, swaying and falling were observed in addition to the enhanced locomotor activity. Backpedaling and rotation were observed in 10 mg/kg PCP-treated rats. IP injection of PCP caused a dose-related increase in the levels of DA and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the medial frontal cortex (MFC) and anterior cingulate cortex (ant.CC) without any changes in the nucleus accumbens (NAc) or striatum. CCK-LI in the MFC, ant.CC and NAc was decreased in a dose-dependent manner following IP injection of PCP. These findings support the evidence that PCP selectively activates the mesocortical DA systems. Furthermore, our results indicate a functional relationship between the mesocortical DA neurons and intrinsic CCK containing cortical neurons, and the change in the activity of the intrinsic CCK-containing cortical neurons in these two areas, perhaps due to an alteration in DA transmission, might be involved in behavioral changes after PCP injection.
Introduction
tory or circulatory depression. Although it is no longer in clinical use (1) because it induces agitation, bizarre behavior and hallucination in about 30% of the patients (2), it is currently the most popular abused drug in the United State (3). Luby et al (4) and Davies and Beech (5) observed a consistent syndrome of neurological and psychological changes in normal subjects intravenously
Phencyclidine (phencyclohexyl piperidine; PCP) was originally developed as an intravenous anesthetic that induces analgesia without respira-
Date received 23 October 1990 Date accepted 22 January 1991
77
NEUROPEPTIDES
78
given 0.075-0.1 mg/kg of PCP. The neurological changes include nystagmus, ataxia, and diminished pain, touch and position sense. The first psychological symptom after drug administration is a distortion of body image, followed by feelings disorganization of thought of estrangement, including tangentiality and blocking, hostility and negativismus, and apathy. Furthermore, PCP exacerbated schizophrenic symptoms and some PCP abusers developed schizophrenic form behaviors after chronic use (6). The similarity between schizophrenia and PCP-induced psychosis suggests that PCP is a better drug model of psychosis than the currently used amphetamine-related drugs (4-7). This similarity also suggests that PCP interacts with an endogenous system involved in the pathogenesis of schizophrenia. Thus, the study on the mechanism of central action of PCP will help to explore pathogenic factors and mechanism of schizophrenia. PCP causes diverse behavioral and neurochemical changes in animals. Low doses of PCP increase locomotor activity (8-9), and interfere with cognitive processes such as memory of previously learned responses (10-12). High doses produce ataxia (13-14), disruption of motor reflexes (II), and stereotyped behaviours consisting of head weaving, rearing, nondirected mouth movements and circling (14-18). The PCP-induced hyperactivity and stereotypy can be blocked by neuroleptic dopaminne (DA) receptor blockers (17-20), suggesting that PCP-induced behavioral changes are mediated by central dopaminergic mechanisms. Recently, systemic administration of PCP has been demonstrated to cause a marked increased in the levels of DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) , in the mesocortical and mesolimbic regions without significant changes in the nigrostriatal pathway (21-24). This suggests that PCP preferentially enhances DA releases in these regions. Previously, we have reported that the DA innervations to the medial frontal cortex (MFC) and anterior cingulate cortex (ant.CC) may be functionally related to the intrinsic cholecystokinin (CCK)-containing cortical neurons, and that CCK containing neurons within these two cortical areas may play an important role in the behavioral sensitization induced by chronic administration of
methamphetamine (25). Thus, the present study investigated effects of PCP on central DA systems and CCK concentrations in the rat brain in connection with PCP-induced behavioral changes. Materials and Methods Male Wistar rats weighing 200-250g were used throughout the experiments. The animals were housed in groups of two rats per cage in a temperature controlled room (22 + 2°C) with a 12/12h light/dark cycle (lights on at 08.00 a.m.) with free access to commercial food and water. Behavioral measurements a) Locomotor activity. The locomotor
activity was measured using a behavioral tracing analyzer (Model BTA-1: Muromachi Kikai Co., Tokyo, Japan), a photocell apparatus consisting of X-Y and Z sensor units, as described previously (26). The X-Y position detected by the BTA-1 indicates the center of the animal body. In addition, a movement of more than 2cm of the X-Y position during a 300ms period is defined as ambulation. Thus, locomotor activity is represented by the distance of the animal movement. Rearing is detected by the beams placed within the Z unit. The status of all the beams within the X-Y and Z units was stored in a computer (PC-9801 VX, NEC Co., Tokyo, Japan) every 300ms during sampling. In the present study, the animals was placed individually in the test cage for a 60 min habituation period. Then, the locomotor activity was determined for 60 min after intraperitoneal (IP) injection of PCP to the rat. Each test session was 10 min. b) Other behavioral
changes
induced
by PCP.
