Ventral Midbrain NTS1 Receptors Mediate Conditioned Reward Induced by the Neurotensin Analog, D-Tyr[11]neurotensin

The present study was aimed at characterizing the mechanisms by which neurotensin (NT) is acting within the ventral midbrain to induce a psychostimulant-like effect. In a first experiment, we determine which subtype(s) of NT receptors is/are involved in the reward-inducing effect of ventral midbrain microinjection of NT using the conditioned place-preference (CPP) paradigm. In a second study, we used in vitro patch clamp recording technique to characterize the NT receptor subtype(s) involved in the modulation of glutamatergic neurotransmission (excitatory post-synaptic current, EPSC) in ventral tegmental neurons that expressed (Ih+), or do not express (Ih-), a hyperpolarization-activated cationic current. Behavioral studies were performed with adult male Long-Evans rats while electrophysiological recordings were obtained from brain slices of rat pups aged between 14 and 21 days. Results show that bilateral ventral midbrain microinjections of 1.5 and 3 nmol of D-Tyr[11]NT induced a CPP that was respectively attenuated or blocked by co-injection with 1.2 nmol of the NTS1/NTS2 antagonist, SR142948, and the preferred NTS1 antagonist, SR48692. In electrophysiological experiments, D-Tyr[11]NT (0.01-0.5 μM) attenuated glutamatergic EPSC in Ih+ but enhanced it in Ih- neurons. The attenuation effect (Ih+ neurons) was blocked by SR142948 (0.1 μM) while the enhancement effect (Ih- neurons) was blocked by both antagonists (0.1 μM). These findings suggest that (i) NT is acting on ventral midbrain NTS1 receptors to induce a rewarding effect and (ii) that this psychostimulant-like effect could be due to a direct action of NT on dopamine neurons and/or an enhancement of glutamatergic inputs to non-dopamine (Ih-) neurons.


