Abstract
In the present study, we investigated the effects of selective activation or inhibition of ventral tegmental area (VTA) adenylate cyclase (AC) and protein kinase A (PKA) on long-term sensitization induced by repeated intra-VTA or peripheral amphetamine (AMPH). Selective inhibition of AC by SQ 22,536 (9-(tetrahydro-2-furanyl)-9H-purin-6-amine; 100 nmol/side bilateral into VTA) had no effect on acute basal locomotion but attenuated the locomotor stimulation induced by acute i.p. AMPH (1.5 mg/kg). Coinjection of SQ 22,536 (100 nmol/side) fully blocked the sensitization induced by repeated intra-VTA AMPH (15 nmol/side) but had no detectable effect on the sensitization induced by repeated i.p. AMPH. Persistent activation of AC by intra-VTA cholera toxin (500 ng/side) modestly increased acute locomotion and induced a robust sensitization to i.p. AMPH challenge 10 days after the last of three repeated VTA microinjections. Selective inhibition of PKA by Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine (Rp-cAMPS; 25 nmol/side) had no effect on acute basal or AMPH-stimulated locomotion. Coinjection of Rp-cAMPS (25 nmol/side) fully blocked the sensitization induced by repeated intra-VTA AMPH but had no effect on sensitization induced by repeated i.p. AMPH. Intra-VTA microinjection of the selective PKA activator Sp-adenosine-3′,5′-cyclic monophosphothioate triethylamine (Sp-cAMPS; 25–100 nmol/side) dose-dependently stimulated acute locomotion and exerted synergistic effects on locomotor activity when coinfused into the VTA with AMPH but had no detectable effect on acute i.p. AMPH-induced locomotion. Repeated intra-VTA Sp-cAMPS did not induce sensitization to AMPH challenge but potentiated the sensitization induced by repeated i.p. AMPH. These results suggest that VTA cAMP signal transduction is necessary for the induction of persistent sensitization to intra-VTA amphetamine and that peripheral and intra-VTA AMPH may not induce behavioral sensitization by identical mechanisms.
The development of enhanced behavioral sensitivity to psychostimulants with repeated administration has been well documented (reviewed in Robinson and Becker, 1986; Kalivas and Stewart, 1991; Kalivas, 1995; Pierce and Kalivas, 1997). Behavioral sensitization, typically characterized by augmented motor responsiveness to cocaine or amphetamine (AMPH) challenge, is long lasting and reportedly can increase susceptibility to stimulant self-administration in animals (Piazza et al., 1989, 1990;Horger et al., 1990, 1992; Robinson and Berridge, 1993). A large and growing body of evidence suggests that alterations in mesolimbic dopaminergic neurotransmission play a critical role in initiation and expression of behavioral sensitization (Robinson and Becker, 1986;Kalivas and Stewart, 1991; Kalivas, 1995). Ventral midbrain nuclei containing dopamine neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex have been implicated as the site of initiation of sensitization because repeated microinjections of AMPH into the VTA, but not into axon terminal regions of the nucleus accumbens, can induce behavioral sensitization in rats (Hitzmann et al., 1980; Dougherty and Ellinwood, 1981; Kalivas and Weber, 1988; Vezina and Stewart, 1990; Cador et al., 1995; Vezina, 1996). Moreover, intra-VTA administration of selected classes of pharmacological antagonists can attenuate or block the induction of sensitization by cocaine or AMPH (Stewart and Vezina, 1989; Kalivas and Alesdatter, 1993; Sorg and Ulibarri, 1995; Bijou et al., 1996; Vezina, 1996).
Although the cellular events that mediate the initiation of AMPH sensitization in the VTA are poorly understood, several lines of evidence implicate alterations in neural second messenger systems in this process. Components of the cAMP signal transduction cascade may be especially important in drug-induced neuroplasticity (reviewed inNestler et al., 1993; Nestler and Aghajanian, 1997). Behavioral sensitization results from treatments that either 1) reduce tonic inhibition of adenylate cyclase (AC) in the VTA by the inhibitory G protein subunit Gi or 2) increase activation of AC in the VTA by the stimulatory G protein subunit Gs. For example, chronic exposure to stimulants has been shown to reduce both the function and density of the Gi protein in the VTA (Nestler et al., 1990;Terwilliger et al., 1991; Striplin and Kalivas, 1992). Persistent inactivation of Gi in the VTA by a single microinjection of pertussis toxin induces a robust and lasting sensitization to AMPH or cocaine challenge (Steketee and Kalivas, 1991). Conversely, repeated activation of the D1dopamine receptor, which stimulates cAMP production by activating AC via the Gs protein, with intra-VTA microinjections of a D1 agonist has been shown to induce sensitization to systemic stimulant challenge (Pierce et al., 1996), whereas intra-VTA antagonism of the Gs-coupled D1 receptor is known to block the induction of AMPH sensitization (Stewart and Vezina, 1989; Bijou et al., 1996; Vezina, 1996).