PCP-induced behavioral changes, such as sniffing, licking, gnawing, grooming, swaying and falling, were assessed using the method as described by Koek et al (27). Each rat was sequentially observed for 3s per 10 min test session and the occurrence of any of the aforementioned behaviors was recorded. Drug
PCP was provided from Shionogi Research Laboratories (Osaka, Japan). PCP was dissolved
INTRAPERITONEALLY
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in physiological saline and freshly prepared just before use. Each animal was allowed 60 min to become acclimatized to the test cage. Then, saline or PCP (2.5,5.0,10 mg/kg) was IP injected to each animal.
Biochemical assays
The rat was sacrificed by microwave irradiation immediately after behavioral observation. The brain was then quickly removed from the skull and stored at -80°C until assay for DA, DOPAC, CCK-like immunoreactivity (CCK-LI). Each brain was dissected into 4 discrete brain regions, such as the MFC, ant.CC, nucleus accumbens (NAc) and striatum (STR), according to the method as described Marley et al (28).
a) Dopamine and 3,4-dihydroxyphenylacetic
acid.
DA and DOPAC were determined using high performance liquid chromatography (HPLC) with an electrochemical detector. The method for the measurement of DA and DOPAC was described in detail previously (26). In brief, dissected brain tissues were weighed and homogenized with an ultrasonic homogenizer (Branson Sonic Power Co., Conn., USA) in 2ml of ice-cold 0.4N perchloric acid (PCA) containing 0.01 mM EDTA, 20ng 3,4_dihydroxybenzylamine (DHBA). After the homogenates were centrifuged at 13000 rpm using a refrigerated centrifuge for 20 min at 4°C the supernatant was transferred to another tube and adjusted to pH 8.5 with 0.4M K&03. The mixture was centrifuged at 3000 rpm for 10 min at 4°C. All of the supernatants were passed through glass columns packed with 1Omg aluminum oxide conditioned by the method of Anton and Sayre. The catechols were eluted with 0.5ml of 0.6N HCl. A 50~1 portion of the elute was used for the determination of DA and DOPAC. The mobile phase for the separation of DA, DOPAC and DHBA (internal standard) consisted of 0.05M KH2P04, 17% MeOH, 2mM CH3(CH&,S03Na and O.OlnM EDTA (pH 3.6). The column temperature was maintained at 27°C. The flow rate was 0.6 mh’min. The overall recoveries of DA and DOPAC were 98.74 + 0.83% and 96.20 + 10.72%, respectively.
NERVOUS
SYSTEM
b) EIA procedure for CCK-LI
79 immunoreactivity.
Synthetic CCK-8 and non-sulfated CCK-8 were purchased from the Peptide Research Foundation (Mino, Japan). Rabbit antiserum against non-sulfated CCK-8 (first antibody) was kindly supplied by Dr T. Higuchi (Saitama Medical College, Saitama, Japan). The EIA procedure for CCK-LI was fundamentally the same as that described by Arai et al (29). Briefly, after dissected brain tissues were homogenized by an ultrasonic homogenizer in 2 ml of 0.1 N acetic acid. the homogenates were boiled for 10 min and then centrifuged at 3000 rpm for 20 min (at 4°C). The supernatants were transferred to another tube and centrifuged again. Aliquots of the supernatants were lyophilized and reconstituted in the assay buffer (as described below) for EIA. The well was coated with 250~1 of carbonate-bicarbonate buffer (pH 9.6) containing 5 kg/ml of second antibody. After overnight incubation at 4”C, each well was washed 5 times with wasing buffer (0.02M phosphate-buffered saline pH 7.4 with 0.05% Tween 20). Then, lOOl~,lassay buffer (0.14M phosphate buffer pH 7.4 containing 25mM EDTA, 0.5% bovine serum albumin and 0.02% Tween 20), 5011.1standard or unknown sample prepared in the assay buffer and 50 ~1 first antibody (1:2500 diluted with the assay buffer) were added to each well. After overnight incubation at 4”C, 50~1 HRP-CCK-8 conjugate (the HRP conjugated with CCK-8 by a periodate oxidation method (1:400 diluted with assay buffer) was added and allowed to react at 4°C for 4 h. Each well was then washed 5 times with washing buffer and finally incubated with 250~1 of substrate (36.8mg 0-phenylendiamine and 50~1 of 3% Hz02 dissolved in 50ml of 0.02M citrate-phosphate buffer pH 5.2) at room temperature for 40 min. The reaction was stopped by adding 50~~1of 5N HzS04 to each well. The absorbance of the resulting chromogen was read in a Microplate Photometer (MTP-22. Corona Electric, Ibaragi, Japan) at 492nm. Each plate contained standard (B), enzyme blank (no first antibody, non-specific binding (NSB)), and wells without free antigen (Bo). After the substraction of NSB values, the results were expressed as the plot of logarithm of CCK-8 concentration against the B/B0 ratio.
