286
Brain Research, 533 (1990) 286-291 Elsevier
BRES 16054
Hemispheric asymmetry in behavioral response to D1 and agonists in the nucleus accumbens
D 2
receptor
Iren Belcheva 1, Joseph B. Bryer 1, Sergio E. Starkstein 1'3, Marc Honig 1, Timothy H. Moran 1 and Robert G. Robinson 1'2'3 1Departments of Psychiatry and Behavioral Sciences, 2Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) and 3Department of Psychiatry, University of lowa, Iowa City, IA 52242 (U.S.A.) (Accepted 5 June 1990) Key words: Dopamine; Locomotion; Nucleus accumbens; Asymmetry; Lateralization
The present study examined the locomotor response of rats to unilateral injections of the mixed D1/D2 agonist apomorphine, the D2 agonist quinpirole, and the D, agonist SKF 38393 into the left or right nucleus accumbens (NA) of male Sprague-Dawley rats. There were 2 main findings. First, unilateral (left or right) injections of apomorphine, quinpirole, or SKF 38393 all provoked locomotor hyperactivity. The second and more important finding was that, at specific dosages, apomorphine and SKF 38393 injections into the right NA produced significantly more locomotor hyperactivity than identical injections into the left NA. These findings suggest the presence of asymmetries in the NA which may involve quantitative differences in the distribution of D~ and D 2 receptors.
INTRODUCTION The role of the nucleus accumbens ( N A ) in locomotion in the rat has received considerable attention in recent years. Lesions of the N A , as well as local administration of drugs that affect transmitter activity, have been shown to influence l o c o m o t o r activity. For example, bilateral electrolytic or radio-frequency lesions of the N A result in spontaneous hyperactivity ~2"15 while bilateral d o p a m i n e ( D A ) lesions utilizing 6-hydroxydopamine produce hypoactivity 11'~3'~5'2~. The nucleus accumbens contains high densities of both dopamine 1 (D 0 and dopamine 2 (D2) receptors ~'2'9'1°'z° and bilateral N A injections of either the dopamine D1 agonist S K F 38393 (refs. 7, 9), or the D 2 agonist quinpirole (LY 171555) (refs. 4, 7) have been shown to induce dose-related increases in locomotor activity. We have recently d e m o n s t r a t e d an asymmetrical behavioral response to unilateral lesions in the N A of male S p r a g u e - D a w l e y rats z5'28. Right electrolytic N A lesions p r o d u c e d a 50% increase in spontaneous locomotor activity while identical left N A lesions did not significantly alter l o c o m o t o r activity. These results suggest that there may be hemispheric asymmetries involving the N A which play an important role in the neural circuits controlling locomotion.
The present experiments were designed to d e t e r m i n e whether a s y m m e t r y in the N A could also be demonstrated in response to the local administration of dopamine agonists. That is, does the behavioral response to unilateral administration of d o p a m i n e r g i c agonists into the N A d e p e n d upon the hemisphere of injection? To address this question, we e x a m i n e d the animals' activity following unilateral injections of the mixed D1/D 2 D A agonist a p o m o r p h i n e , the D1 agonist S K F 38393, and the D 2 agonist quinpirole into the right and left N A . Previous work using these dopaminergic agonists has involved either bilateral injections or injections in only one hemisphere 7.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats weighing 225-250 g at the time of surgery were housed individually in polypropylene boxes with free access to food and water, except during experimentation. The animals were maintained in a constant temperature environment of 21 + 2 °C with a regular 12 h/12 h light/dark day/night cycle. Stereotaxic implantation and drug admin&tration into the NA The surgical procedure was similar to that described by Crawley et al. 6. Animals were anesthetized with Nembutal, 50 mg/kg i.p., and placed in a stereotaxic apparatus with the incisor bar 5 mm
Correspondence: R.G. Robinson, Department of Psychiatry, University of Iowa, Iowa City, IA 52242, U,S.A. (~)6-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
