Neurotensin in the ventral pallidum increases extracellular gamma-aminobutyric acid and differentially affects cue- and cocaine-primed reinstatement

Abstract

Cocaine-primed reinstatement is an animal model of drug relapse. The neurocircuitry underlying cocaine-primed reinstatement includes a decrease in GABA in the ventral pallidum (VP) that is inhibited by a mu opioid receptor antagonist, suggesting that opioid peptides colocalized with GABA in the projection from the nucleus accumbens to the VP may mediate this effect. Neurotensin is also colocalized with GABA and has been shown to increase GABA release in several brain regions. Therefore, the present study determined whether neurotensin increases GABA release in the VP, antagonizes cocaine-induced decreases in GABA, and prevents reinstatement of cocaine seeking. In vivo microdialysis revealed that the neurotensin agonist neurotensin peptide fragment 8-13 [NT(8-13)] increased GABA in the VP in a neurotensin receptor and tetrodotoxin-dependent manner and blocked the cocaine-induced decrease in GABA. NT(8-13) (3 nmol) microinjected into the VP prevented cue-induced reinstatement without affecting cocaine self-administration. In contrast, 3 nmol NT(8-13) potentiated cocaine-primed reinstatement. The neurotensin antagonist SR142948 (2-[[[5-(2,6-dimethoxyphenyl)-1-[4-[[[3-(dimethylamino)propyl]methylamino]carbonyl]-2-(1-methylethyl)phenyl]-1H -pyrazol-3-yl]carbonyl]amino]-tricyclo-[3.3.1.13,7]decane-2-carboxylic acid) had no effect on any behavioral measure when infused in the VP at the dose tested but attenuated cocaine-primed reinstatement when administered systemically. In contrast to reinstatement, NT(8-13) did not alter the motor response to acute cocaine or the development of motor sensitization by chronic cocaine. Three conclusions can be drawn from these data: 1) neurotensin promotes GABA release in the VP and correspondingly inhibits cue-induced reinstatement, 2) neurotensin and cocaine interact in a manner that countermands the neurotensin-induced increase in GABA and promotes reinstatement, and 3) endogenous release of neurotensin in the VP is not necessary for reinstatement.

Fig. 1

Effect of NT(8–13) and SR48692 on extracellular concentrations of GABA in the VP. A and C, effect of increasing doses of NT(8–13) (n = 7) (A) or SR48692 (n = 5) (C) over time. The arrows represent where the drug was added, and dose was increased. The amount of GABA is expressed as a percentage of the average of the baseline samples. The average basal concentration of GABA in the VP was 0.59 ± 0.04 pmol/40 μl in A and B and 0.56 ± 0.05 pmol/40 μl in C and D. B and D, dose-effect representation of the effect of NT(8–13) (B) and SR48692 (D) on the concentration of GABA in the VP where the baseline bar represents the average of the last hour of baseline samples collected, and each dose represents the average of the last two samples collected at that dose.**, p < 0.01, compared with baseline using a Dunnett’s post hoc analysis.

Fig. 2

Blockade of the increase in VP GABA produced by 100 μM NT(8–13) by SR48692 and TTX. A and C, time course of the amount of GABA collected as a percentage of baseline, where the first arrow represents the addition of 10 μM SR48692 (n = 7) (A) or 1 μM TTX (n = 5) (C) to the dialysis buffer, and the second arrow represents the addition of 100 μM NT(8–13) to the dialysis buffer containing SR48692 or TTX (which were kept in the dialysis buffer for the duration of the experiment). The basal concentration of GABA in the VP was 1.2 ± 0.23 pmol/40 μl sample in A and B and 0.53 ± 0.06 pmol/40 μl sample in C and D. B and D, bar graph representation of the average of the last hour of baseline compared with the average of the last two samples collected after each change to SR48692 (B) or TTX (D) alone or in combination with 100 μM NT(8–13).

Fig. 3

Effect of increasing doses of NT(8–13) on extracellular GABA in the VP of rats chronically treated with cocaine for 7 days and withdrawn for 21 to 28 days. A, effect of NT(8–13) (n = 6) over time. The arrows represent where the drug was added, and dose was increased. The amount of GABA is expressed as a percentage of the average of the baseline samples. The average basal concentration of GABA in this experiment was 0.43 ± 0.03 pmol/40 μl sample. B, dose-effect representation of the increase in GABA produced by NT(8–13) in the VP.*, p < 0.05;**, p < 0.01, comparing NT(8–13) with baseline.

