Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

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 in Nestler 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 G_i or 2) increase activation of AC in the VTA by the stimulatory G protein subunit G_s. For example, chronic exposure to stimulants has been shown to reduce both the function and density of the G_i protein in the VTA (Nestler et al., 1990; Terwilliger et al., 1991; Striplin and Kalivas, 1992). Persistent inactivation of G_i 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 D₁ dopamine receptor, which stimulates cAMP production by activating AC via the G_s 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 G_s-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 G_s 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

Top Abstract Introduction Materials and Methods Results Discussion References

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 (see Experimental 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-Whitney U 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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

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 (F_1,13 = 0.10, p = .76) or when coadministered with 100 nmol/side of the AC inhibitor SQ 22,536 (F_3,22 = 0.39, p = .76; Fig. 1). SQ 22,536 did not stimulate acute locomotion when administered alone at this dose (F_1,11 = 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 (F_1,16 = 4.57, p < .05), although no significant differences in locomotor response (F_2,22 = 1.35, p = .28) were found when intra-VTA AMPH-treated rats were included in the analysis.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of acute bilateral intra-VTA drug infusion on locomotor activity in 60 min after microinjection. All animals also received an i.p. injection of saline immediately before behavioral monitoring began. Data are presented as mean ± S.E.M. A, cumulative locomotor response to intra-VTA AMPH, AMPH plus cAMP agent, or cAMP agent alone. All microinjections were made in a volume of 0.5 µl/side. AMPH was administered at 5 µg/side alone and in all coadministration groups. Microinjections of cAMP drugs were made at the following doses: SQ 22,536 (100 nmol/side), Rp-cAMPS (25 nmol/side), CTX (0.5 µg/side), and Sp-cAMPS (25 and 100 nmol/side). Numbers inside the bars represent the sample size for each treatment group. *Significantly different from intra-VTA saline by one-way ANOVA comparison of saline, AMPH, AMPH plus cAMP drug, and drug alone (p < .05, Newman-Keuls). #Significantly different from intra-VTA saline by Student's t test (p < .05). B, time course of locomotor response to intra-VTA SQ 22,536 or Rp-cAMPS in 10-min intervals from groups depicted in top. The first 60 min correspond to basal locomotion during habituation period, whereas the last 60 min correspond to locomotor activity immediately after microinjection. C, time course of locomotor response to intra-VTA CTX or Sp-cAMPS from groups depicted in top. *Significantly different from intra-VTA saline (p < .05). Significantly different from 100 nmol/side Sp-cAMPS alone (p < .05).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of pretreatment with intra-VTA SQ 22,536 (100 nmol/side, n = 12), Rp-cAMPS (25 nmol/side, n = 6), Sp-cAMPS (100 nmol/side, n = 6), or saline (n = 6) on the locomotor response to acute systemic AMPH (1.5 mg/kg i.p.). All data are presented as mean ± S.E.M. A, time course of locomotor activity in 10-min intervals. The first 60 min correspond to basal locomotion during habituation period. The last 60 min correspond to locomotor activity after i.p. AMPH, given 2 min after the intra-VTA microinjection. *Significantly different from intra-VTA saline (p < .05). B, cumulative locomotor responses over 60 min from groups depicted in left. **Significantly different from intra-VTA saline (p < .005).

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 (F_1,24 = 0.03, p = .87; Fig. 3a). Likewise, repeated microinjections of SQ 22,536 (F_1,22 = 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 (F_1,32 = 10.92, p < .005) was significantly enhanced on days 3 and 5 relative to day 1 (day effect: F_2,32 = 3.54, p < .05; Fig. 3a). However, it should be noted that CTX administration also elevated basal locomotion (F_1,32 = 13.67, p < .005), an effect that was observed even after a single bilateral microinjection on day 1 (Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A, locomotor activity in 60 min after each of three repeated intra-VTA microinjections of AMPH or drugs acting on the cAMP pathway. All animals also received an i.p. injection of saline immediately before behavioral monitoring began on each day tested. Day 1 data are reproduced from Fig. 1. Microinjection volume, doses, and sample sizes are as described in legend to Fig. 1 for acute data in the same rats. Data are presented as mean ± S.E.M. *Significantly different from intra-VTA saline on corresponding day (p < .05). B, effect of intra-VTA pretreatment on locomotion induced by each of three repeated systemic AMPH injections. Day 1 data are reproduced from Fig. 2. *Significantly different from intra-VTA saline pretreatment on day 1 (p < .05).

