Psychopharmacology (1992) 108:141-146
Psychopharmacology © Springer-Verlag 1992
Dopamine and endogenous opioid regulation of picrotoxin-induced locomotion in the ventral pallidum after dopamine depletion in the nucleus accumbens Lynn Churchill, Mark C. Austin*, and Peter W. Kalivas Alcohol and Drug Abuse Program and Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA Received June 26, 1991 / Final version December 2, 1991
Abstract. Microinjection of picrotoxin or the g-opioid agonist, Tyr-D-Ala-Gly-NmePhe-Gly-OH (DAMGO), into the ventral pallidum (VP) produces an increase in locomotor activity that is antagonized by dopamine receptor blockade. To investigate the regulation of VPinduced locomotion by the dopaminergic innervation of the nucleus accumbens (NA) and the role of opioid receptors in this regulation, dopamine innervation of the NA was bilaterally lesioned with 6-hydroxydopamine (6-OHDA). The lesions resulted in an 89-97% depletion of tissue dopamine levels in the nucleus accumbens compared with sham-lesioned rats. Dopamine depletion in the NA failed to significantly antagonize picrotoxin or DAMGO injected into the VP. However, the dopamine receptor antagonist, haloperidol (0.1 mg/kg, IP), blocked the picrotoxin-initiated increase in horizontal photocell counts in both sham- and 6-OHDA-lesioned rats. The opioid receptor antagonist, naloxone (1.0 mg/kg, SC), also blocked the picrotoxin-induced locomotion in 6-OHDA-lesioned rats but did not block locomotion in the sham-lesioned rats. At a higher dose (3.0 mg/kg, SC), naloxone blocked picrotoxin-induced locomotion in both sham- and 6-OHDA-lesioned rats. These results indicate that although dopamine depletion in the NA does not affect the permissive role of dopamine transmission on locomotion elicited from the VP, it results in an increased sensitivity to enkephalinergic transmission. Key words: Mesolimbic dopamine system- Opioid receptor - Haloperidol - Naloxone
The NA receives dopaminergic innervation from the ventral tegmental area in the mesencephalon (Gerfen et al. 1987). The mesoaccumbens dopamine projection has been characterized as a system important in the modula* Current address: Western Psychiatric Institute and Clinic, Univer-
sity of Pittsburgh, Pittsburgh, PA 15213, USA Offprint requests to: L. Churchill
tion of spontaneous and pharmacologically-stimulated locomotion (Kelly et al. 1975; Dells et al. 1990). Neurons within the NA project densely to the VP (Haber et al. 1985; Heimer et al. 1991) and contain both 7-aminobutyric acid (GABA) and the opioid peptide, met-enkephalin (Zahm et al. 1985). The accumbal-pallidal projection appears to mediate dopamine-dependent locomotion (Swerdlow et al. 1986), since microinjection of the GABAA agonist, muscimol, into the VP prevents locomotion elicited by dopamine receptor stimulation in the NA (Mogenson and Nielsen, 1983; Austin and Kalivas 1988). Also, injections of opioid agonists or GABAA antagonists into the VP will increase locomotion (Austin and Kalivas 1990). Behavioral, anatomical and neurophysiological data suggest that the projection of the VP neurons to the pedunculopontine tegmental area, mediodorsal thalamus, and/or prefrontal cortex may also regulate locomotor activity (Haber et al. 1985; Mogenson et al. 1985, 1987; Vives and Mogenson 1985; Swerdlow and K o o b 1987). Although this multisynaptic locomotor system proceeds to spinal motor generators via the pedunculopontine tegmental area, there is also the potential for feedback regulation onto the mesoaccumbens dopamine system or the accumbens-pallidal GABA system via the mediodorsal thalamus and prefrontal cortex. Recently, Austin and Kalivas (1991) found that motor stimulation elicited by microinjection of the indirect GABAA antagonist, picrotoxin, or the opioid agonist, DAMGO, into the VP was prevented by blocking dopamine receptors in the NA. These data suggest that locomotion initiated from the VP may feedback onto the mesoaccumbens dopamine projection and that this feedback is an important regulatory component for VP-induced locomotion. To further evaluate the possibility that mesoaccumbens dopamine transmission is permissive to the induction of motor activity from the VP, dopamine innervation to the NA was lesioned with 6 0 H D A and the motor stimulant response to picrotoxin or DAMGO injection into the VP examined. Since these lesions were
