Behavioural Brahl Research, 49 (1992) 225-230 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92/$05.00
225
BBR01300
Short Communications
6-Hydroxydopamine lesions of the medial prefrontal cortex fail to influence cocaine-induced place conditioning S.E. Hereby ~, G.H. Jones 2, D.B. Neill a n d J.B. Justice Jr. Departments of Ps)vhology and Chemistry, Emory University, Atlanta, GA (USA) (Received 3 October 1991) (Revised version received 22 January 1992) (Accepted 13 March 1992)
Key n'ords: Medial prefrontal cortex; Dopamine; Place conditioning; Cocaine; 6-Hydroxydopamine; Reward; Neurochemistry; Rat
This study investigated the involvement of medial prefrontal cortex (mPFC) dopamine in cocaine place conditioning using a totally balanced place conditioning design. Presynaptic dopamine terminals of the mPFC were lesioned by bilaterally infusing the selective neurotoxin 6-hydroxydopamine (6-OHDA). These lesions significantly depleted dopamine ( - 8 3 % ) and .norepmephrme . . . ( - 7 0 % ) in the mPFC but there were no significant reductions in either the nucleus accumbens or in the caudate-putamen compared with sham-operated controls. Furthermore, serotonin levels were not affected in any of the brain regions investigated. These lesions failed to attenuate place conditioning induced by the intraperitoneal (i.p. 10 mg/kg) administration of cocaine when compared to sham lesioned controls. In addition, there were no significant differences in spontaneous locomotor activity between the two groups during the precondition!ng phase or the test phase. These results suggest that 6-OHDA lesions which produced profound depletions of dopamine and norcpinephrine in the mPFC did not alter the rewarding efficacy of cocaine as measured by the place conditioning paradigm.
The mesocorticolimbic dopamine pathway is considered to be a critical substrate for the incentivemotivational properties of rewarding stimuli v. This pathway originates in the ventral tegmental area (VTA) and projects to several forebrain regions including the olfactory tubercles, the ventral anterior striatum/ nucleus accumbens (NACC), and the medial prefrontal cortex (mPFC). Destruction of the cell bodies or of some terminal areas of this pathway have been reported to attenuate or abolish the rewarding properties of psychotropic compounds as well as a variety of natural reinforcers 25. The place conditioning and self-administration par-
Present address: Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Drive, Winston-Salem, NC 27103, USA. 2 Present address: Department ofNeuropsychopharmacology, Schering AG, Mallerstrasse 170-178, D-1000 Berlin 65, Germany. Correspondence: S.E. Hereby, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Dr., Winston-Salem, NC 27103, USA.
adigms have been the most widely used methods of assessing the rewarding efficacy of abused drugs. In addition, most drugs that induce place conditioning are also self-administered t. With respect to cocaine, both paradigms have revealed that central dopamine systems are important neural substrates for the mediation of cocaine reward 6,w'13,t9. Six-hydroxydopamine (6OHDA) lesions of the VTA or the NACC, either of which significantly deplete forebrain dopamine, particularly in the N A C C or m P F C 2~ attenuate intravenous (i.v.) cocaine self-administration xs'w. However, some subjects with near complete depletion of N A C C dopamine maintained near normal rates ofcocaine selfadministration 18"w implying that other forebrain dopamine terminal regions may be important. Although place conditioning induced by intraperitoneal (i.p.) cocaine is reportedly not affected by 6-OHDA lesions of the N A C C 22, the ability of neuroleptic pretreatment to attenuate place conditioning induced by i.v. 23 or intracerebroventrieular (i.c.v.) t3 infusions of cocaine implicates a central dopaminergic component. Since the demonstration that rats will self-administer