80
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o---10
0 0
20---30
lo---20
PCP Smg/kg
S~llno (ns2S) PCP l.Smg/kg
30---40
(n=26)
m
PCP lOmg/kg
(n=26) (n=25)
40---so *
P < 0.05
**
P < 0.01
w*
P a 0.001
50---60
(mln)
Pig. 1 The effects of PCP injection (IP) on locomotor activity in rats. The mean distances of the animal movements at 10,20,30, 40,50 and 60 min for the immediately proceeding 10 min intervals following IP administration of physiological saline or PCP are shown. Each value represents the mean f S.E. The numbers in parentheses indicate the number of rats tested. Abbreviations: PCP; phencyclidine, IP; intraperitoneally.
c) Protein assay. Protein concentrations were determined by the method of Lowry et al (3) using bovine serum albumin as the standard. Statistical analysis
The data were analyzed by Student’s t-test (twotailed) or Fisher’s exact or Chi-square test (twotailed). A value less than 0.05 is considered to be statistically significant. Results Behavioral experiments a) Effects of IP administered PCP on locomotor activity. Figure 1 shows the mean distances of the
animal movement at 10,20,30,40,50 and 60 min for the immediately proceeding 10 min interval following IP administration of saline or PCP. PCP
increased locomotor activity in a dose dependent manner. The mean moving distances of the 5 an 10 mg/kg PCP-treated groups at all 10 min intervals were significantly greater than those of the 2.5 mg/kg PCP-treated and control groups. Moreover, there was a significant difference in the moving distances between the 5 and 10 mg/kg PCP-treated groups. However, the time course for maximal effect and decline in locomotor activity of the 5mg/kg PCP-treated rats were similar to those of the 10 mg/kg PCP-treated rats. b) Other behaviors induced by IP administration of PCP. Figure 2 shows the mean numbers of rearing
in all the groups for a 10 min test session during a 60 min observation period. When administered at a dose of 10 mg/kg, the time course for maximal effect and decline in rearing was similar to those
INTRAPERIT’ONEALLY
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81
ON THE CENTRAL NERVOUS SYSTEM
observed in locomotor activity. However, there was no dose-related increase or decrease in rearing, which suggest that rearing is not a predominant response to the dosage of PCP ranging from 2.5 to 10 mg/kg tested. Figure 3 shows the percentages of rats showing sniffing, swaying, falling, licking, grooming and gnawing during 60 min observation period in all the groups. The percentages of the 10 mg/kg PCP-treated rats showing sniffing, swaying and falling were greater than those of the controls, 2.5 mg/mg and 5.0 mg/kg PCP-treated rats. The percentages of rats showing licking during the last three test sessions were greater in the 10 mg/kg PCP-treated groups than in the other three groups. There were significant differences in the percentages of rats showing sniffing between the 5 mg/kg PCP-treated, 2.5 mg/kg PCP-treated and the con-
trol groups only during the first three test sessions. Neither grooming nor gnawing were observed in any of the groups. In addition to aforementioned stereotyped behaviors, two other behaviors such as backpedaling and rotation were observed following IP administration of PCP. The percentages of the rats showing backpedaling behavior in the 10 mg/kg PCP-treated group were 16, 20, 20, 12,8 and 0% in the six test sessions, whereas 3.8% of the 5 mg/kg PCP-treated rats exhibited backpedaling behavior only during the first test session. Backpedaling behavior was not observed in the 2.5 mg/kg PCP-treated and control groups during any test session. Rotating behavior was observed only in the 10 mg/kg PCP-treated rats during the first three test sessions and the percentages were 12,12 and 4%, respectively. Anesthesia and straub tail were not observed in the present study. Further-
kamu 80 -
70 .
=O-
T r*i
l-T
T
T
1*
T
Ilfil&dL r-*1
*
.,..+L
o---10
Fig. 2
1 o---20
0
sdm(n=2P
q
PCP25mwkabk26)
20---30
30---40
PcP8mm~h=26) m
PCPlGng/kg(n=20
eo---so
;.::j:
SO---e0
*
PeQw
**
P-O.01
Q
(mln)
Changes in PCP-induced rearing. The mean numbers of rearing in all the groups for a 10 min test session during a 60 min observation period are shown. Each value represents the mean + S.D. The numbers in parentheses indicate the number of rats tested.