Recent studies using the selective neurotoxin cholera toxin (CTX), which persistently activates AC via ADP-ribosylation of the Gs protein, have provided direct evidence for the involvement of VTA cAMP signal transduction in sensitization (Tolliver et al., 1996; Byrnes et al., 1997). These studies demonstrated that a single bilateral microinjection of CTX into the VTA induces a robust sensitization to systemic AMPH or cocaine challenge (Tolliver et al., 1996; Byrnes et al., 1997) and that both CTX- and intra-VTA AMPH-induced sensitization can be attenuated by coadministration of an inhibitor of cAMP-dependent protein kinase (Tolliver et al., 1996). The present experiments address several issues unresolved in our previous report (Tolliver et al., 1996). First, this earlier study examined sensitization induced only by a single intra-VTA AMPH or CTX microinjection administered to rats under pentobarbital anesthesia. Thus, the previous experiments were unable to evaluate the acute effects of intra-VTA administration of the drugs on behavior, to characterize any effects of repeated administration, to assess the effects of cAMP drugs on sensitization induced by systemic AMPH, and to rule out potential drug/anesthetic interactions. In addition, the involvement of protein kinase A (PKA) in CTX- and AMPH-induced sensitization was inferred from attenuation by the nonselective protein kinase inhibitor H8 (Tolliver et al., 1996), which also inhibits protein kinase C and cGMP-dependent protein kinase (Hidaka et al., 1984; Saitoh et al., 1987). In the current study, microinjection cannulas were implanted into the VTA to assess both the acute and chronic behavioral effects of highly selective inhibitors and activators of AC and PKA, administered alone or with AMPH (both systemic and intra-VTA), in awake, freely moving rats. Finally, the previous studies demonstrated that VTA cAMP systems are involved in AMPH sensitization at early (3-day) withdrawal time points (Tolliver et al., 1996; Byrnes et al., 1997). Because neurophysiological adaptations associated with AMPH sensitization at early withdrawal time points are different than those after longer (10–14 days) withdrawal (Wolf et al., 1993), it is possible that signal transduction mechanisms involved in early withdrawal sensitization do not mediate late-onset neural and behavioral adaptations. Therefore, the current study examined the effects of cAMP agents on the induction of AMPH sensitization at a late withdrawal period to extend our previous studies of sensitization at early withdrawal points.
Materials and Methods
Animals and Drugs.
Male Sprague-Dawley rats (Simonsen, Gilroy, CA) weighing between 200 and 350 g were housed on a 12-h light/dark cycle and received food and water ad libitum throughout the experiment. Rats were housed individually after surgery. Cholera toxin (Research Biochemicals Inc., Natick, MA) was dissolved in isotonic saline at 1.0 μg/μl. SQ 22,536 (9-(tetrahydro-2-furanyl)-9H-purin-6-amine; Research Biochemicals Inc.) was dissolved in isotonic saline at 41 μg/μl. Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine (Rp-cAMPS) (Research Biochemicals Inc.) was dissolved in isotonic saline at 22.3 μg/μl. Its diastereomer, Sp-adenosine-3′,5′-cyclic monophosphothioate triethylamine (Sp-cAMPS) (Research Biochemicals Inc.) was dissolved in isotonic saline at 22.3 and 89.2 μg/μl.d-AMPH sulfate (Sigma Chemical Co., St. Louis, MO) was dissolved in isotonic saline at 1.5 mg/ml for i.p. administration (at 1 ml/kg b.wt.) and at 10 μg/μl for microinjections.
Surgical Procedures.
Rats were anesthetized with ketamine/xylazine (80 and 12 mg/kg i.p., respectively) and placed in a stereotaxic instrument (Kopf, Tujunga, CA). Bilateral 26-gauge stainless steel guide cannulas fitted with 33-gauge stainless steel obturators (Plastics One, Roanoke, VA) were implanted into the VTA [A/P, −5.3 mm; M/L, ±0.6 mm; D/V, −8.3 mm from bregma according to the atlas of Paxinos and Watson (1986)] and affixed to the skull using stainless steel skull screws and dental acrylic. After surgery, dust caps were attached to the guide cannulas, and rats were allowed to recover for at least 1 week before beginning the experiment. During this time, all rats were habituated daily to handling.
VTA Microinjections and Acute Behavioral Tests.
On days 1, 3, and 5, rats were habituated to the behavioral test chambers (50 × 50 cm, equipped with 15 photocell beams in each direction located 4 cm off the cage floor; Opto-Varimex Minor; Columbus Instruments, Columbus, OH) for 1 h and then underwent bilateral microinjections into the VTA. Dust caps and obturators were removed and replaced with 33-gauge injection cannulas (Plastics One) connected to 10-μl Hamilton syringes. A total volume of 0.5 μl of saline or drug solution was infused over 60 s per side (volumes were confirmed by tracing the movement of an air bubble introduced into each injection line approximately 10 μl distal to the injection cannulas). Approximately 2 min were allowed for diffusion of the drugs before removal of the injection cannulas and obturator replacement. All rats then received an i.p. injection of saline or 1.5 mg/kg AMPH (seeExperimental Design and Statistical Analysis) and were returned to the test chambers, and locomotor activity was recorded in 10-min bins for 1 h. Rats were returned to home cages after treatment.
Behavioral Sensitization Tests.
After a 10-day withdrawal after the last intra-VTA microinjection, rats were challenged on day 15 with i.p. saline and 0.5 mg/kg i.p. AMPH. On the test day, rats were habituated to the chambers for 1 h while being monitored for baseline locomotor activity. At the end of this period, all rats received an i.p. injection of saline (1 ml/kg) and were returned to the testing apparatus for 1 h. At the end of the saline test period, rats received an i.p. injection of d-AMPH and were monitored for locomotor activity for 2 h. During the saline and drug test periods, all animals were scored for frequency and intensity of stereotyped behavior by a trained observer (who was usually, but not always, blinded to the treatment conditions) for 10 s every 10 min according to the scale of Steketee and Kalivas (1991): 1) asleep or still; 2) inactive, grooming, mild licking; 3) locomotion, rearing or sniffing; 4) any combination of two of locomotion, rearing, or sniffing; 5) continuous sniffing for 10 s without locomotion or rearing; 6) continuous sniffing for 10 s with locomotion or rearing; 7) patterned sniffing for 5 s; 8) patterned sniffing for 10 s; 9) continuous gnawing; and 10) bizarre dyskinetic movements or seizures. All rats were euthanized for histological analysis of microinjection tract location on completion of testing.