226 cocaine directly into the mPFC but not into the NACC 5, prefrontal dopamine terminals have been considered a critical substrate for cocaine reward 6. In agreement with the studies of intracranial self-administration of cocaine, microinfusions of cocaine into the NACC are not rewarding as measured by place preference conditioning 8. To our knowledge the effect of intra-prefrontal cocaine infusions on place conditioning has not been reported. Selective depletion of mPFC dopamine has been reported to block the responding for intraprefrontal cocaine in the self-administration paradigm 6, although responding for i.v. cocaine has been reported to either increase z~ or remain unaffected ~2. Aspirative ablations of the mPFC have been shown to block place conditioning induced by systemic cocaine ~~ However, complete removal of this cortical region eliminates preand post-synaptic elements as well as fibers en passant and therefore does not provide information as to the neurophysiological substrate for cocaine place conditioning. The present experiment was designed to determine if presynaptic dopamine terminals in the mPFC have a functional role in place conditioning induced by i.p. cocaine. Male Wistar rats (n = 26; Sasco/King, Inc.) weighing 275-325 g were housed three per cage in a light- and temperature-controlled environment. Food and water were available in the home cage ad libitum throughout the experiment. Subjects were on a 12 h light/dark cycle with lights on from 7 a.m. to 7 p.m. Behavioral testing was conducted during the light phase of the cycle. Subjects were randomly assigned to two groups to receive either sham (n = 9) or 6-OHDA lesions (n = 17) of the mPFC. Subjects were pretreated with pargylinehydrochloride (25 mg/kg, i.p.; Sigma) and desipraminehydrochloride (50 mg/kg, i.p.; Sigma) 45 min prior to being anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.) and placed in a Kopf stereotaxic frame. The lesion group received bilateral infusions of 6-OHDA-HBr (4 lag/lal/site) dissolved in a solution of 0 . 9 ~ saline containing 0.02~ ascorbic acid while the sham-lesioned group was infused with equivalent volumes of vehicle at each site. Infusion coordinates were: (1) A + 1.0 mm anterior to bregma, L 0.8 to midline, V - 2 . 0 m m to dura; (2) A + 2 . 4 , L0.8, V - 1 . 5 ; (3) A + 2.4, L 0.8, V - 4.0; (4) A + 4.0, L 0.8, V - 3.0 xS. Infusion cannulae, constructed of 30 ga. hypodermic tubing, were connected via polyethylene tubing to 10 lal Hamilton syringes mounted on an infusion pump (Sage Instruments, Cambridge, MA). Left and right coordinates were simultaneously infused over a 1-min period with an additional 2 min allowed for diffusion. Following surgery, subjects were allowed 10 days to recover before behavioral testing.
The day immediately preceeding the initial exposure to the place conditioning apparatus, spontaneous locomotor activity was measured for both groups. The locomotor apparatus was a clear rectangular plexiglas box ( 4 1 c m • 2 1 5 high) placed inside an Omnitech Digiscan optical activity monitor (Columbus, OH). The monitor was equipped with 8 optical sensors spaced 4.5 mm apart on two perpendicular sides. Locomotor activity counts required the breaking of two beams consecutively. Subjects were placed in the activity chamber for 15 min and data was recorded in 5-min increments on a microcomputer. The spontaneous locomotor activity scores were subjected to an analysis of variance (ANOVA) 24 with one repeated measure, Time. The place conditioning apparatus has been described previously8'22. Briefly, the apparatus consisted of two main conditioning compartments (35 cm x 25 cm) separated by a neutral compartment (10 cm x 25 cm). The neutral compartment, used as the point of introduction on the preconditioning and test days, had sheet aluminum flooring and gray walls and was separated from the main compartments by removable guillotine doors. One 0 f the main compartments had black walls and flooring of steel dowels (6.4 mm diameter) spaced 2.5 mm apart whereas the alternate compartment had white walls and metal grid flooring (6 m m spacing of 1 mm ~,vires). A 1.5~ acetic acid solution was wiped on the flooring and