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10
20
so
40
50
50
10
20
20
40
so
60
10
20
50
40
50
50
10
20
20
40
so
60(mh.)
(mln3
llcklng
10
20
so
-
&ILwh=25)
40
50
50 -PCP
2.6m~/kd(n=26)
-
PCP Smg/k2h=SI)
-
PCP lO~/k2(n=26)
Fig. 3 Changes in stereotyped behaviors elicited by PCP. The maximum percentages of rats exhibiting sniffing, licking, gnawing, grooming, swaying and falling during 30s per 10 min session are shown. Each value represents the mean calculated from the numbers of rats shown in parentheses.
more, it was difficult to recognize headdown according to the definition as described by Fray et al (31). Biochemical assay a) Effects of ZP adminstered PCP on DA and DOPAC concentrations in various discrete brain regions. Figures 4 and 5 show DA and DOPAC
concentrations in all the brain regions studied. IP injection of PCP to rats increased DA and DOPAC concentrations in the MFC and ant.CC, but not in the NAc and STR, in a dose-dependent manner, which suggest that PCP preferentially causes enhancement of DA turnover rate in these two brain regions. b) Validation of the EZA for CCK-LI. The antiserum against non-sulfated CCK-8 was used at
a final dilution of 1:2500. Figure 6 shows a typical standard curve demonstrating the inhibition of the HRP-conjugates to the solid-phase by soluble CCK-8. Using this antiserum, the assay sensitivity was 15 pg/well at a 80% B/B0 level. The NSB of the assay was 4.6 + 0.4%. The intra- and interassay coefficients of variation were 3.6-5.2 and 5.4-9.8%, respectively. When various dilutions of homogenates of the MFC were measured by the EIA, the inhibition curve was the same as that of synthetic CCK-8 (Fig. 6). c) Effects of IP administered PCP on CCK-LI in various discrete brain regions. Figure 7 shows
CCK-LI in all the brain regions studied. CCK-LI in the MFC, ant.CC and NAc were significantly decreased in the 5 and 10 mg/kg PCP-treated groups as compared to those in the corresponding
INTRAPERITONEALLY
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ON THE CENTRAL
brain regions of the 2.5 mg/kg PCP-treated rats and controls. There were no differences in the reduction in CCK-LI between the 5 and 10 mg/kg PCP-treated groups. Discussion PCP has been well known to induce a complex syndrome of behavior including locomotor activity, stereotyped behaviors and ataxia in rodents. Using a cumulative dosing procedure, Koek et al (27) demonstrated that PCP induces locomotion, rearing, sniffing, head down, swaying, falling, loss of righting and straub tail in a dose-related manner. The maximum drug effect on locomotion, rearing, sniffing, headdown and swaying occurred at low doses less than 10 mg/kg; falling, loss of righting and straub tail were induced at higher doses. In the present study, the predominant
NERVOUS
83
SYSTEM
behavioral change elicited by 2.5 mg/kg PCP was locomotion, while with higher doses (5 and 10 mg/kg) sniffing, swaying and falling were observed in addition to the enhanced locomotor activity. In addition, the animals receiving 10 mg/kg PCP displayed backpedaling and rotating. These behavioral profiles are in good agreement with previously reported results (14, 16, 18-19, 32-34). Consistent with earlier reports (14, l&32-34), the dose-related time course for aforementioned behaviors elicited by PCP was observed in the present study. However, with respect to the effect of PCP on rearing, varying results were reported (9, 33-34). Recently, Greenberg and Segal (34) have reported that while rearings were decreased in rats injected with the 1.25-5 mg/kg dose range of PCP (33), the incidence of rearing during a 210 min observation period was significantly increased at a dose of 5mg/kg which was the highest dose tested
r**i
WC
NAc
ant.CC (n=io)
0
Sailno
m
PCP P.Sm!Hku
PCP 5mg/kg (n=lO)
m
PCP lOmg/ke
belo) (n=lO)
str *
PCO.05
**
PCO.01
Fig. 4 The effects of IP injected PCP on DA concentrations in 4 discrete regions of the rat brain. Each value represents the mean f S.E. The numbers in parentheses indicate the number of rats tested. Abbreviations: DA; dopamine, MFC; medial frontal cortex, ant.CC; anterior cingulate cortex, NAc; nucleus accumbens.