Histology.
On completion of behavioral experiments, animals were overdosed with ketamine (100 mg/kg), perfused via the ascending aorta with 4% paraformaldehyde in 0.1 M phosphate buffer, and decapitated. Whole brains were removed and stored in paraformaldehyde in 0.1 M phosphate buffer until sectioned using a vibratome (Lancer, St. Louis, MO). Coronal sections (100 μm) were mounted on gel-coated slides, stained with 2% cresyl violet, and examined under a light microscope. Microinjection tract location and probe placement were determined according to the atlas of Paxinos and Watson (1986).
Experimental Design and Statistical Analysis.
Three experiments were designed and conducted as follows. In each experiment, acute data were collected on day 1, repeated data were collected on days 3 and 5, and sensitization (AMPH challenge) data were collected on day 15. All locomotor data were analyzed by ANOVA as described below. All stereotypy data were analyzed by Kruskal-Wallis and Mann-WhitneyU tests and were confirmed by one-way ANOVA. The purpose of experiment 1 was to test the effects of inhibition of AC and either inhibition or activation of PKA on acute locomotion and behavioral sensitization induced by systemic AMPH. Doses of cAMP analogs were chosen based on previous studies involving intracranial microinjections of related compounds (Miserendino and Nestler 1995). Rats received bilateral VTA microinjections of saline, SQ 22,536 (100 nmol/side), Rp-cAMPS (25 nmol/side), or Sp-cAMPS (100 nmol/side) 2 min before an i.p. injection of saline or 1.5 mg/kg AMPH, and locomotor activity was assessed for 1 h as described above. Statistical analyses were conducted separately for SQ 22,536, Rp-cAMPS, and Sp-cAMPS. For each compound acute, repeated, and sensitization data were compared with respective data from VTA saline-pretreated rats. Acute VTA pretreatment effects of each drug on AMPH-induced locomotion were determined by one-way ANOVA comparison (VTA drug versus VTA saline) in rats challenged with i.p. AMPH on day 1. The acute effects of each VTA drug treatment on basal locomotion were assessed by one-way ANOVA comparison in rats challenged with i.p. saline on day 1. The effects of repeated VTA drug treatments on basal and AMPH-induced locomotor activity on days 3 and 5 were compared with those on day 1 by mixed-factor ANOVA (between-subject measure = VTA drug, within-subject measure = day). Finally, the effects of previous repeated VTA drug treatments on late-withdrawal behavioral sensitization were assessed by two-way between-subject ANOVA (factor 1 = repeated VTA pretreatment, factor 2 = repeated i.p. treatment) of locomotor responses to AMPH challenge on day 15. Time course data were analyzed by mixed-factor (between subject = VTA treatment, within subject = time) ANOVA. Corresponding two-way ANOVAs of basal locomotion and locomotor responses to saline challenge on day 15 were used to assess nonspecific VTA pretreatment effects. For all acute, repeated, and sensitization data, Newman-Keuls and Dunnett’s post-hoc tests were used after ANOVAs where appropriate.
The purpose of experiment 2 was to test the effects of inhibition of AC and either inhibition or activation of PKA on acute locomotion and behavioral sensitization induced by intra-VTA AMPH. Rats received bilateral intra-VTA injections of AMPH (5 μg/side) or AMPH plus the test drug (same drugs and doses as described in experiment 1) 2 min before i.p. saline injections on days 1, 3, and 5. Groups from experiment 1 that received VTA saline or test drug alone and i.p. saline injections were included in all analyses in experiment 2. All statistical analyses of AMPH plus test drug coadministration treatment groups were conducted by one-way ANOVA comparing VTA saline, VTA AMPH, VTA AMPH plus test drug, and VTA test drug alone. Acute VTA pretreatment effects on basal (after i.p. saline) locomotion were assessed by one-way ANOVA followed by Newman-Keuls and Dunnett’s post-hoc tests where appropriate. The effects of repeated VTA drug treatments on locomotor activity on days 3 and 5 were compared with those on day 1 by mixed-factor ANOVA (between-subject measure = VTA drug, within-subject measure = day). The effects of previous repeated VTA drug treatments on basal locomotion and late-withdrawal behavioral sensitization were analyzed by one-way ANOVA of locomotor responses on day 15. Time course data were analyzed by mixed-factor (between subject = VTA treatment, within subject = time) ANOVAs.
Previous work in our laboratory has shown that a single intra-VTA infusion of CTX under pentobarbital anesthesia induces a robust but transient sensitization to AMPH (Tolliver et al., 1996). The purpose of experiment 3 was to evaluate whether CTX can induce a lasting (at least 10 days) sensitization to AMPH when infused repeatedly into the VTA of awake rats. Rats received bilateral intra-VTA infusions of CTX (500 ng/side) 2 min before i.p. saline injections on days 1, 3, and 5. Statistical comparisons were made between CTX-treated rats and rats that received VTA saline pretreatments before i.p. saline injections (experiment 1). Acute, repeated, and sensitization data were analyzed by one-way and mixed-factor ANOVA as described above.
Results
Role of AC
Acute Locomotor Effects.
A single bilateral infusion of 5 μg/side AMPH into the VTA did not elicit locomotor activity in the 60 min after the microinjection when administered alone (F1,13 = 0.10, p = .76) or when coadministered with 100 nmol/side of the AC inhibitor SQ 22,536 (F3,22 = 0.39,p = .76; Fig. 1). SQ 22,536 did not stimulate acute locomotion when administered alone at this dose (F1,11 = 0.01,p = .93; Fig. 1, a and b). Activation of AC with CTX generally stimulated acute locomotion when injected into the VTA (Fig.1, a and c). Direct comparison by Student’s t test of rats that received intra-VTA CTX with those that received intra-VTA saline revealed modestly enhanced locomotion after acute CTX (F1,16 = 4.57, p < .05), although no significant differences in locomotor response (F2,22 = 1.35, p = .28) were found when intra-VTA AMPH-treated rats were included in the analysis.