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: F_1,23 = 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 F_3,22 = 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 (F_1,14 = 6.75, p < .03; Fig. 5).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effects of prior repeated intra-VTA saline (n = 6), SQ 22,536 (n = 12), Rp-cAMPS (n = 6), or Sp-cAMPS (n = 6) on systemic AMPH-induced sensitization 10 days after the last of three AMPH injections. Data are presented as mean ± S.E.M. and correspond to the locomotor response over 120 min to 0.5 mg/kg i.p. AMPH challenge in rats treated previously with repeated i.p. saline or i.p. AMPH (acute and repeated data presented in Figs. 2 and 3). *Significantly different from rats treated previously with repeated i.p. saline in corresponding VTA pretreatment group (p < .05). #Significantly different from intra-VTA saline pretreatment in rats treated with repeated i.p. saline (p < .05). Significantly different from intra-VTA saline pretreatment in rats treated with repeated i.p. AMPH (p < .05).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Sensitization induced by previous repeated intra-VTA AMPH (n = 7 versus intra-VTA saline, n = 8) and effects of coadministration of SQ 22,536 (100 nmol/side, n = 6), Rp-cAMPS (25 nmol/side, n = 6), or Sp-cAMPS (100 nmol/side, n = 6). Sensitization induced by repeated prior intra-VTA CTX (n = 8) is included for comparison. Data are presented as mean ± S.E.M. and correspond to basal locomotion (open bars, 60-min habituation period) and the locomotor response to 0.5 mg/kg i.p. AMPH challenge (filled bars, 120 min). *Significantly different from repeated intra-VTA saline treatment (p < .05). Significantly different from repeated intra-VTA AMPH treatment (p < .05).

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 (F_1,12 = 0.53, p = .48) or when coadministered with 5 µg/side AMPH (F_3,22 = 0.98, p = .42), although Rp-cAMPS was found to elicit locomotor activity (F_1,11 = 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 (F_2,21 = 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's t test revealed slight but statistically significant locomotor activation by this dose of Sp-cAMPS (F_1,15 = 6.71, p < .05). When coadministered with 5 µg/side AMPH, both the 25 nmol/side dose (F_3,26 = 4.27, p < .02) and 100 nmol/side dose (F_3,23 = 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: F_15,130 = 1.79, p < .05) and 100 nmol/side (time × treatment interaction: F_15,120 = 3.86, p < .0001) Sp-cAMPS (Fig. 1c). Neither Rp-cAMPS (F_1,11 = 0.11, p = .75) nor Sp-cAMPS (F_1,11 = 0.26, p = .62) altered the acute locomotor response to i.p. AMPH (Fig. 2, a and b).

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 (F_1,22 = 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 (F_2,28 = 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: F_2,12 = 1.03, p = .39) or in comparison with VTA saline or 25 nmol/side Sp-cAMPS (day effect: F_2,28 = 0.41, p = .67). Similarly, the locomotor stimulation resulting from repeated coadministration of Sp-cAMPS with 5 µg/side AMPH (F_2,34 = 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 (F_1,26 = 0.07, p = .80) nor repeated Sp-cAMPS (F_1,22 = 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: F_1,22 = 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 (overall F_3,22 = 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: F_1,21 = 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 (F_1,10 = 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 (F_1,11 = 0.02, p = .90; Fig. 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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.

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).

View larger version (52K): [in this window] [in a new window]   Fig. 6.   A, representative tract locations of microinjection cannulas in rats treated with intra-VTA saline or 25 nmol/side Rp-cAMPS. Points shown represent accepted cannula placements in the VTA from 4.80 mm to 6.00 mm from bregma according to the atlas of Paxinos and Watson (1986). Animals with tract placements rostral or caudal to these coordinates, or otherwise outside the VTA, were excluded from the behavioral analysis. B, photomicrograph (20×) of a 100-µm section stained with cresyl violet indicating the location and tissue damage associated with bilateral cannulas implantation into the VTA. The tissue was harvested 24 h after the final behavioral test from a rat treated with three prior microinjections of 100 nmol/side SQ 22,536.