84
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NAc
ant-CC
IJ
S8lino
a
PCP 2.5mWkg
PCP Smg/kg
(n=lO) (n=lO)
=
str
(n do)
PCP lOmg/kg
(n=lO)
*
PC 0.05
**
P
The Effects of Intraperitoneally Administered Phencyclidine on the Central Nervous System: Behavioral and’Neurochemical Studies Q. YANG*,t,
T. MOROJI”,
Y. TAKAMATSU”,
Y. HAGINO”
and M. OKUWA*,S
*Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156 Japan; tDepartment of Psychiatry, West China University of Medical Science, Chengdu, Sichuan, China; *New Product Development Department, Shionogi & Company Ltd, Osaka, Japan (Reprint requests to TM)
Abstract-The effects of intraperitoneally (IP) injected phencyclidine (phencyclohexyl piperidine; PCP) on the metabolism of dopamine (DA) and cholecystokinin-like immunoreactivity (CCK-LI) in the rat brain were investigated in connection with PCP-induced behavioral changes. The predominant behavior change elicited by 2.5 mg/kg PCP was locomotion, while with higher doses (5 and 10 mg/kg) sniffing, swaying and falling were observed in addition to the enhanced locomotor activity. Backpedaling and rotation were observed in 10 mg/kg PCP-treated rats. IP injection of PCP caused a dose-related increase in the levels of DA and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the medial frontal cortex (MFC) and anterior cingulate cortex (ant.CC) without any changes in the nucleus accumbens (NAc) or striatum. CCK-LI in the MFC, ant.CC and NAc was decreased in a dose-dependent manner following IP injection of PCP. These findings support the evidence that PCP selectively activates the mesocortical DA systems. Furthermore, our results indicate a functional relationship between the mesocortical DA neurons and intrinsic CCK containing cortical neurons, and the change in the activity of the intrinsic CCK-containing cortical neurons in these two areas, perhaps due to an alteration in DA transmission, might be involved in behavioral changes after PCP injection.
Introduction
tory or circulatory depression. Although it is no longer in clinical use (1) because it induces agitation, bizarre behavior and hallucination in about 30% of the patients (2), it is currently the most popular abused drug in the United State (3). Luby et al (4) and Davies and Beech (5) observed a consistent syndrome of neurological and psychological changes in normal subjects intravenously
Phencyclidine (phencyclohexyl piperidine; PCP) was originally developed as an intravenous anesthetic that induces analgesia without respira-
Date received 23 October 1990 Date accepted 22 January 1991
77
NEUROPEPTIDES
78
given 0.075-0.1 mg/kg of PCP. The neurological changes include nystagmus, ataxia, and diminished pain, touch and position sense. The first psychological symptom after drug administration is a distortion of body image, followed by feelings disorganization of thought of estrangement, including tangentiality and blocking, hostility and negativismus, and apathy. Furthermore, PCP exacerbated schizophrenic symptoms and some PCP abusers developed schizophrenic form behaviors after chronic use (6). The similarity between schizophrenia and PCP-induced psychosis suggests that PCP is a better drug model of psychosis than the currently used amphetamine-related drugs (4-7). This similarity also suggests that PCP interacts with an endogenous system involved in the pathogenesis of schizophrenia. Thus, the study on the mechanism of central action of PCP will help to explore pathogenic factors and mechanism of schizophrenia. PCP causes diverse behavioral and neurochemical changes in animals. Low doses of PCP increase locomotor activity (8-9), and interfere with cognitive processes such as memory of previously learned responses (10-12). High doses produce ataxia (13-14), disruption of motor reflexes (II), and stereotyped behaviours consisting of head weaving, rearing, nondirected mouth movements and circling (14-18). The PCP-induced hyperactivity and stereotypy can be blocked by neuroleptic dopaminne (DA) receptor blockers (17-20), suggesting that PCP-induced behavioral changes are mediated by central dopaminergic mechanisms. Recently, systemic administration of PCP has been demonstrated to cause a marked increased in the levels of DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) , in the mesocortical and mesolimbic regions without significant changes in the nigrostriatal pathway (21-24). This suggests that PCP preferentially enhances DA releases in these regions. Previously, we have reported that the DA innervations to the medial frontal cortex (MFC) and anterior cingulate cortex (ant.CC) may be functionally related to the intrinsic cholecystokinin (CCK)-containing cortical neurons, and that CCK containing neurons within these two cortical areas may play an important role in the behavioral sensitization induced by chronic administration of
methamphetamine (25). Thus, the present study investigated effects of PCP on central DA systems and CCK concentrations in the rat brain in connection with PCP-induced behavioral changes. Materials and Methods Male Wistar rats weighing 200-250g were used throughout the experiments. The animals were housed in groups of two rats per cage in a temperature controlled room (22 + 2°C) with a 12/12h light/dark cycle (lights on at 08.00 a.m.) with free access to commercial food and water. Behavioral measurements a) Locomotor activity. The locomotor
activity was measured using a behavioral tracing analyzer (Model BTA-1: Muromachi Kikai Co., Tokyo, Japan), a photocell apparatus consisting of X-Y and Z sensor units, as described previously (26). The X-Y position detected by the BTA-1 indicates the center of the animal body. In addition, a movement of more than 2cm of the X-Y position during a 300ms period is defined as ambulation. Thus, locomotor activity is represented by the distance of the animal movement. Rearing is detected by the beams placed within the Z unit. The status of all the beams within the X-Y and Z units was stored in a computer (PC-9801 VX, NEC Co., Tokyo, Japan) every 300ms during sampling. In the present study, the animals was placed individually in the test cage for a 60 min habituation period. Then, the locomotor activity was determined for 60 min after intraperitoneal (IP) injection of PCP to the rat. Each test session was 10 min. b) Other behavioral
changes
induced
by PCP.