Although the AC inhibitor SQ 22,536 had no detectable effects on locomotion in i.p. saline-treated rats (Fig. 1), bilateral intra-VTA pretreatment with 100 nmol/side SQ 22,536 significantly attenuated the acute locomotor response to 1.5 mg/kg i.p. AMPH (F1,16 = 12.74, p < .003; Fig. 2). In this experiment, statistically significant attenuation of AMPH responsiveness was observed in each of two independent groups (n = 6 each) of rats pretreated with SQ 22,536 relative to those pretreated with saline (data not shown). Although the inhibitory effect of SQ 22,536 was most pronounced during the first 30 min after AMPH injection, the AC inhibitor blunted AMPH-induced locomotion throughout the 60-min test period without altering its time course (pretreatment × time interaction: F5,80 = 1.10,p = .37; Fig. 2a).
Repeated Administration.
As was the case after an acute microinjection on day 1, no locomotor activation was induced by repeated intra-VTA AMPH on days 3 and 5 (F1,24 = 0.03, p = .87; Fig. 3a). Likewise, repeated microinjections of SQ 22,536 (F1,22 = 0.33, p = .58) did not elicit locomotion on any of days 1, 3, or 5 when administered alone (Fig. 3a) or in combination with 5 μg/side AMPH (days 3 and 5; data not shown). Thus, no detectable sensitization or tolerance developed to the effects of intra-VTA AMPH or SQ 22,536. In contrast, the locomotor response to repeated microinjections of 500 ng/side CTX (F1,32 = 10.92, p < .005) was significantly enhanced on days 3 and 5 relative to day 1 (day effect: F2,32 = 3.54,p < .05; Fig. 3a). However, it should be noted that CTX administration also elevated basal locomotion (F1,32 = 13.67, p < .005), an effect that was observed even after a single bilateral microinjection on day 1 (Table 1).
Although intra-VTA pretreatment with SQ 22,536 continued to attenuate i.p. AMPH-induced locomotion across days 1, 3, and 5 relative to intra-VTA saline (F1,34 = 5.30,p < .05; Fig. 3b), locomotor responses to i.p. AMPH were elevated in both saline- and SQ 22,536-pretreated rats on day 5 relative to day 1 (day effect: F2,34 = 6.81, p < .005), with no drug × day interaction present (F2,34 = 1.88,p = .17). Within-subject ANOVA revealed a significant effect of day (F2,22 = 8.34,p < .005) on the locomotor response to i.p. AMPH in SQ 22,536-pretreated rats (Fig. 3b), suggesting tolerance to the inhibitory effect of SQ 22,536 on AMPH-induced locomotion, sensitization to repeated AMPH, or both.
AMPH Sensitization.
Whether repeatedly administered i.p. (Fig.4) or microinjected into the VTA (Fig.5), three prior exposures to AMPH on days 1, 3, and 5 induced a significant behavioral sensitization to i.p. AMPH challenge on day 15. However, the sensitization that resulted from intra-VTA AMPH was differentially modulated by intra-VTA administration of SQ 22,536 relative to the sensitization induced by i.p. AMPH. Two-way ANOVA (repeated VTA pretreatment × repeated i.p. treatment) indicated that SQ 22,536 pretreatment 2 min before each of three i.p. AMPH injections on days 1, 3, and 5 was unable to alter i.p. AMPH-induced behavioral sensitization on day 15 (VTA × i.p. interaction: F1,23 = 0.02,p = .89; Fig. 4). In contrast, repeated coadministration of SQ 22,536 with each of three intra-VTA infusions of 5 μg/side AMPH fully blocked the VTA AMPH-induced sensitization on day 15 (overall F3,22 = 4.23,p < .02; Newman-Keuls p < .05; Fig.5). Conversely, repeated intra-VTA infusions of CTX resulted in a pronounced sensitization to AMPH challenge (F1,14 = 6.75, p < .03; Fig. 5).
In general, repeated pretreatment with these cAMP system drugs exerted minimal effects on basal locomotion on day 15 before AMPH challenge (Fig. 5). Across all VTA pretreatment groups, rats that had been treated with i.p. AMPH exhibited greater baseline locomotor activity than i.p. saline-treated rats during habituation on day 15 (F1,41 = 9.36, p < .005), but no effect of VTA pretreatment on basal locomotion was found by two-way ANOVA (F3,41 = 1.94,p > .14; data not shown). It should be noted that three previous microinjections of CTX tended to result in enhanced baseline locomotion (Fig. 5) although with only marginal statistical significance (F1,14 = 4.25,p = .058).
Role of PKA
Acute Locomotor Effects.