142
ineffective, two further experiments were conducted. In one experiment, rats were pretreated with haloperidol to determine if the dopamine remaining in the NA or unlesioned dopamine terminal fields was sufficient to maintain the motor response to picrotoxin. In the second experiment, the regulation of VP-induced locomotion by enkephalinergic transmission was evaluated. Previous studies have shown that opioid-induced locomotion was augmented after accumbal dopamine lesions indicating a postsynaptic upregulation of enkephalinergic transmission (Kalivas and Bronson 1985; Stinus et al. 1985; Churchill and Kalivas 1992). Other studies have shown that dopamine lesions increase preproenkephalin m R N A and enkephalin immunoreactivity, indicating a presynaptic regulation of enkephalin transmission (Thal et al. 1983; Voorn et al. 1987; Vernier et al. 1988). Thus, rats were pretreated with the opioid antagonist, naloxone, to determine if the lesion-induced increase in enkephalin transmission may play a role in the locomotor response elicited by picrotoxin in the VP.
0.5 ml of mobile phase (0.1 M trichloroacetic acid, 0.1 M sodium acetate, 0.1 M EDTA, 18% methanol (pH 4) and 2 x 10 .7 M isoproterenol as an internal standard), sonicated and centrifuged (13000 g) for 2 min. The supernatant was removed for analysis on the HPLC, while the pellet was assayed for protein using the Lowry method. The biogenic amines were separated on an ODS C-18 reversed-phase column and oxidized at 0.7 V. The concentration of biogenic amines was calculated from a standard curve (10-12_ 10 -1 o tool). The detection limit was about 3 x 10-is tool.
Statistics. All rats with any of the cannula tips outside the VP or with dopamine depletions < 70% in the N A were excluded from the data analyses. Total horizontal photocell counts were statistically evaluated with a 2-way repeated measures analysis of variance followed by a Newman-Keuls multiple comparison test. The time course of the horizontal photocell counts was statistically evaluated for either the sham- or 6-OHDA-lesioned rats with a two-way repeated measures analysis of variance followed by a least significant difference multiple comparison test as described by Milliken and Johnson (1984) if the interaction was significant, or by a Newman-Keuls test.
Results
Materials and methods
Effect of picrotoxin or DAMGO microinjections
Animal housing, surgery and microinjections. Male Sprague-Dawley
into the VP on locomotion in sham- or 6-OHDA-lesioned rats
rats (250-350 g; Laboratory Animal Research Center, Pullman, WA or Bantin & Kingman, San Francisco, CA) were injected with desmethylimipramine (25 mg/ml, IP) at least 20 min prior to anesthetizing with Equithesin (1.8 ml/kg). Bilateral 6 - O H D A lesions of the N A were performed by injecting 3 gl of 6~OHDA (4 pg/gl free base in 0.25 mg/mi ascorbic acid in sterile saline) through 30 gauge cannulae inserted in the N A (A/P 9.0; M/L 1.7; D/V - 0 . 4 ram, relative to the interaural line; Pellegrino et al. 1979) over 12 min. Following a 3 min delay for absorption, the 30 gauge cannulae were removed and 26 gauge guide cannulae were implanted in the VP (A/P 7.2; M/L 2.2; D/V -0.9). The cannulae were secured to the skull with dental acrylate and stainless steel screws, and protected from obstruction by 33 gauge obturators. The rats were allowed to recover for a minimum of 10 days after surgery. Simultaneous bilateral microinjections were performed in the VP in unrestrained rats by inserting a 33 gauge needle into each guide cannulae, as described elsewhere (Austin and Kalivas 1990). Picrotoxin (0.17 nmol/0.5 pl sterile saline/side), D A M G O (0.03 nmol/0.5 gl/side) or saline was microinjected into the VP. The opioid antagonist, naloxone (1 or 3 mg/kg, SC in sterile saline) or haloperidol (0.1 mg/kg, IP, in 0.5 mg/ml ascorbic acid) was injected 4-5 rain prior to saline or picrotoxin microinjections. Twenty seconds after injection, the needles were removed and the obturators replaced.