PCP-induced behavioral changes, such as sniffing, licking, gnawing, grooming, swaying and falling, were assessed using the method as described by Koek et al (27). Each rat was sequentially observed for 3s per 10 min test session and the occurrence of any of the aforementioned behaviors was recorded. Drug
PCP was provided from Shionogi Research Laboratories (Osaka, Japan). PCP was dissolved
INTRAPERITONEALLY
ADMINISTERED
PHENCYCLIDINE
ON THE CENTRAL
in physiological saline and freshly prepared just before use. Each animal was allowed 60 min to become acclimatized to the test cage. Then, saline or PCP (2.5,5.0,10 mg/kg) was IP injected to each animal.
Biochemical assays
The rat was sacrificed by microwave irradiation immediately after behavioral observation. The brain was then quickly removed from the skull and stored at -80°C until assay for DA, DOPAC, CCK-like immunoreactivity (CCK-LI). Each brain was dissected into 4 discrete brain regions, such as the MFC, ant.CC, nucleus accumbens (NAc) and striatum (STR), according to the method as described Marley et al (28).
a) Dopamine and 3,4-dihydroxyphenylacetic
acid.
DA and DOPAC were determined using high performance liquid chromatography (HPLC) with an electrochemical detector. The method for the measurement of DA and DOPAC was described in detail previously (26). In brief, dissected brain tissues were weighed and homogenized with an ultrasonic homogenizer (Branson Sonic Power Co., Conn., USA) in 2ml of ice-cold 0.4N perchloric acid (PCA) containing 0.01 mM EDTA, 20ng 3,4_dihydroxybenzylamine (DHBA). After the homogenates were centrifuged at 13000 rpm using a refrigerated centrifuge for 20 min at 4°C the supernatant was transferred to another tube and adjusted to pH 8.5 with 0.4M K&03. The mixture was centrifuged at 3000 rpm for 10 min at 4°C. All of the supernatants were passed through glass columns packed with 1Omg aluminum oxide conditioned by the method of Anton and Sayre. The catechols were eluted with 0.5ml of 0.6N HCl. A 50~1 portion of the elute was used for the determination of DA and DOPAC. The mobile phase for the separation of DA, DOPAC and DHBA (internal standard) consisted of 0.05M KH2P04, 17% MeOH, 2mM CH3(CH&,S03Na and O.OlnM EDTA (pH 3.6). The column temperature was maintained at 27°C. The flow rate was 0.6 mh’min. The overall recoveries of DA and DOPAC were 98.74 + 0.83% and 96.20 + 10.72%, respectively.
NERVOUS
SYSTEM
b) EIA procedure for CCK-LI
79 immunoreactivity.
