The accepted value for significance was P < 0.05 (Statistica V5.0, StatSoft; IBM SPSS Version 23). RESULTS Histology Location of the injection sites for each group of animals that were included in the behavioral and the neurochemical analyses are shown in Figure 1 and ?and22 respectively. D-Tyr[11]Neurotensin (NT, n = 7). Each bar represents the mean ( s.e.m) total activity measured over the two-hour test period. Stars indicate a statistically significant difference with VEH (*p < 0.05; ***p < 0.001). NIHMS6591-supplement-Suppl__2.pdf (5.5K) GUID:?6D87817D-090E-46A4-AF7B-6AB36B0C3ED9 Abstract Previous studies have shown that activation of ventral midbrain NMDA receptors is required for the initiate of sensitization to amphetamine. In view of the recent evidence that neurotensin modulates ventral midbrain glutamate neurotransmission, we tested the hypothesis that neurotensin is acting upstream to glutamate to initiate sensitization to the behavioral and neurochemical effects of amphetamine. During a first testing phase, adult male rats implanted with bilateral Triamcinolone hexacetonide VM cannulae were injected every second day for three days with D-[Tyr11]neurotensin (1.5 nmol/side), the preferred NMDA GluN2A/B antagonist, RS-CPP (40 or 120 pmol/side), the selective GluN2B antagonist, Ro04-5595 (200 or 1200 pmol/side), RS-CPP (40 or 120 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) or Ro04-5595 (200 or 1200 pmol/side) + D-[Tyr11]neurotensin (1.5 nmol/side) and locomotor activity was measured immediately after the injection. Five days after the last central injection, the locomotor response or the expression of phosphorylated extracellular signal-regulated kinase (pERK1/2) in neurons of different limbic nuclei was measured following a systemic injection of amphetamine sulfate (0.75 mg/kg, ip). Results show that amphetamine induced significantly stronger locomotor activity and pERK1/2 expression in the nucleus accumbens shell and infralimbic cortex in neurotensin pre-exposed animals than in controls (vehicle pre-exposed). These sensitization effects initiated by neurotensin were prevented by RS-CPP, but not Ro04-5595. These results support the hypothesis that neurotensin is stimulating glutamate neurotransmission to initiate neural changes that sub-serve amphetamine sensitization and that glutamate is acting on NMDA receptors that are mostly likely composed of GluN2A, but not GluN2B, subunits. (Ellisville, MI, USA) and [D-Tyr11]neurotensin-(1-13) from Bachem (Sunnydale, CA, USA). They were dissolved in sterile 0.9% saline and stored frozen at ?20C in 40C50 l aliquots. Peptide and drug solutions were thawed just before Triamcinolone hexacetonide testing and used only once. Amphetamine sulphate was dissolved in saline and injected intraperitoneally in a volume of 1 ml/kg. Drug and peptide doses were chosen on the basis of previous studies (Bauco and Rompr, 2004; Bergeron and Rompr, 2013; Cador et al, 1999; Kalivas and Duffy, 1990). Statistical analysis Measures of locomotor activity (distance traveled, time of non-ambulatory activity and vertical counts) and number of pERK positive neurons, were totaled for all subjects and group means were calculated. Data were analyzed with a two-way ANOVA (treatment x day) with repeated measures on day (training phase) or a one-way ANOVA (sensitization test and immunohistochemistry results) and differences among means were determined with Duncans multiple range tests. Student T-test was used to compare means of a given treatment obtained at different days. The accepted value for significance was P < 0.05 (Statistica V5.0, StatSoft; IBM SPSS Version 23). RESULTS Histology Location of the injection sites for each group of animals that were included in the behavioral and the neurochemical analyses are shown in Figure 1 and ?and22 respectively. Sites were located within the ventral tegmental area between a rostro-caudal region extending between 5.0 and 5.8 mm posterior to bregma, a region Triamcinolone hexacetonide that is densely innervated by NT-containing terminals and NT receptors (Geisler and Zahm, 2006; Szigethy and Beaudet, 1989). Open in a separate window Figure 1 Location of injection sites for each animal included in the behavioral study. Filled circles indicate the location of the injections for those animals that were injected with the vehicle, CPP or Ro04-5595 alone (left panels). Filled stars indicate the location of the injections for those animals that were injected with the neurotensin (NT), CPP + NT or Ro04-5595 + NT alone (right panels). Illustrations are modified drawings from Paxinos and Watson (1986; numbers in the middle represent the anterior-posterior distance from bregma. Open in a separate window Figure 2 Location of injection sites for each animal included in the immunohistochemical study. See legend of figure 1 for details. Training Phase Microinjections of NT into the VM produced a significant increase in ambulatory and non ambulatory activity, an effect that was stronger after the third injection on RHOJ day 5 (Figure 3, top and middle panels). The ANOVA yielded a significant treatment by day interaction (Ambulatory activity, F5,87 = 9.0 p < 0.001; non-ambulatory activity,.