Behavioral measurements. Motor activity was measured using a photoceU apparatus (Omnitech Electronics, Columbus, OH) operated via an Apple IIe computer, as described elsewhere (Austin and Kalivas 1990). The rats were preadapted to both cages and injection procedure 24 h prior to starting the experiment by adapting them to the cages for 30 min, making sham injections and returning them to the photocell cages for another hour. When injections were to be made, the rats were adapted to the cages for 1 h prior to injection, and behavioral measurements were conducted for 2 h after the injection. After the behavioral analyses, the rats were returned to their home cages for a minimum of 3 days. Each rat received a maximum of four treatments in random order. Measurement of biooenie amines. Dopamine and serotonin were measured using high pressure liquid chromatography (HPLC) with electrochemical detection. The NA, ventromedial and dorsolateral striatum, and prefrontal cortex were dissected on an ice-cooled glass plate. After dissection, bilateral tissue punches were placed in
Microinjections of either picrotoxin (0.17 nmol/side) or D A M G O (0.03 nmol/side) into the VP produced a significant increase in horizontal photocell counts in both sham- and 6--OHDA-lesioned rats (Fig. 1). No significant differences were observed between the locomotor responses in the sham- versus 6-OHDA-lesioned rats. Dopamine depletion in the N A of these rats was greater than 96.8 % (Table 1). Serotonin was also depleted in the NA, but only by 39%. The lesions were only partially evident in the dorsolateral caudate (35% depletion).
Effect of blocking dopamine receptors on picrotoxin-induced locomotion from the VP
Injections of halopefidol (0.1 mg/kg, IP) produced a significant decrease in horizontal photocell counts after
eq ~z ,~,~ ~
20000 j 15000 ]
10000] 5000 ] 0 ~
Sham 6-OHDA Treatment
Fig. 1. Effect ofpicrotoxin or D A M G O on VP-induced locomotion in sham- or 6qDHDA-lesioned rats. Total horizontal photocell counts for 2 h after microinjections of either D A M G O (0.03 nmol/ side) or picrotoxin (0.17 nmol/side) into the VP. F values were surgical treatment F(1,9) = 0.004, P = 0.9497; repeated measure F(2,18) = 22.869, P < 0.0001 ; interaction F(2,18) = 1.14, P = 0.3419. * P < 0.05 comparing picrotoxin or D A M G O with saline using a 2-way repeated measures analysis of variance (ANOVA) followed by a Newman-Keuls multiple comparison test. (m) Saline; ([]) picrotoxin; ([]) D A M G O
143 Table 1, Dopamine and serotonin concentrations in the nucleus accumbens and surrounding brain areas followingsham or 6-OHDA injectionsinto the nucleus accumbens. N= the number of rats
Brain region
Treatment
N
Concentration (pmol/mgprotein) Dopamine Serotonin
Picrotoxin-DAMGO experiment Nucleus accumbens Dorsolateral caud.