Synthetic CCK-8 and non-sulfated CCK-8 were purchased from the Peptide Research Foundation (Mino, Japan). Rabbit antiserum against non-sulfated CCK-8 (first antibody) was kindly supplied by Dr T. Higuchi (Saitama Medical College, Saitama, Japan). The EIA procedure for CCK-LI was fundamentally the same as that described by Arai et al (29). Briefly, after dissected brain tissues were homogenized by an ultrasonic homogenizer in 2 ml of 0.1 N acetic acid. the homogenates were boiled for 10 min and then centrifuged at 3000 rpm for 20 min (at 4°C). The supernatants were transferred to another tube and centrifuged again. Aliquots of the supernatants were lyophilized and reconstituted in the assay buffer (as described below) for EIA. The well was coated with 250~1 of carbonate-bicarbonate buffer (pH 9.6) containing 5 kg/ml of second antibody. After overnight incubation at 4”C, each well was washed 5 times with wasing buffer (0.02M phosphate-buffered saline pH 7.4 with 0.05% Tween 20). Then, lOOl~,lassay buffer (0.14M phosphate buffer pH 7.4 containing 25mM EDTA, 0.5% bovine serum albumin and 0.02% Tween 20), 5011.1standard or unknown sample prepared in the assay buffer and 50 ~1 first antibody (1:2500 diluted with the assay buffer) were added to each well. After overnight incubation at 4”C, 50~1 HRP-CCK-8 conjugate (the HRP conjugated with CCK-8 by a periodate oxidation method (1:400 diluted with assay buffer) was added and allowed to react at 4°C for 4 h. Each well was then washed 5 times with washing buffer and finally incubated with 250~1 of substrate (36.8mg 0-phenylendiamine and 50~1 of 3% Hz02 dissolved in 50ml of 0.02M citrate-phosphate buffer pH 5.2) at room temperature for 40 min. The reaction was stopped by adding 50~~1of 5N HzS04 to each well. The absorbance of the resulting chromogen was read in a Microplate Photometer (MTP-22. Corona Electric, Ibaragi, Japan) at 492nm. Each plate contained standard (B), enzyme blank (no first antibody, non-specific binding (NSB)), and wells without free antigen (Bo). After the substraction of NSB values, the results were expressed as the plot of logarithm of CCK-8 concentration against the B/B0 ratio.
80
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0 0
20---30
lo---20
PCP Smg/kg
S~llno (ns2S) PCP l.Smg/kg
30---40
(n=26)
m
PCP lOmg/kg
(n=26) (n=25)
40---so *
P < 0.05
**
P < 0.01
w*
P a 0.001
50---60
(mln)
Pig. 1 The effects of PCP injection (IP) on locomotor activity in rats. The mean distances of the animal movements at 10,20,30, 40,50 and 60 min for the immediately proceeding 10 min intervals following IP administration of physiological saline or PCP are shown. Each value represents the mean f S.E. The numbers in parentheses indicate the number of rats tested. Abbreviations: PCP; phencyclidine, IP; intraperitoneally.
c) Protein assay. Protein concentrations were determined by the method of Lowry et al (3) using bovine serum albumin as the standard. Statistical analysis
The data were analyzed by Student’s t-test (twotailed) or Fisher’s exact or Chi-square test (twotailed). A value less than 0.05 is considered to be statistically significant. Results Behavioral experiments a) Effects of IP administered PCP on locomotor activity. Figure 1 shows the mean distances of the
animal movement at 10,20,30,40,50 and 60 min for the immediately proceeding 10 min interval following IP administration of saline or PCP. PCP
increased locomotor activity in a dose dependent manner. The mean moving distances of the 5 an 10 mg/kg PCP-treated groups at all 10 min intervals were significantly greater than those of the 2.5 mg/kg PCP-treated and control groups. Moreover, there was a significant difference in the moving distances between the 5 and 10 mg/kg PCP-treated groups. However, the time course for maximal effect and decline in locomotor activity of the 5mg/kg PCP-treated rats were similar to those of the 10 mg/kg PCP-treated rats. b) Other behaviors induced by IP administration of PCP. Figure 2 shows the mean numbers of rearing
in all the groups for a 10 min test session during a 60 min observation period. When administered at a dose of 10 mg/kg, the time course for maximal effect and decline in rearing was similar to those
INTRAPERIT’ONEALLY
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observed in locomotor activity. However, there was no dose-related increase or decrease in rearing, which suggest that rearing is not a predominant response to the dosage of PCP ranging from 2.5 to 10 mg/kg tested. Figure 3 shows the percentages of rats showing sniffing, swaying, falling, licking, grooming and gnawing during 60 min observation period in all the groups. The percentages of the 10 mg/kg PCP-treated rats showing sniffing, swaying and falling were greater than those of the controls, 2.5 mg/mg and 5.0 mg/kg PCP-treated rats. The percentages of rats showing licking during the last three test sessions were greater in the 10 mg/kg PCP-treated groups than in the other three groups. There were significant differences in the percentages of rats showing sniffing between the 5 mg/kg PCP-treated, 2.5 mg/kg PCP-treated and the con-
trol groups only during the first three test sessions. Neither grooming nor gnawing were observed in any of the groups. In addition to aforementioned stereotyped behaviors, two other behaviors such as backpedaling and rotation were observed following IP administration of PCP. The percentages of the rats showing backpedaling behavior in the 10 mg/kg PCP-treated group were 16, 20, 20, 12,8 and 0% in the six test sessions, whereas 3.8% of the 5 mg/kg PCP-treated rats exhibited backpedaling behavior only during the first test session. Backpedaling behavior was not observed in the 2.5 mg/kg PCP-treated and control groups during any test session. Rotating behavior was observed only in the 10 mg/kg PCP-treated rats during the first three test sessions and the percentages were 12,12 and 4%, respectively. Anesthesia and straub tail were not observed in the present study. Further-
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Fig. 3 Changes in stereotyped behaviors elicited by PCP. The maximum percentages of rats exhibiting sniffing, licking, gnawing, grooming, swaying and falling during 30s per 10 min session are shown. Each value represents the mean calculated from the numbers of rats shown in parentheses.