No locomotor stimulation was observed after acute bilateral administration of 25 nmol/side of the PKA inhibitor Rp-cAMPS either alone (F1,12= 0.53, p = .48) or when coadministered with 5 μg/side AMPH (F3,22 = 0.98,p = .42), although Rp-cAMPS was found to elicit locomotor activity (F1,11 = 11.78,p < .01) and pronounced dyskinesia at the higher 100 nmol/side dose (data not shown). A single bilateral infusion of PKA activator Sp-cAMPS robustly and dose-dependently stimulated acute locomotion (F2,21 = 5.36,p < .02; Fig. 1a). This behavioral stimulant effect was maximal in the first 10 min after microinjection and persisted less than 40 min (Fig. 1c). Overall one-way ANOVA followed by Newman-Keuls and Dunnett’s post-hoc analysis revealed that significant locomotor stimulation was induced by 100 nmol/side but not by 25 nmol/side Sp-cAMPS (Newman-Keuls p < .05). However, direct comparison of 25 nmol/side Sp-cAMPS with VTA saline by Student’st test revealed slight but statistically significant locomotor activation by this dose of Sp-cAMPS (F1,15 = 6.71, p < .05). When coadministered with 5 μg/side AMPH, both the 25 nmol/side dose (F3,26 = 4.27, p< .02) and 100 nmol/side dose (F3,23= 5.06, p < .008) elicited locomotor activation. The tendency for enhanced locomotion in groups treated with intra-VTA AMPH plus Sp-cAMPS relative to those treated only with Sp-cAMPS (Fig. 1a) did not reach statistical significance at either dose of Sp-cAMPS (Newman-Keuls p > .05), but coadministration of AMPH into the VTA significantly prolonged the locomotor responses to both 25 nmol/side (time × treatment interaction:F15,130 = 1.79, p < .05) and 100 nmol/side (time × treatment interaction:F15,120 = 3.86, p < .0001) Sp-cAMPS (Fig. 1c). Neither Rp-cAMPS (F1,11 = 0.11, p = .75) nor Sp-cAMPS (F1,11 = 0.26,p = .62) altered the acute locomotor response to i.p. AMPH (Fig. 2, a and b).
It is noteworthy that at high doses (100 nmol/side), both Rp-cAMPS and Sp-cAMPS produced postural abnormalities, stereotyped behavior, and, in several animals, bizarre dyskinesias that began within 5 to 10 min and persisted for 25 to 30 min after acute microinjection into the VTA. Stereotyped behaviors induced by Rp-cAMPS and Sp-cAMPS were qualitatively indistinguishable. Diskinesias were characterized by persistent tilting of the head, side-to-side and backward locomotion, tight circling, and “pirouetting” on the hind feet while rearing. These effects were induced by Rp-cAMPS only at 100 nmol/side, but Sp-cAMPS exerted some postural and mild behavioral effects even at 25 nmol/side.
Repeated Administration.
Repeated microinjections of Rp-cAMPS into the VTA did not elicit locomotion on any of days 1, 3, or 5 when administered alone (F1,22 = 2.12,p = .17; Fig. 3a) or in combination with 5 μg/side AMPH (data for days 3 and 5 not shown). Conversely, the PKA activator Sp-cAMPS remained active as a locomotor stimulant across days 1, 3, and 5 (F2,28 = 11.17, p < .002). Although the locomotor response to 100 nmol/side Sp-cAMPS tended to be greater on day 5 relative to days 1 and 3 (Fig. 3a), this effect was not significant when this treatment group was analyzed alone (day effect: F2,12 = 1.03,p = .39) or in comparison with VTA saline or 25 nmol/side Sp-cAMPS (day effect: F2,28= 0.41, p = .67). Similarly, the locomotor stimulation resulting from repeated coadministration of Sp-cAMPS with 5 μg/side AMPH (F2,34 = 16.14, p< .0001) was unchanged across days (data for days 3 and 5 not shown). Thus, no detectable sensitization or tolerance developed to the effects of intra-VTA Rp-cAMPS or Sp-cAMPS when administered repeatedly on days 1, 3, and 5, although Rp-cAMPS altered basal locomotion on days 3 and 5 (Table 1). Neither repeated Rp-cAMPS (F1,26 = 0.07, p = .80) nor repeated Sp-cAMPS (F1,22 = 0.06, p = .81) pretreatment exerted detectable effects on i.p. AMPH-induced locomotion across days 1, 3, and 5 (Fig. 3b).
AMPH Sensitization.
As was the case with SQ 22,536, the sensitization that resulted from intra-VTA AMPH was differentially modulated by drugs acting on PKA relative to the sensitization induced by i.p. AMPH. Two-way ANOVA (repeated VTA pretreatment × repeated i.p. treatment) indicated that Rp-cAMPS pretreatment on days 1, 3, and 5 was unable to alter i.p. AMPH-induced behavioral sensitization on day 15 (VTA × i.p. interaction:F1,22 = 1.53, p = .21; Fig. 4). In contrast, repeated coadministration of Rp-cAMPS with each of three intra-VTA infusions of 5 μg/side AMPH fully blocked the VTA AMPH-induced sensitization on day 15 (overallF3,22 = 6.52, p < .005; Newman-Keuls p < .05; Fig. 5). In addition, Sp-cAMPS augmented only the sensitization induced by repeated i.p. AMPH (VTA × i.p. interaction: F1,21 = 10.31, p < .005). Secondary analysis by one-way ANOVA revealed that repeated pretreatment with 100 nmol/side Sp-cAMPS 2 min before each of three i.p. AMPH injections on days 1, 3, and 5 clearly potentiated i.p. AMPH-induced sensitization on day 15 relative to VTA saline pretreatment (F1,10 = 18.05,p < .002; Fig. 4), whereas coadministration of Sp-cAMPS with intra-VTA AMPH had no effect on VTA AMPH-induced sensitization (Newman-Keuls p > .05; Fig. 5). Despite potentiating i.p. AMPH-induced sensitization, repeated intra-VTA Sp-cAMPS did not itself induce sensitization to AMPH challenge when administered alone (F1,11 = 0.02,p = .90; Fig. 4).
The effects of Rp-cAMPS and Sp-cAMPS on responsiveness to AMPH challenge do not appear to result from alterations in basal locomotion (Fig. 5). Relative to VTA saline, Rp-cAMPS had no significant effect on day 15 basal locomotion (Dunnett’s p > .05). Although prior intra-VTA coadministration of Sp-cAMPS with 5 μg/side AMPH tended to elevate day 15 basal locomotion (Fig. 5), this effect did not reach statistical significance (F3,22= 2.76, p = .07).