287 above the interaural line. Burr holes were drilled left and right of midline at the following coordinates relative to bregma: anterior +3.4 ram; lateral + 1.8 mm. Stainless steel guide cannulae (length 16,0 ram, external diameter 0.559 ram, internal diameter 0.305 ram) were positioned vertically with their tips at a depth of 6.2 mm below the dura. Small brass screws were inserted in the skull and plastic tubing (length I cm and internal diameter 7 mm) was placed around the screws and cannulae and was fixed with dental acrylic. A stainless steel trocar which extended 1 mm beyond the tip of the cannula was inserted into the guide cannula. After surgery the animals were allowed 7-10 days to recover before beginning the behavioral studies. During the recovery period, the rats were handled daily. Three groups of rats were tested: Group 1 was given apomorphine; Group 2 was given SKF 38393 and Group 3 was given quinpirole. All compounds were injected into the NA. Apomorphine hydrochloride (Sigma) was dissolved in distilled water containing 0.2 mg/mi ascorbic acid as an antioxidant; R(+)SKF 38393 [hydrochloride 1, 2, 3, 4, 5-tetrahydro7, 8-dihydroxy-l-phenyl-lH-3-benzazepine HCL] (RBI, U.S.A.) and quinpirole hydrochloride [trans (-)-4aR-4, 4a, 5, 6, 7, 8, 8a, 9-octahydro-5-propyl-lH-pyrazolo [3,4-g] quinoline LY171555] (Eli Lilly, U.S.A.), were both dissolved in distilled water. The concentration of drug was varied for each dose so that a constant volume (1/zl) would be injected at each dose. After removing the trocar, the injection cannula was inserted into the implanted guide cannula. All drugs were injected through an injection cannula connected by polyethylene tubing with a constant rate microsyringe (Hamilton, Reno, NV). O n e / A of the drug solution was then injected over a period of 1 min and the injection cannula was left in place for an additional 30 s. Apomorphine, SKF 38393 and quinpirole were injected into the NA in dosages of 0.32, 1.0, 3.2 and 10/~g. Rats from each group were injected unilaterally in the NA every other day with randomly selected dosages of drug or 1 /zl sterile saline (0.9%) until each rat received 5 injections in the left NA and 5 in the right NA. Drug dose and injection side were individually randomized for each rat. Immediately prior to killing, the animals were injected with 1/~l of 2% Fastgreen dye through the injection cannula. Injection sites were then verified histologically postmortem in 25/zm coronal brain sections cut through the NA by a rater blind to the behavioral results. Animals were excluded if either cannula placement was outside the borders of the NA as depicted in Paxinos and Watson ~9. This resulted in exclusion of 11 of 20 rats in the apomorphine experiment, 12 of 20 in the SKF 38393 experiment and 9 of 20 in the quinpirole experiment.
RESULTS
Apomorphine injections Injections of apomorphine
i n t o t h e r i g h t o r left N A
p r o d u c e d s i g n i f i c a n t i n c r e a s e s in a c t i v i t y o n a v a r i e t y o f m e a s u r e s in a d o s e - d e p e n d e n t
fashion with the magni-
tude of response and shape of the dose-response
rela-
t i o n s h i p s d i f f e r i n g f o r r i g h t a n d left N A i n j e c t i o n s . T h e analysis of the effects of apomorphine
on total distance
APOMORPHINE Left NA Right NA
°" 0 700.
~.32
~io
312 ~o'.o
tw
o
t~
Behavioral study Locomotion was recorded in computerized Digiscan activity monitors (Omnitech Electronics, Columbus, OH). Monitors consisted of a clear plexiglass cage measuring 40 x 40 x 30.5 cm with infrared horizontal sensors, mounted every 5 cm along the perimeter, and vertical sensors mounted 10 cm above the floor. Data were collected by a Digiscan computer connected to an Apple IIe computer for data storage and subsequent analysis. Behavioral variables were recorded during 2-h test sessions at 20-rain sampling intervals. Variables measured included total distance, movement time, number of horizontal movements, mean distance per movement, mean speed of movements, and number of stereotypic movements. The stereotypic movement variable is sensitive to, but not specific for, stereotypic movements. Behavioral testing was carried out in the light cycle with water but not food present. Before the rats received an injection of drug or placebo, they were placed individually in the Omnitech cages for a 5-min period of habituation.