more, it was difficult to recognize headdown according to the definition as described by Fray et al (31). Biochemical assay a) Effects of ZP adminstered PCP on DA and DOPAC concentrations in various discrete brain regions. Figures 4 and 5 show DA and DOPAC
concentrations in all the brain regions studied. IP injection of PCP to rats increased DA and DOPAC concentrations in the MFC and ant.CC, but not in the NAc and STR, in a dose-dependent manner, which suggest that PCP preferentially causes enhancement of DA turnover rate in these two brain regions. b) Validation of the EZA for CCK-LI. The antiserum against non-sulfated CCK-8 was used at
a final dilution of 1:2500. Figure 6 shows a typical standard curve demonstrating the inhibition of the HRP-conjugates to the solid-phase by soluble CCK-8. Using this antiserum, the assay sensitivity was 15 pg/well at a 80% B/B0 level. The NSB of the assay was 4.6 + 0.4%. The intra- and interassay coefficients of variation were 3.6-5.2 and 5.4-9.8%, respectively. When various dilutions of homogenates of the MFC were measured by the EIA, the inhibition curve was the same as that of synthetic CCK-8 (Fig. 6). c) Effects of IP administered PCP on CCK-LI in various discrete brain regions. Figure 7 shows
CCK-LI in all the brain regions studied. CCK-LI in the MFC, ant.CC and NAc were significantly decreased in the 5 and 10 mg/kg PCP-treated groups as compared to those in the corresponding
INTRAPERITONEALLY
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ON THE CENTRAL
brain regions of the 2.5 mg/kg PCP-treated rats and controls. There were no differences in the reduction in CCK-LI between the 5 and 10 mg/kg PCP-treated groups. Discussion PCP has been well known to induce a complex syndrome of behavior including locomotor activity, stereotyped behaviors and ataxia in rodents. Using a cumulative dosing procedure, Koek et al (27) demonstrated that PCP induces locomotion, rearing, sniffing, head down, swaying, falling, loss of righting and straub tail in a dose-related manner. The maximum drug effect on locomotion, rearing, sniffing, headdown and swaying occurred at low doses less than 10 mg/kg; falling, loss of righting and straub tail were induced at higher doses. In the present study, the predominant
NERVOUS
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SYSTEM
behavioral change elicited by 2.5 mg/kg PCP was locomotion, while with higher doses (5 and 10 mg/kg) sniffing, swaying and falling were observed in addition to the enhanced locomotor activity. In addition, the animals receiving 10 mg/kg PCP displayed backpedaling and rotating. These behavioral profiles are in good agreement with previously reported results (14, 16, 18-19, 32-34). Consistent with earlier reports (14, l&32-34), the dose-related time course for aforementioned behaviors elicited by PCP was observed in the present study. However, with respect to the effect of PCP on rearing, varying results were reported (9, 33-34). Recently, Greenberg and Segal (34) have reported that while rearings were decreased in rats injected with the 1.25-5 mg/kg dose range of PCP (33), the incidence of rearing during a 210 min observation period was significantly increased at a dose of 5mg/kg which was the highest dose tested
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Fig. 4 The effects of IP injected PCP on DA concentrations in 4 discrete regions of the rat brain. Each value represents the mean f S.E. The numbers in parentheses indicate the number of rats tested. Abbreviations: DA; dopamine, MFC; medial frontal cortex, ant.CC; anterior cingulate cortex, NAc; nucleus accumbens.
84
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