walls of the black compartment before each test to negate the unconditioned preference for the black compartment ~4. The procedure for assessing cocaine-induced place conditioning consisted of three distinct phases: preconditioning, conditioning, and test. In the preconditioning phase, each subject was placed in the neutral compartment, the guillotine doors were removed, and the subject was allowed free access to the entire apparatus for 15 min in order to assess unconditioned preferences. The amount of time spent in each compartment was recorded. Following the preconditioning phase, the 6-OHDAand the sham-lesioned groups were counterbalanced such that halfofeach group had cocaine paired with the initially preferred compartment while the other half received saline in the preferred compartment. Within each group, half of the subjects received cocaine on the second and fourth days and halfon the third and fifth days. Saline injections were administered on alternate days. Rats were injected with either cocaine-HCl (10 mg/kg, i.p.; NIDA) or saline and immediately placed in the appropriate compartment for 30 rain. On the~test dab', subjects were placed in the neutral compartment and the guillotine doors removed thus giving free access to
227 tic acid (DOPAC), homovanillic acid (HVA), 5hydroxyindoleacetic acid (5-HIAA), and serotonin (5HT) concentrations. Quantification was achieved by using standards of known concentration. The HPLC consisted of an Isco LC-5000 syringe pump, an Adsorbosphere column (4.6mm i.d.x 150 mm, 5 la C-18 ODS silica; Alltech), an amperometric detector (model LC-4B; Bioanalytical Systems), and a dual carbon working electrode (model MF-100; Bioanalytical Systems). The mobile phase consisted of 15 mM sodium acetate, 152 mM citric acid, 5.0 mM triethylamine, 1.98 mM sodium octyl sulfate, 0.1 mM EDTA, and 89/0 methanol with the pH adjusted to 3.6 il. The flow rate was 1.25 ml/min and the applied potential was + 700 mV against an Ag/AgCI reference electrode (model MW-2021; Bioanalytical Systems). The biochemical results were analyzed by Student's ttest. The results of 6-OHDA lesions of the mPFC on biogenic amine levels in the mPFC, NACC, anterior caudate-putamen, and posterior caudate-putamen are presented in Table I. These lesions significantly depleted dopamine (t = 8.92, P < 0.0001) to 17~o of control levels and norepinephrine (t= 8.88, P
225
BBR01300
Short Communications
6-Hydroxydopamine lesions of the medial prefrontal cortex fail to influence cocaine-induced place conditioning S.E. Hereby ~, G.H. Jones 2, D.B. Neill a n d J.B. Justice Jr. Departments of Ps)vhology and Chemistry, Emory University, Atlanta, GA (USA) (Received 3 October 1991) (Revised version received 22 January 1992) (Accepted 13 March 1992)
Key n'ords: Medial prefrontal cortex; Dopamine; Place conditioning; Cocaine; 6-Hydroxydopamine; Reward; Neurochemistry; Rat
This study investigated the involvement of medial prefrontal cortex (mPFC) dopamine in cocaine place conditioning using a totally balanced place conditioning design. Presynaptic dopamine terminals of the mPFC were lesioned by bilaterally infusing the selective neurotoxin 6-hydroxydopamine (6-OHDA). These lesions significantly depleted dopamine ( - 8 3 % ) and .norepmephrme . . . ( - 7 0 % ) in the mPFC but there were no significant reductions in either the nucleus accumbens or in the caudate-putamen compared with sham-operated controls. Furthermore, serotonin levels were not affected in any of the brain regions investigated. These lesions failed to attenuate place conditioning induced by the intraperitoneal (i.p. 10 mg/kg) administration of cocaine when compared to sham lesioned controls. In addition, there were no significant differences in spontaneous locomotor activity between the two groups during the precondition!ng phase or the test phase. These results suggest that 6-OHDA lesions which produced profound depletions of dopamine and norcpinephrine in the mPFC did not alter the rewarding efficacy of cocaine as measured by the place conditioning paradigm.