Statistical analysis Data were analyzed by a 2-factor repeated measures analysis of variance (ANOVA). The factors were drug dosage (saline and 4 drug levels) and side of injection (right or left). Findings from the ANOVA were further analyzed using planned comparison t-tests where appropriate. All P-values are 2-tailed.
o.32 1~o 312 lo.o DOSE (~0 in 1 ~J)
Fig. 1. The effects of apomorphine on total distance, average distance per movement and average speed of movement, which were variables for which there was a significant effect of side of injection. There were significant effects for the side of injection (F1.32 = 8.11, P = 0.02) and dose (F4,32 = 9.34, P < 0.001) on total distance, as well as a significant side-dose interaction (F4.a2= 5.76, P < 0.001). There were also significant effects of side and dose of apomorphine injection on average distance per movement (side: F1.a = 6.37, P = 0.036; dose: F4.a~ = 8.09, P < 0.0001; side x dose: F4,32 = 5.72, P = 0.0014) and average speed of movement (side: F1,a = 8.56, P = 0.019, dose: F4.a~ = 9.54, P < 0.0001, side x dose: F4,32 = 4,37, P = 0.0062). Asterisks depict t-comparisons of locomotor activity following left vs. right injections: *P < 0.05; **P < 0.0001.
288 revealed that left N A injections produced a linearly increasing dose-response relationship, while low dosages of apomorphine into the right N A produced greater levels of activity than higher dosages. Planned t-test comparisons demonstrated that injections of apomorphine into the N A produced significantly greater total distance when injected into the right N A as compared with the left N A at doses of 0.32 ~g (t = 5.34, df = 32, P < 0.0001) and 1.0 pg (t = 2.51, df = 32, P < 0.05) (Fig. 1). There were similar significant effects of apomorphine dose and side of injection on the behavioral variables of average distance per movement and average speed of movement. After right N A injections of 0.32/~g (t =
SKF38393
SKF 38393 injections
:i
----o---
0 ~
(J~e z
Brain Research, 533 (1990) 286-291 Elsevier
BRES 16054
Hemispheric asymmetry in behavioral response to D1 and agonists in the nucleus accumbens
D 2
receptor
Iren Belcheva 1, Joseph B. Bryer 1, Sergio E. Starkstein 1'3, Marc Honig 1, Timothy H. Moran 1 and Robert G. Robinson 1'2'3 1Departments of Psychiatry and Behavioral Sciences, 2Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) and 3Department of Psychiatry, University of lowa, Iowa City, IA 52242 (U.S.A.) (Accepted 5 June 1990) Key words: Dopamine; Locomotion; Nucleus accumbens; Asymmetry; Lateralization
The present study examined the locomotor response of rats to unilateral injections of the mixed D1/D2 agonist apomorphine, the D2 agonist quinpirole, and the D, agonist SKF 38393 into the left or right nucleus accumbens (NA) of male Sprague-Dawley rats. There were 2 main findings. First, unilateral (left or right) injections of apomorphine, quinpirole, or SKF 38393 all provoked locomotor hyperactivity. The second and more important finding was that, at specific dosages, apomorphine and SKF 38393 injections into the right NA produced significantly more locomotor hyperactivity than identical injections into the left NA. These findings suggest the presence of asymmetries in the NA which may involve quantitative differences in the distribution of D~ and D 2 receptors.
INTRODUCTION The role of the nucleus accumbens ( N A ) in locomotion in the rat has received considerable attention in recent years. Lesions of the N A , as well as local administration of drugs that affect transmitter activity, have been shown to influence l o c o m o t o r activity. For example, bilateral electrolytic or radio-frequency lesions of the N A result in spontaneous hyperactivity ~2"15 while bilateral d o p a m i n e ( D A ) lesions utilizing 6-hydroxydopamine produce hypoactivity 11'~3'~5'2~. The nucleus accumbens contains high densities of both dopamine 1 (D 0 and dopamine 2 (D2) receptors ~'2'9'1°'z° and bilateral N A injections of either the dopamine D1 agonist S K F 38393 (refs. 7, 9), or the D 2 agonist quinpirole (LY 171555) (refs. 4, 7) have been shown to induce dose-related increases in locomotor activity. We have recently d e m o n s t r a t e d an asymmetrical behavioral response to unilateral lesions in the N A of male S p r a g u e - D a w l e y rats z5'28. Right electrolytic N A lesions p r o d u c e d a 50% increase in spontaneous locomotor activity while identical left N A lesions did not significantly alter l o c o m o t o r activity. These results suggest that there may be hemispheric asymmetries involving the N A which play an important role in the neural circuits controlling locomotion.