Sham 6-OHDA Sham 6-OHDA
5 6 5 6
572.6± 35.7 18.3:t: 5.8* 824.6+ 58.4 534.6+51.7"
33~3± 2.6 20"3±3.6* 11.6+ 0.9 10.94-1.3
11 7 11 7 11 7
549.2+ 23.7 59.4:t:16.2" 839.2+ 58.2 411.0±94.8" 7.4± 1.5 4.5+ 2.8
36.3 + 3.2 32.6:t:3.4 20.8 ± 1.8 20.4±4.0 44.2±3.7 33~0±4.7
Haloperidol-Picrotoxin experiment Nucleus accumbens Ventromedial caud. Prefrontal cortex
Sham 6-OHDA Sham 6-OHDA Sham 6-OHDA
* P
Psychopharmacology © Springer-Verlag 1992
Dopamine and endogenous opioid regulation of picrotoxin-induced locomotion in the ventral pallidum after dopamine depletion in the nucleus accumbens Lynn Churchill, Mark C. Austin*, and Peter W. Kalivas Alcohol and Drug Abuse Program and Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA Received June 26, 1991 / Final version December 2, 1991
Abstract. Microinjection of picrotoxin or the g-opioid agonist, Tyr-D-Ala-Gly-NmePhe-Gly-OH (DAMGO), into the ventral pallidum (VP) produces an increase in locomotor activity that is antagonized by dopamine receptor blockade. To investigate the regulation of VPinduced locomotion by the dopaminergic innervation of the nucleus accumbens (NA) and the role of opioid receptors in this regulation, dopamine innervation of the NA was bilaterally lesioned with 6-hydroxydopamine (6-OHDA). The lesions resulted in an 89-97% depletion of tissue dopamine levels in the nucleus accumbens compared with sham-lesioned rats. Dopamine depletion in the NA failed to significantly antagonize picrotoxin or DAMGO injected into the VP. However, the dopamine receptor antagonist, haloperidol (0.1 mg/kg, IP), blocked the picrotoxin-initiated increase in horizontal photocell counts in both sham- and 6-OHDA-lesioned rats. The opioid receptor antagonist, naloxone (1.0 mg/kg, SC), also blocked the picrotoxin-induced locomotion in 6-OHDA-lesioned rats but did not block locomotion in the sham-lesioned rats. At a higher dose (3.0 mg/kg, SC), naloxone blocked picrotoxin-induced locomotion in both sham- and 6-OHDA-lesioned rats. These results indicate that although dopamine depletion in the NA does not affect the permissive role of dopamine transmission on locomotion elicited from the VP, it results in an increased sensitivity to enkephalinergic transmission. Key words: Mesolimbic dopamine system- Opioid receptor - Haloperidol - Naloxone
The NA receives dopaminergic innervation from the ventral tegmental area in the mesencephalon (Gerfen et al. 1987). The mesoaccumbens dopamine projection has been characterized as a system important in the modula* Current address: Western Psychiatric Institute and Clinic, Univer-
sity of Pittsburgh, Pittsburgh, PA 15213, USA Offprint requests to: L. Churchill
tion of spontaneous and pharmacologically-stimulated locomotion (Kelly et al. 1975; Dells et al. 1990). Neurons within the NA project densely to the VP (Haber et al. 1985; Heimer et al. 1991) and contain both 7-aminobutyric acid (GABA) and the opioid peptide, met-enkephalin (Zahm et al. 1985). The accumbal-pallidal projection appears to mediate dopamine-dependent locomotion (Swerdlow et al. 1986), since microinjection of the GABAA agonist, muscimol, into the VP prevents locomotion elicited by dopamine receptor stimulation in the NA (Mogenson and Nielsen, 1983; Austin and Kalivas 1988). Also, injections of opioid agonists or GABAA antagonists into the VP will increase locomotion (Austin and Kalivas 1990). Behavioral, anatomical and neurophysiological data suggest that the projection of the VP neurons to the pedunculopontine tegmental area, mediodorsal thalamus, and/or prefrontal cortex may also regulate locomotor activity (Haber et al. 1985; Mogenson et al. 1985, 1987; Vives and Mogenson 1985; Swerdlow and K o o b 1987). Although this multisynaptic locomotor system proceeds to spinal motor generators via the pedunculopontine tegmental area, there is also the potential for feedback regulation onto the mesoaccumbens dopamine system or the accumbens-pallidal GABA system via the mediodorsal thalamus and prefrontal cortex. Recently, Austin and Kalivas (1991) found that motor stimulation elicited by microinjection of the indirect GABAA antagonist, picrotoxin, or the opioid agonist, DAMGO, into the VP was prevented by blocking dopamine receptors in the NA. These data suggest that locomotion initiated from the VP may feedback onto the mesoaccumbens dopamine projection and that this feedback is an important regulatory component for VP-induced locomotion. To further evaluate the possibility that mesoaccumbens dopamine transmission is permissive to the induction of motor activity from the VP, dopamine innervation to the NA was lesioned with 6 0 H D A and the motor stimulant response to picrotoxin or DAMGO injection into the VP examined. Since these lesions were