The mesocorticolimbic dopamine pathway is considered to be a critical substrate for the incentivemotivational properties of rewarding stimuli v. This pathway originates in the ventral tegmental area (VTA) and projects to several forebrain regions including the olfactory tubercles, the ventral anterior striatum/ nucleus accumbens (NACC), and the medial prefrontal cortex (mPFC). Destruction of the cell bodies or of some terminal areas of this pathway have been reported to attenuate or abolish the rewarding properties of psychotropic compounds as well as a variety of natural reinforcers 25. The place conditioning and self-administration par-
Present address: Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Drive, Winston-Salem, NC 27103, USA. 2 Present address: Department ofNeuropsychopharmacology, Schering AG, Mallerstrasse 170-178, D-1000 Berlin 65, Germany. Correspondence: S.E. Hereby, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Dr., Winston-Salem, NC 27103, USA.
adigms have been the most widely used methods of assessing the rewarding efficacy of abused drugs. In addition, most drugs that induce place conditioning are also self-administered t. With respect to cocaine, both paradigms have revealed that central dopamine systems are important neural substrates for the mediation of cocaine reward 6,w'13,t9. Six-hydroxydopamine (6OHDA) lesions of the VTA or the NACC, either of which significantly deplete forebrain dopamine, particularly in the N A C C or m P F C 2~ attenuate intravenous (i.v.) cocaine self-administration xs'w. However, some subjects with near complete depletion of N A C C dopamine maintained near normal rates ofcocaine selfadministration 18"w implying that other forebrain dopamine terminal regions may be important. Although place conditioning induced by intraperitoneal (i.p.) cocaine is reportedly not affected by 6-OHDA lesions of the N A C C 22, the ability of neuroleptic pretreatment to attenuate place conditioning induced by i.v. 23 or intracerebroventrieular (i.c.v.) t3 infusions of cocaine implicates a central dopaminergic component. Since the demonstration that rats will self-administer
226 cocaine directly into the mPFC but not into the NACC 5, prefrontal dopamine terminals have been considered a critical substrate for cocaine reward 6. In agreement with the studies of intracranial self-administration of cocaine, microinfusions of cocaine into the NACC are not rewarding as measured by place preference conditioning 8. To our knowledge the effect of intra-prefrontal cocaine infusions on place conditioning has not been reported. Selective depletion of mPFC dopamine has been reported to block the responding for intraprefrontal cocaine in the self-administration paradigm 6, although responding for i.v. cocaine has been reported to either increase z~ or remain unaffected ~2. Aspirative ablations of the mPFC have been shown to block place conditioning induced by systemic cocaine ~~ However, complete removal of this cortical region eliminates preand post-synaptic elements as well as fibers en passant and therefore does not provide information as to the neurophysiological substrate for cocaine place conditioning. The present experiment was designed to determine if presynaptic dopamine terminals in the mPFC have a functional role in place conditioning induced by i.p. cocaine. Male Wistar rats (n = 26; Sasco/King, Inc.) weighing 275-325 g were housed three per cage in a light- and temperature-controlled environment. Food and water were available in the home cage ad libitum throughout the experiment. Subjects were on a 12 h light/dark cycle with lights on from 7 a.m. to 7 p.m. Behavioral testing was conducted during the light phase of the cycle. Subjects were randomly assigned to two groups to receive either sham (n = 9) or 6-OHDA lesions (n = 17) of the mPFC. Subjects were pretreated with pargylinehydrochloride (25 mg/kg, i.p.; Sigma) and desipraminehydrochloride (50 mg/kg, i.p.; Sigma) 45 min prior to being anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.) and placed in a Kopf stereotaxic frame. The lesion group received bilateral infusions of 6-OHDA-HBr (4 lag/lal/site) dissolved in a solution of 0 . 9 ~ saline containing 0.02~ ascorbic acid while the sham-lesioned group was infused with equivalent volumes of vehicle at each site. Infusion coordinates were: (1) A + 1.0 mm anterior to bregma, L 0.8 to midline, V - 2 . 0 m m to dura; (2) A + 2 . 4 , L0.8, V - 1 . 5 ; (3) A + 2.4, L 0.8, V - 4.0; (4) A + 4.0, L 0.8, V - 3.0 xS. Infusion cannulae, constructed of 30 ga. hypodermic tubing, were connected via polyethylene tubing to 10 lal Hamilton syringes mounted on an infusion pump (Sage Instruments, Cambridge, MA). Left and right coordinates were simultaneously infused over a 1-min period with an additional 2 min allowed for diffusion. Following surgery, subjects were allowed 10 days to recover before behavioral testing.