Discussion
The current results support and extend previous evidence that cAMP signal transduction in the VTA is involved in the induction of behavioral sensitization to AMPH (Tolliver et al., 1996; Byrnes et al., 1997). The present experiments demonstrate that the cAMP cascade plays an integral role not only in early-withdrawal sensitization (Tolliver et al., 1996) but also in the induction of behavioral adaptations evident at later withdrawal points. Repeated intra-VTA administration of CTX, presumably acting to increase intracellular cAMP production after each infusion, was sufficient to induce a lasting hyperresponsiveness to AMPH challenge. Moreover, the sensitization induced by repeated systemic AMPH was potentiated by repeated activation of PKA with intra-VTA Sp-cAMPS. Conversely, persistent behavioral sensitization induced by repeated intra-VTA AMPH was fully blocked by coadministration of either an inhibitor of AC or an inhibitor of PKA. In addition, the current results suggest that VTA AC and PKA also influence acute AMPH-induced and spontaneous locomotor behavior.
Acute Effects of cAMP System Drugs on Locomotor Activity.
Drugs that presumably elevated cAMP (CTX) or activated PKA (Sp-cAMPS) exerted stimulatory effects on acute locomotor activity when infused bilaterally into the VTA. This result was also observed in preliminary studies using a high dose of 8-Br-cAMP, another phosphodiesterase-resistant analog of cAMP (B. K. Tolliver and S. P. Berger, unpublished observations). In contrast, intra-VTA inhibition of AC with SQ 22,536 had no detectable effect on spontaneous locomotion. Unlike SQ 22,536, the PKA inhibitor Rp-cAMPS was not behaviorally inert when microinjected into the VTA, even at the 25 nmol/side dose. This compound altered basal locomotion even after a single intra-VTA exposure, and repeated administration of Rp-cAMPS alone actually induced sensitization to AMPH on day 15. Paradoxically, the acute postural effects and pronounced dyskinesias that resulted from 100 nmol/side Rp-cAMPS were qualitatively indistinguishable from those produced by its diastereomer Sp-cAMPS. This suggests that either the inhibitory and excitatory stereoisomers share similar nonspecific actions that affect motor behavior or that at a sufficiently high dose, Rp-cAMPS may share the stimulatory effects of the Sp isomer on PKA in vivo. Future experiments using other highly selective PKA inhibitors may be useful in addressing this question.
The neuroanatomical mechanisms by which activators of VTA AC and PKA stimulate acute locomotor activity remain speculative. Although it is reasonable to assume that actions at neuronal rather than nonneuronal cells are responsible for the observed behavioral effects, it is unknown whether CTX and Sp-cAMPS are acting on dopaminergic or nondopaminergic cell bodies within the VTA, on terminals of afferent projections to the VTA, or some combination thereof. Regardless of the cell types affected, the intracellular events that translate elevated cAMP or PKA activity into behavioral stimulation are unclear. Considering the importance of mesolimbic dopamine transmission in spontaneous and stimulant-induced locomotor activity (Roberts et al., 1975; Kelly and Iversen, 1976; Costall and Naylor, 1977; Beninger, 1983) and the high density of dopamine cell bodies in the VTA (Oades and Halliday, 1987; Kalivas, 1993), it is plausible that CTX or Sp-cAMPS microinjection ultimately leads to enhanced firing of mesoaccumbens dopamine neurons, either directly or via complex interactions of VTA neural circuitry. Previous work suggests at least one direct mechanism by which activation of the cAMP cascade in dopamine neurons could stimulate dopamine neurotransmission. Dopamine cell firing is under tonic control by impulse-regulating D2-type dopamine autoreceptors that bind somatodendritically released dopamine (Kamata and Rebec, 1984; White and Wang, 1984a, b; Lacey et al., 1987). Interestingly, PKA-mediated phosphorylation of the D2 dopamine receptor reduces its binding affinity for dopamine (Elazar and Fuchs, 1991). Accordingly, electrophysiological studies using VTA slices have shown that activation of PKA with the cAMP analog 8-bromo-cAMP can increase basal firing rates and significantly decreases the ability of dopamine to inhibit dopamine neuron firing in vitro (Shi and Bunney, 1992). These investigators further demonstrated that inhibition of PKA by the kinase inhibitor H8 or inhibition of AC by SQ 22,536 potentiates the inhibitory effect of dopamine without altering basal firing rate of dopamine neurons in vitro (Shi and Bunney, 1992). Although such a slice preparation lacks intact afferents to the VTA and thus is not fully representative of the synaptic regulation of dopamine neurons in the whole animal, these in vitro results are consistent with the acute locomotor stimulant effects of CTX and Sp-cAMPS observed in the present study.
Currently unknown are whether AMPH itself acutely activates the cAMP cascade in the VTA and, if so, the cell types and receptors and neurotransmitter systems involved. Unlike CTX or Sp-cAMPS, intra-VTA AMPH did not stimulate acute locomotor activity, a result that is consistent with previous studies using a range of AMPH doses (Kalivas and Weber, 1988; Perugini and Vezina, 1994; Cador et al., 1995; Bijou et al., 1996). However, intra-VTA inhibition of AC with SQ 22,536 attenuated the locomotor response to acute systemic AMPH without any effects on basal locomotion. Thus, even if AMPH does not activate the cAMP cascade directly, as does CTX or Sp-cAMPS, AC activity in the VTA appears to be necessary for full expression of acute AMPH-induced locomotor stimulation. Because SQ 22,536 potentiates the inhibitory effect of dopamine without altering basal firing rate of dopamine neurons in vitro (Shi and Bunney, 1992), it is possible that the attenuation of AMPH-induced locomotion by SQ 22,536 is mediated through enhanced autoinhibition of dopamine neurons by somatodendritically released dopamine. This may be difficult to reconcile with the fact that AMPH-induced dopamine release in the nucleus accumbens is not impulse-dependent (McMillen, 1983). Paradoxically, however, stimulation of autoreceptors in the VTA has been shown to inhibit the locomotor stimulant effect of systemic AMPH but not that of cocaine (Steketee and Kalivas, 1992), which exerts impulse-dependent effects on dopamine neurotransmission (Carboni et al., 1989; Nomikos et al., 1990). Future experiments using selective autoreceptor agonists will be useful to address whether tonic AC activity modulates autoinhibition and how this influences AMPH-induced behavioral stimulation.