Fig. 4

NT(8–13) reverses the cocaine-induced decrease in GABA in the VP of rats chronically treated with cocaine. A, extracellular concentration of GABA in the VP is expressed as a percentage of baseline, where the first arrow represents a switch to vehicle (n = 11) or 3 μM NT(8–13) (n = 11), and the second arrow represents the point where all animals received an injection of cocaine (15 mg/kg i.p.). The basal concentration of GABA in the VP for these experiments was 63.4 ± 8.7 pmol/40 μl sample for the vehicle-treated group and 47.3 ± 5.3 pmol/40 μl sample for the NT(8–13)-treated group. B, bar graph representation of the data shown in A, showing that 1 and 2 h after cocaine, the 3 μM NT(8–13)-treated group had a significantly higher concentration of GABA than the aCSF group.**, p < 0.01;***, p < 0.001, comparing between the two groups using a Dunnett’s post-hoc test.

Fig. 5

Effect of NT(8–13) micro-injected into the VP on cocaine self-administration and reinstatement related behaviors. A, during the maintenance phase of self-administration, rats received a microinjection of vehicle (n = 4) or 3 nmol/side NT(8–13) (n = 4) into the VP, and the number of cocaine infusions self-administered was recorded (test) and compared with the average number of infusions self-administered on the 2 days before the test (pretest) and the 2 days after the test (post-test). B, after microinjection of vehicle (n = 8) or 3 nmol/side NT(8–13) (n = 6) in the VP, rats were tested for cue-primed reinstatement.*, p < 0.05. C, 3 nmol/side NT(8–13) (n = 5, n = 7, n = 6, respectively) or vehicle (n = 6, n = 9, n = 6, respectively) was microinjected into the VP before a 3, 10, or 30 mg/kg i.p. injection of cocaine. D, several doses of NT(8–13) (n = 4–7) or vehicle (n = 5) were administered into the VP, and the amount of active lever presses during extinction was recorded.*, p < 0.05;***, p < 0.001, compared with extinction pressing; ##, p < 0.01, comparing Reinst-3 nmol NT with Reinst-aCSF.

Fig. 6

Effect of SR142948 i.p. and microinjected into the VP on cocaine self-administration and reinstatement related behaviors. A, 1 nmol/side SR142948 (n = 6) or vehicle (n = 8) was microinjected into the VP, and the number of cocaine infusions self-administered was recorded (test) and compared with the average number of infusions self-administered on the 2 days before the test (pretest) and the 2 days after the test (post-test). B, 1 nmol/side SR142948 (n = 9) or vehicle (n = 9) was microinjected into the VP before placing the animal in the operant chamber for day 1 of extinction to determine whether SR142948 could alter extinction pressing. C, 1 nmol/side SR142948 (n = 9) or vehicle (n = 9) was microinjected into the VP before a 10 mg/kg i.p. cocaine injection to determine whether SR142948 could prevent cocaine-primed reinstatement. D, 10 μg/kg SR142948 (n = 6) or vehicle (n = 6) was injected i.p. 1 h. before a 10 mg/kg cocaine-priming injection.*, p < 0.05;**, p < 0.01, compared with extinction pressing.

Fig. 7

Effect of NT(8–13) on locomotor activity induced by a saline injection, 15 mg/kg cocaine, and on the expression of cocaine-induced locomotor sensitization. A, animals were microinjected with vehicle (n = 8) or 0.3 (n = 4) or 3 (n = 8) nmol NT(8–13) into the VP, followed by an i.p. injection of saline. Locomotor activity is shown as the total distance traveled in 10-min bins. B, experiment was carried out as described in A, except that animals received a 15 mg/kg injection of cocaine instead of saline (aCSF; n = 7, 0.3 nmol, n = 4; 3 nmol, n = 7). C, animals were given a sensitizing regimen of cocaine for 7 days, where locomotor activity was measured on day 1 of cocaine administration. After 21 to 28 days of withdrawal, animals were tested for the expression of locomotor sensitization after microinjection of vehicle (n = 6) or 0.3 (n = 7) or 3 (n = 6) nmol/side NT(8–13) into the VP, followed by an i.p. injection of 15 mg/kg cocaine. The graph shows the amount of locomotor activity in the 2 h after the cocaine injection expressed as the sensitization magnitude where the amount of locomotor activity produced on day 1 of cocaine was subtracted from the amount of locomotor activity measured on the sensitization test day in each of the 10-min bins. All values above zero are an increase or sensitization in the amount of locomotor activity produced by cocaine. NT(8–13) had no significant effect on sensitization magnitude.

Fig. 8

Location of all microdialysis probes and microinjections sites used for data analysis. A, illustrations based upon the atlas of Paxinos and Watson (1986) showing the placement of the active zone of the dialysis probes for all experiments. One brain hemisphere was used for each dialysis experiment, but in some animals, the other hemisphere was used for a second experiment. B, illustrative representation of the micro-injection sites for all experiments. Animals with placements outside of the VP were not included in the data analysis and are not shown.