The present experiments were designed to d e t e r m i n e whether a s y m m e t r y in the N A could also be demonstrated in response to the local administration of dopamine agonists. That is, does the behavioral response to unilateral administration of d o p a m i n e r g i c agonists into the N A d e p e n d upon the hemisphere of injection? To address this question, we e x a m i n e d the animals' activity following unilateral injections of the mixed D1/D 2 D A agonist a p o m o r p h i n e , the D1 agonist S K F 38393, and the D 2 agonist quinpirole into the right and left N A . Previous work using these dopaminergic agonists has involved either bilateral injections or injections in only one hemisphere 7.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats weighing 225-250 g at the time of surgery were housed individually in polypropylene boxes with free access to food and water, except during experimentation. The animals were maintained in a constant temperature environment of 21 + 2 °C with a regular 12 h/12 h light/dark day/night cycle. Stereotaxic implantation and drug admin&tration into the NA The surgical procedure was similar to that described by Crawley et al. 6. Animals were anesthetized with Nembutal, 50 mg/kg i.p., and placed in a stereotaxic apparatus with the incisor bar 5 mm
Correspondence: R.G. Robinson, Department of Psychiatry, University of Iowa, Iowa City, IA 52242, U,S.A. (~)6-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
287 above the interaural line. Burr holes were drilled left and right of midline at the following coordinates relative to bregma: anterior +3.4 ram; lateral + 1.8 mm. Stainless steel guide cannulae (length 16,0 ram, external diameter 0.559 ram, internal diameter 0.305 ram) were positioned vertically with their tips at a depth of 6.2 mm below the dura. Small brass screws were inserted in the skull and plastic tubing (length I cm and internal diameter 7 mm) was placed around the screws and cannulae and was fixed with dental acrylic. A stainless steel trocar which extended 1 mm beyond the tip of the cannula was inserted into the guide cannula. After surgery the animals were allowed 7-10 days to recover before beginning the behavioral studies. During the recovery period, the rats were handled daily. Three groups of rats were tested: Group 1 was given apomorphine; Group 2 was given SKF 38393 and Group 3 was given quinpirole. All compounds were injected into the NA. Apomorphine hydrochloride (Sigma) was dissolved in distilled water containing 0.2 mg/mi ascorbic acid as an antioxidant; R(+)SKF 38393 [hydrochloride 1, 2, 3, 4, 5-tetrahydro7, 8-dihydroxy-l-phenyl-lH-3-benzazepine HCL] (RBI, U.S.A.) and quinpirole hydrochloride [trans (-)-4aR-4, 4a, 5, 6, 7, 8, 8a, 9-octahydro-5-propyl-lH-pyrazolo [3,4-g] quinoline LY171555] (Eli Lilly, U.S.A.), were both dissolved in distilled water. The concentration of drug was varied for each dose so that a constant volume (1/zl) would be injected at each dose. After removing the trocar, the injection cannula was inserted into the implanted guide cannula. All drugs were injected through an injection cannula connected by polyethylene tubing with a constant rate microsyringe (Hamilton, Reno, NV). O n e / A of the drug solution was then injected over a period of 1 min and the injection cannula was left in place for an additional 30 s. Apomorphine, SKF 38393 and quinpirole were injected into the NA in dosages of 0.32, 1.0, 3.2 and 10/~g. Rats from each group were injected unilaterally in the NA every other day with randomly selected dosages of drug or 1 /zl sterile saline (0.9%) until each rat received 5 injections in the left NA and 5 in the right NA. Drug dose and injection side were individually randomized for each rat. Immediately prior to killing, the animals were injected with 1/~l of 2% Fastgreen dye through the injection cannula. Injection sites were then verified histologically postmortem in 25/zm coronal brain sections cut through the NA by a rater blind to the behavioral results. Animals were excluded if either cannula placement was outside the borders of the NA as depicted in Paxinos and Watson ~9. This resulted in exclusion of 11 of 20 rats in the apomorphine experiment, 12 of 20 in the SKF 38393 experiment and 9 of 20 in the quinpirole experiment.