The day immediately preceeding the initial exposure to the place conditioning apparatus, spontaneous locomotor activity was measured for both groups. The locomotor apparatus was a clear rectangular plexiglas box ( 4 1 c m • 2 1 5 high) placed inside an Omnitech Digiscan optical activity monitor (Columbus, OH). The monitor was equipped with 8 optical sensors spaced 4.5 mm apart on two perpendicular sides. Locomotor activity counts required the breaking of two beams consecutively. Subjects were placed in the activity chamber for 15 min and data was recorded in 5-min increments on a microcomputer. The spontaneous locomotor activity scores were subjected to an analysis of variance (ANOVA) 24 with one repeated measure, Time. The place conditioning apparatus has been described previously8'22. Briefly, the apparatus consisted of two main conditioning compartments (35 cm x 25 cm) separated by a neutral compartment (10 cm x 25 cm). The neutral compartment, used as the point of introduction on the preconditioning and test days, had sheet aluminum flooring and gray walls and was separated from the main compartments by removable guillotine doors. One 0 f the main compartments had black walls and flooring of steel dowels (6.4 mm diameter) spaced 2.5 mm apart whereas the alternate compartment had white walls and metal grid flooring (6 m m spacing of 1 mm ~,vires). A 1.5~ acetic acid solution was wiped on the flooring and walls of the black compartment before each test to negate the unconditioned preference for the black compartment ~4. The procedure for assessing cocaine-induced place conditioning consisted of three distinct phases: preconditioning, conditioning, and test. In the preconditioning phase, each subject was placed in the neutral compartment, the guillotine doors were removed, and the subject was allowed free access to the entire apparatus for 15 min in order to assess unconditioned preferences. The amount of time spent in each compartment was recorded. Following the preconditioning phase, the 6-OHDAand the sham-lesioned groups were counterbalanced such that halfofeach group had cocaine paired with the initially preferred compartment while the other half received saline in the preferred compartment. Within each group, half of the subjects received cocaine on the second and fourth days and halfon the third and fifth days. Saline injections were administered on alternate days. Rats were injected with either cocaine-HCl (10 mg/kg, i.p.; NIDA) or saline and immediately placed in the appropriate compartment for 30 rain. On the~test dab', subjects were placed in the neutral compartment and the guillotine doors removed thus giving free access to
227 tic acid (DOPAC), homovanillic acid (HVA), 5hydroxyindoleacetic acid (5-HIAA), and serotonin (5HT) concentrations. Quantification was achieved by using standards of known concentration. The HPLC consisted of an Isco LC-5000 syringe pump, an Adsorbosphere column (4.6mm i.d.x 150 mm, 5 la C-18 ODS silica; Alltech), an amperometric detector (model LC-4B; Bioanalytical Systems), and a dual carbon working electrode (model MF-100; Bioanalytical Systems). The mobile phase consisted of 15 mM sodium acetate, 152 mM citric acid, 5.0 mM triethylamine, 1.98 mM sodium octyl sulfate, 0.1 mM EDTA, and 89/0 methanol with the pH adjusted to 3.6 il. The flow rate was 1.25 ml/min and the applied potential was + 700 mV against an Ag/AgCI reference electrode (model MW-2021; Bioanalytical Systems). The biochemical results were analyzed by Student's ttest. The results of 6-OHDA lesions of the mPFC on biogenic amine levels in the mPFC, NACC, anterior caudate-putamen, and posterior caudate-putamen are presented in Table I. These lesions significantly depleted dopamine (t = 8.92, P < 0.0001) to 17~o of control levels and norepinephrine (t= 8.88, P