142
ineffective, two further experiments were conducted. In one experiment, rats were pretreated with haloperidol to determine if the dopamine remaining in the NA or unlesioned dopamine terminal fields was sufficient to maintain the motor response to picrotoxin. In the second experiment, the regulation of VP-induced locomotion by enkephalinergic transmission was evaluated. Previous studies have shown that opioid-induced locomotion was augmented after accumbal dopamine lesions indicating a postsynaptic upregulation of enkephalinergic transmission (Kalivas and Bronson 1985; Stinus et al. 1985; Churchill and Kalivas 1992). Other studies have shown that dopamine lesions increase preproenkephalin m R N A and enkephalin immunoreactivity, indicating a presynaptic regulation of enkephalin transmission (Thal et al. 1983; Voorn et al. 1987; Vernier et al. 1988). Thus, rats were pretreated with the opioid antagonist, naloxone, to determine if the lesion-induced increase in enkephalin transmission may play a role in the locomotor response elicited by picrotoxin in the VP.
0.5 ml of mobile phase (0.1 M trichloroacetic acid, 0.1 M sodium acetate, 0.1 M EDTA, 18% methanol (pH 4) and 2 x 10 .7 M isoproterenol as an internal standard), sonicated and centrifuged (13000 g) for 2 min. The supernatant was removed for analysis on the HPLC, while the pellet was assayed for protein using the Lowry method. The biogenic amines were separated on an ODS C-18 reversed-phase column and oxidized at 0.7 V. The concentration of biogenic amines was calculated from a standard curve (10-12_ 10 -1 o tool). The detection limit was about 3 x 10-is tool.
Statistics. All rats with any of the cannula tips outside the VP or with dopamine depletions < 70% in the N A were excluded from the data analyses. Total horizontal photocell counts were statistically evaluated with a 2-way repeated measures analysis of variance followed by a Newman-Keuls multiple comparison test. The time course of the horizontal photocell counts was statistically evaluated for either the sham- or 6-OHDA-lesioned rats with a two-way repeated measures analysis of variance followed by a least significant difference multiple comparison test as described by Milliken and Johnson (1984) if the interaction was significant, or by a Newman-Keuls test.
Results
Materials and methods
Effect of picrotoxin or DAMGO microinjections
Animal housing, surgery and microinjections. Male Sprague-Dawley
into the VP on locomotion in sham- or 6-OHDA-lesioned rats
rats (250-350 g; Laboratory Animal Research Center, Pullman, WA or Bantin & Kingman, San Francisco, CA) were injected with desmethylimipramine (25 mg/ml, IP) at least 20 min prior to anesthetizing with Equithesin (1.8 ml/kg). Bilateral 6 - O H D A lesions of the N A were performed by injecting 3 gl of 6~OHDA (4 pg/gl free base in 0.25 mg/mi ascorbic acid in sterile saline) through 30 gauge cannulae inserted in the N A (A/P 9.0; M/L 1.7; D/V - 0 . 4 ram, relative to the interaural line; Pellegrino et al. 1979) over 12 min. Following a 3 min delay for absorption, the 30 gauge cannulae were removed and 26 gauge guide cannulae were implanted in the VP (A/P 7.2; M/L 2.2; D/V -0.9). The cannulae were secured to the skull with dental acrylate and stainless steel screws, and protected from obstruction by 33 gauge obturators. The rats were allowed to recover for a minimum of 10 days after surgery. Simultaneous bilateral microinjections were performed in the VP in unrestrained rats by inserting a 33 gauge needle into each guide cannulae, as described elsewhere (Austin and Kalivas 1990). Picrotoxin (0.17 nmol/0.5 pl sterile saline/side), D A M G O (0.03 nmol/0.5 gl/side) or saline was microinjected into the VP. The opioid antagonist, naloxone (1 or 3 mg/kg, SC in sterile saline) or haloperidol (0.1 mg/kg, IP, in 0.5 mg/ml ascorbic acid) was injected 4-5 rain prior to saline or picrotoxin microinjections. Twenty seconds after injection, the needles were removed and the obturators replaced.