RESULTS
Apomorphine injections Injections of apomorphine
i n t o t h e r i g h t o r left N A
p r o d u c e d s i g n i f i c a n t i n c r e a s e s in a c t i v i t y o n a v a r i e t y o f m e a s u r e s in a d o s e - d e p e n d e n t
fashion with the magni-
tude of response and shape of the dose-response
rela-
t i o n s h i p s d i f f e r i n g f o r r i g h t a n d left N A i n j e c t i o n s . T h e analysis of the effects of apomorphine
on total distance
APOMORPHINE Left NA Right NA
°" 0 700.
~.32
~io
312 ~o'.o
tw
o
t~
Behavioral study Locomotion was recorded in computerized Digiscan activity monitors (Omnitech Electronics, Columbus, OH). Monitors consisted of a clear plexiglass cage measuring 40 x 40 x 30.5 cm with infrared horizontal sensors, mounted every 5 cm along the perimeter, and vertical sensors mounted 10 cm above the floor. Data were collected by a Digiscan computer connected to an Apple IIe computer for data storage and subsequent analysis. Behavioral variables were recorded during 2-h test sessions at 20-rain sampling intervals. Variables measured included total distance, movement time, number of horizontal movements, mean distance per movement, mean speed of movements, and number of stereotypic movements. The stereotypic movement variable is sensitive to, but not specific for, stereotypic movements. Behavioral testing was carried out in the light cycle with water but not food present. Before the rats received an injection of drug or placebo, they were placed individually in the Omnitech cages for a 5-min period of habituation.
Statistical analysis Data were analyzed by a 2-factor repeated measures analysis of variance (ANOVA). The factors were drug dosage (saline and 4 drug levels) and side of injection (right or left). Findings from the ANOVA were further analyzed using planned comparison t-tests where appropriate. All P-values are 2-tailed.
o.32 1~o 312 lo.o DOSE (~0 in 1 ~J)
Fig. 1. The effects of apomorphine on total distance, average distance per movement and average speed of movement, which were variables for which there was a significant effect of side of injection. There were significant effects for the side of injection (F1.32 = 8.11, P = 0.02) and dose (F4,32 = 9.34, P < 0.001) on total distance, as well as a significant side-dose interaction (F4.a2= 5.76, P < 0.001). There were also significant effects of side and dose of apomorphine injection on average distance per movement (side: F1.a = 6.37, P = 0.036; dose: F4.a~ = 8.09, P < 0.0001; side x dose: F4,32 = 5.72, P = 0.0014) and average speed of movement (side: F1,a = 8.56, P = 0.019, dose: F4.a~ = 9.54, P < 0.0001, side x dose: F4,32 = 4,37, P = 0.0062). Asterisks depict t-comparisons of locomotor activity following left vs. right injections: *P < 0.05; **P < 0.0001.
288 revealed that left N A injections produced a linearly increasing dose-response relationship, while low dosages of apomorphine into the right N A produced greater levels of activity than higher dosages. Planned t-test comparisons demonstrated that injections of apomorphine into the N A produced significantly greater total distance when injected into the right N A as compared with the left N A at doses of 0.32 ~g (t = 5.34, df = 32, P < 0.0001) and 1.0 pg (t = 2.51, df = 32, P < 0.05) (Fig. 1). There were similar significant effects of apomorphine dose and side of injection on the behavioral variables of average distance per movement and average speed of movement. After right N A injections of 0.32/~g (t =
SKF38393
SKF 38393 injections
:i
----o---
0 ~
(J~e z