Behavioral Sensitization.
Although previous studies reported that the sensitization induced by CTX alone was transient, observed at 1 to 3 days but not 14 to 18 days after a single bilateral infusion of CTX into the VTA (Tolliver et al., 1996; Byrnes et al., 1997), the current results demonstrate that repeated elevation of cAMP production with three intra-VTA microinjections of CTX was sufficient to induce a long-lasting sensitization to i.p. AMPH challenge. In contrast, repeated activation of PKA with intra-VTA Sp-cAMPS was not sufficient to induce behavioral sensitization 10 days after the last microinjection, although repeated Sp-cAMPS pretreatment potentiated the sensitization induced by systemic AMPH. It is potentially of mechanistic importance to understand why repeated CTX but not Sp-cAMPS was able to induce sensitization. On the one hand, it is possible that both drugs ultimately activated PKA, either directly (Sp-cAMPS) or subsequent to elevated cAMP (CTX), but only CTX exhibited a sufficiently long duration of action to induce sensitization. Consistent with this is our previous observation that the sensitization induced by a single intra-VTA injection of CTX was blocked by coadministration of the PKA inhibitor H8 (Tolliver et al., 1996), suggesting PKA-mediated phosphorylation in CTX-induced sensitization. Moreover, CTX and Sp-cAMPS clearly differ in their time course of action. Whereas the behavioral effects of CTX microinjection into the brain are delayed several hours in onset but persist for days (Miller and Kelly, 1975; Cunningham and Kelley, 1993; Tolliver et al., 1996;Byrnes et al., 1997), the rapidly induced locomotor response to Sp-cAMPS subsided within 40 min of microinjection (Fig.1).
An alternative explanation of these data is that elevation of cAMP after CTX acts independently of PKA on cellular targets to induce lasting alterations in AMPH sensitivity. Evidence of PKA-independent modulation of neuronal cation currents by cAMP in the hippocampus has been reported (Pedarzani and Storm, 1995). More directly relevant is recent electrophysiological evidence of alterations in synaptic regulation of VTA dopamine neurons after chronic exposure to cocaine or morphine that are cAMP dependent but require the metabolism of cAMP to adenosine (Bonci and Williams, 1996). Although in drug-naive rats D1 receptor activation and subsequent cAMP formation in γ-aminobutyric acid (GABA) axon terminals facilitate GABAB inhibitory postsynaptic potentials (IPSPs) in dopamine neurons (Cameron and Williams, 1993), D1 activation or direct cAMP elevation with forskolin reduces GABAB IPSPs in chronically treated animals (Bonci and Williams, 1996). This effect was reversed by the addition of A1 adenosine receptor antagonists, an inhibitor of transmembrane cAMP transport, or adenosine deaminase, which metabolizes adenosine to inosine (Bonci and Williams, 1996). Because cAMP can be metabolized to adenosine (Rosenberg et al., 1994), and adenosine acting at A1 receptors inhibits GABA IPSPs in VTA dopamine neurons (Wu et al., 1995), Bonci and Williams (1996) concluded that cAMP formed in response to D1 activation is transported out of GABA axon terminals and metabolized to adenosine, where it binds to presynaptic A1 receptors to inhibit GABA release, thereby disinhibiting midbrain dopamine neurons. Such a mechanism may predict that persistent activation of AC, but not of PKA, in the VTA would result in persistently altered dopamine neuron firing rates and, therefore, in a lasting enhancement of basal and stimulant-induced locomotion, as observed in the current study. Coadministration of CTX with drugs that affect adenosine tone will be useful in addressing this hypothesis.
Other effects of D1 dopamine receptor activation in the VTA are thought to be involved in stimulant sensitization and may be relevant to the present results. Intra-VTA antagonism of the D1 receptor is known to block the development of AMPH sensitization (Stewart and Vezina, 1989; Bijou et al., 1996;Vezina, 1996), whereas repeated infusions of D1agonists into the VTA increase locomotor sensitivity to psychostimulants (Pierce et al., 1996). These effects may be due to interactions between dopamine and excitatory acid transmission in the ventral mesencephalon (Kalivas, 1995). In addition to regulating GABA release from afferent projections and interneurons within the VTA, D1 dopamine receptors are localized on the axon terminals of cortical glutamatergic afferents (Mansour et al., 1992;Kalivas, 1993) and facilitate glutamate release on binding somatodendritically released dopamine (Kalivas and Duffy, 1995). Because intact glutamatergic afferents to the VTA (Wolf et al., 1995) and VTA glutamate receptor stimulation (Kalivas and Alesdatter, 1993) are necessary for the development of sensitization, the effects of D1 agents on sensitization may be mediated via cAMP-dependent regulation of glutamate release onto dopamine perikerya. It is possible that in the current study CTX may mimic, and SQ 22,536 may inhibit, the elevation of cAMP in VTA glutamatergic axon terminals that results from D1 activation, thereby stimulating (CTX) or inhibiting (SQ 22,536) glutamate release. This question may be addressed in future studies using coadministration of glutamate receptor antagonists with CTX into the VTA.