Behavioral measurements. Motor activity was measured using a photoceU apparatus (Omnitech Electronics, Columbus, OH) operated via an Apple IIe computer, as described elsewhere (Austin and Kalivas 1990). The rats were preadapted to both cages and injection procedure 24 h prior to starting the experiment by adapting them to the cages for 30 min, making sham injections and returning them to the photocell cages for another hour. When injections were to be made, the rats were adapted to the cages for 1 h prior to injection, and behavioral measurements were conducted for 2 h after the injection. After the behavioral analyses, the rats were returned to their home cages for a minimum of 3 days. Each rat received a maximum of four treatments in random order. Measurement of biooenie amines. Dopamine and serotonin were measured using high pressure liquid chromatography (HPLC) with electrochemical detection. The NA, ventromedial and dorsolateral striatum, and prefrontal cortex were dissected on an ice-cooled glass plate. After dissection, bilateral tissue punches were placed in
Microinjections of either picrotoxin (0.17 nmol/side) or D A M G O (0.03 nmol/side) into the VP produced a significant increase in horizontal photocell counts in both sham- and 6--OHDA-lesioned rats (Fig. 1). No significant differences were observed between the locomotor responses in the sham- versus 6-OHDA-lesioned rats. Dopamine depletion in the N A of these rats was greater than 96.8 % (Table 1). Serotonin was also depleted in the NA, but only by 39%. The lesions were only partially evident in the dorsolateral caudate (35% depletion).
Effect of blocking dopamine receptors on picrotoxin-induced locomotion from the VP
Injections of halopefidol (0.1 mg/kg, IP) produced a significant decrease in horizontal photocell counts after
eq ~z ,~,~ ~
20000 j 15000 ]
10000] 5000 ] 0 ~
Sham 6-OHDA Treatment
Fig. 1. Effect ofpicrotoxin or D A M G O on VP-induced locomotion in sham- or 6qDHDA-lesioned rats. Total horizontal photocell counts for 2 h after microinjections of either D A M G O (0.03 nmol/ side) or picrotoxin (0.17 nmol/side) into the VP. F values were surgical treatment F(1,9) = 0.004, P = 0.9497; repeated measure F(2,18) = 22.869, P < 0.0001 ; interaction F(2,18) = 1.14, P = 0.3419. * P < 0.05 comparing picrotoxin or D A M G O with saline using a 2-way repeated measures analysis of variance (ANOVA) followed by a Newman-Keuls multiple comparison test. (m) Saline; ([]) picrotoxin; ([]) D A M G O
143 Table 1, Dopamine and serotonin concentrations in the nucleus accumbens and surrounding brain areas followingsham or 6-OHDA injectionsinto the nucleus accumbens. N= the number of rats
Brain region
Treatment
N
Concentration (pmol/mgprotein) Dopamine Serotonin
Picrotoxin-DAMGO experiment Nucleus accumbens Dorsolateral caud.
Sham 6-OHDA Sham 6-OHDA
5 6 5 6
572.6± 35.7 18.3:t: 5.8* 824.6+ 58.4 534.6+51.7"
33~3± 2.6 20"3±3.6* 11.6+ 0.9 10.94-1.3
11 7 11 7 11 7
549.2+ 23.7 59.4:t:16.2" 839.2+ 58.2 411.0±94.8" 7.4± 1.5 4.5+ 2.8
36.3 + 3.2 32.6:t:3.4 20.8 ± 1.8 20.4±4.0 44.2±3.7 33~0±4.7
Haloperidol-Picrotoxin experiment Nucleus accumbens Ventromedial caud. Prefrontal cortex
Sham 6-OHDA Sham 6-OHDA Sham 6-OHDA
* P