Although repeated activation of PKA by Sp-cAMPS alone was not sufficient to induce a lasting behavioral sensitization to AMPH challenge, PKA activity appears to be necessary for the induction of sensitization by intra-VTA AMPH. The ability of repeated coadministration of Rp-cAMPS to fully block AMPH sensitization, together with previous reports that the protein kinase inhibitors H7 (Steketee, 1994) and H8 (Tolliver et al., 1996) can prevent sensitization to cocaine and AMPH, respectively, strongly suggests the involvement of PKA-mediated phosphorylation events in stimulant-induced sensitization. The cellular targets of PKA phosphorylation that may be important for induction of sensitization remain speculative. PKA is known to phosphorylate a wide range of neuronal proteins, including enzymes involved in neurotransmitter synthesis and release, receptors and ion channels, other signal transduction proteins, and transcription factors (Walaas and Greengard, 1991; Hunter and Karin, 1992; Nestler and Greengard, 1994). A number of PKA-regulated proteins, such as tyrosine hydroxylase (Joh et al., 1978, Kim et al., 1993), D2-type dopamine autoreceptors (Elazar and Fuchs, 1991), and ionotropic glutamate receptors (Wang et al., 1991; Raymond et al., 1993) are of fundamental importance in dopamine neurotransmission. The expression or function of several of these PKA-regulated phosphoproteins in the VTA are altered by chronic stimulant treatment (Kamata and Rebec, 1984; White and Wang, 1984a; Beitner-Johnson and Nestler, 1991; Beitner-Johnson et al., 1992; White et al., 1995; Zhang et al., 1997). Whether these alterations are dependent on PKA activity remains unknown but can be tested systematically in future studies.
Finally, it is noteworthy that although behavioral sensitization was induced by both repeated systemic AMPH and intra-VTA AMPH, inhibitors of AC and PKA were able to block only the sensitization induced by intra-VTA AMPH. One possible explanation of these results is that active concentrations of SQ 22,536 and Rp-cAMPS may have reached only a portion of VTA neurons that were affected by systemic AMPH and that adaptations induced by AMPH in the remaining subpopulation of cells were sufficient to induce sensitization. In contrast, assuming equal diffusion from the microinjection site, coadministered intra-VTA AMPH and SQ 22,536 or Rp-cAMPS would be expected to reach roughly the same populations of cells. Thus, in the case of repeated intra-VTA AMPH, any cAMP-dependent adaptations induced by AMPH that lead to sensitization were effectively prevented. Alternatively, these results may suggest that brain regions other than the VTA may participate in the induction of behavioral sensitization by systemic AMPH. Such an interpretation is weakened by evidence that the sensitization induced by systemic AMPH or cocaine can be fully blocked by intra-VTA pretreatment with several diverse classes of pharmacological agents (Stewart and Vezina, 1989;Kalivas and Alesdatter, 1993; Steketee, 1994; Sorg and Ulibarri, 1995). However, it should be noted that the vast majority of microinjection studies establishing the VTA as the site of induction of behavioral sensitization have examined only the ventral midbrain and nucleus accumbens (Dougherty and Ellinwood, 1981; Kalivas and Weber, 1988;Vezina and Stewart, 1990; Perugini and Vezina, 1994; Cador et al., 1995). Of those that have investigated other brain regions, at least two studies have implicated the amygdala in the initiation of sensitization to AMPH or cocaine (Kalivas and Alesdatter, 1993; Wolf et al., 1995). In any case, the current results suggest that the use of both routes of AMPH administration may be warranted in future studies to determine whether nonidentical mechanisms underlie the sensitization induced by systemic AMPH versus that induced by intra-VTA AMPH.
In conclusion, the current results implicate cAMP signal transduction in the VTA in the acute locomotor stimulant effects of AMPH and in long-term AMPH-induced neuroplasticity. Although AC activity in the VTA is necessary for both the acute locomotion and behavioral sensitization induced by AMPH, PKA activity appears to be necessary but not sufficient for the development of sensitization to AMPH challenge. These results suggest several future research questions that may further our understanding of signal transduction mechanisms in sensitization and, ultimately, of neuropsychiatric disorders such as addiction and schizophrenia that are associated with alterations of mesolimbic dopamine neurotransmission.
Acknowledgments
We thank Dr. F. R. Sharp, Dr. S. S. Panter, and the SFVAMC Neurology Research Service for shared equipment, space, and resources. We also thank A. Franco for assistance with data analysis.
Footnotes
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Send reprint requests to: Bryan K. Tolliver, Ph.D., Department of Psychiatry, University of California, San Francisco/SFVAMC #127, 4150 Clement St., San Francisco, CA 94121. E-mail tollivr{at}itsa.ucsf.edu
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↵1 This work was supported by U.S. Public Health Service Award DA-07376 (S.P.B.) and National Research Service Award DA-05715 (B.K.T.).
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↵2 Present address: Department of Psychiatry, University of Cincinnati, P.O. Box 670559, 231 Bethesda Ave., Cincinnati OH 45267.
- Abbreviations:
- AC
- adenylate cyclase
- AMPH
- amphetamine
- CTX
- cholera toxin
- GABA
- γ-aminobutyric acid
- PKA
- protein kinase A
- Rp-cAMPS
- Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine
- Sp-cAMPS
- Sp-adenosine-3′,5′-cyclic monophosphothioate triethylamine
- IPSP
- inhibitory postsynaptic potential
- SQ 22
- 536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine
- VTA
- ventral tegmental area
- Received August 4, 1998.
- Accepted October 26, 1998.
- The American Society for Pharmacology and Experimental Therapeutics