INTRODUCTION
Neurotensin (NT), a tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) isolated from the hypothalamus more than four decades ago (Carraway and Leeman, 1973), acts as a potent modulator of limbic neurotransmission. Cell bodies and terminals that express NT-like immunoreactivity are found in several limbic brain regions including the amygdala, the nucleus accumbens, the prefrontal cortex, the septum, and the ventral midbrain (Jennes et al., 1982;Hökfelt et al., 1984;Woulfe and Beaudet, 1989;Delle Donne et al., 1996). When released from nerve terminals, NT can activate three receptor sub-types, NTS1, NTS2, and NTS3 (see Vincent et al., 1999). The NTS1 and NTS2 are metabotropic receptors that are coupled to G-proteins linked to different signaling pathways such as cyclic guanosine-monophosphate, phospholipase C and mitogen-activated protein kinase. The NTS3 is a non G-protein coupled receptor that possesses a single transmembrane domain; this receptor appears to be non-selective for NT as its binds several other endogenous ligands (see Mazella and Vincent, 2006). The great majority of the central effects of NT have been attributed to its action on either NTS1 or NTS2 receptors. When administered into the lateral ventricle, for instance, NT produces a dose-dependent hypothermia and analgesia that are prevented by the NTS1/NTS2 antagonist, SR142948, but not the preferred NTS1 antagonist, SR48692, suggesting that they are mediated by the NTS2 receptor (Gully et al., 1997). Central NT injections also attenuate spontaneous and methamphetamine-induced locomotor activity, effects that are prevented by SR48692 suggesting that they are mediated by the NTS1 receptor (Wagstaff et al., 1994;Gully et al., 1995). The behavioral effects of NT are not only dependent upon the receptor sub-type that is activated but also upon the site of action of the peptide within the limbic system. In the ventral midbrain, for instance, NT stimulates dopamine impulse flow and dopamine-dependent behaviors (Kalivas et al., 1981;Holmes and Wise, 1985;Rompré et al., 1992) while in the ventral striatum and the prefrontal cortex, it reduces the postsynaptic effect of dopamine and attenuates dopamine-dependent behaviors (Ervin et al., 1981;Kalivas et al., 1984;Béauregard et al., 1992;Stowe et al., 2005). These findings led to the hypothesis that NT may act as either an endogenous antipsychotic-or psychostimulant-like neuromodulator (Bérod and Rostène, 2002;Kinkead and Nemeroff, 2002). The mechanisms by which NT produces psychostimulant-like effects remain imprecise. The main hypothesis is that NT enhances dopamine release and dopamine-dependent behaviors by stimulating dopamine impulse flow through activation of NTS1 receptors expressed on dopamine cell bodies and dendrites (see Bérod and Rostène, 2002). Consistently, NT induces an increase in dopamine inward current and firing rate that is prevented by SR48692 (St-Gelais et al., 2006). Activation of NTS1 receptors expressed on dopamine neurons also inactivates the dopamine auto-receptor which contributes to enhance dopamine impulse flow (Thibault et al., 2011). We, and others, also reported that NT and its Cterminal fragment, NT-(8-13), enhance excitatory post-synaptic currents (EPSCs) in presumed dopamine neurons, an effect that is blocked by SR48692 (Kempadoo et al., 2013;Bose et al., 2015). Unexpectedly, the increase in ventral striatal dopamine release induced by ventral midbrain application of NT is blocked by SR142948 but not by SR48692 (Steinberg et al., 1994;Leonetti et al., 2002) suggesting that NT is rather stimulating dopamine impulse flow through activation of NTS2 receptors. This latter finding, however, was not supported by another study showing that application of ventral midbrain NT enhances ventral striatal dopamine release in NTS2 but not NTS1 knock-out mice (Leonetti et al., 2004). Neurotensin structure-activity studies have also generated conflicting results regarding the role of NTS1 receptors in the psychostimulant-like effect of NT. For example, the enhancement effect of NT on locomotor activity and on brain stimulation reward is mimicked by  and neuromedin N (Kalivas and Taylor, 1985;Kalivas et al., 1986;Gratton, 1992, 1993), two peptides that bind and activate the NTS1 receptor (Kitabgi et al., 1980;Tanaka et al., 1990). But the induction of a conditioned place-preference (CPP) by repeated ventral midbrain NT microinjections is not mimicked by an equimolar concentration of ; in fact it is mimicked by a NT fragment, NT-(1-11), that fails to interact with the NTS1 receptor (Kitabgi et al., 1980;Glimcher et al., 1984); these results suggest that the conditioned rewarding effect of NT may be mediated by a NT receptor other than NTS1 receptor. In order to clarify this issue, we attempted to determine which ventral midbrain NT receptor is involved in the induction of a CPP using the NT analog, [D-Tyr 11 ]NT and the NT receptor antagonists, SR142948 and SR48692.  ]NT mimicks several behavioral and neurochemical effects of NT. When administered into the cerebral ventricle, for instance, NT and [D-Tyr 11 ]NT enhances brain stimulation reward (Rompré, 1995;Bauco and Rompré, 2001) and sensitizes to the locomotor activating effect of amphetamine (Rompré, 1997). Ventral midbrain infusion of NT and [D-Tyr 11 ]NT stimulates locomotor activity (Bauco and Rompré, 2003) and enhances mesoaccumbens DA release Sotty et al., 2000). But, when injected unilaterally, [D-Tyr 11 ]NT is more effective than NT at inducing circling behavior  and less effective at enhancing mesoprefontal DA release (Sotty et al., 2000). These findings are consistent with previous results showing than  ]NT is a NT agonist that may preferentially activates one sub-type of NT receptors (Kitabgi et al., 1980;Labbé-Jullié et al., 1994); it thus constitutes an useful pharmacological tool to sort out the role of each of these receptors in behavior. Because NT and NT-(8-13) also enhance ventral midbrain glutamatergic neurotransmission, we characterized the effect of [D-Tyr 11 ]NT on glutamatergic EPSCs in putative ventral midbrain dopamine and non-dopamine neurons; the two populations were distinguished by the presence, or the absence, of a hyperpolarization-activated cationic current (Margolis et al., 2006)

Behavioral Experiments
Animals Male Long-Evans rats (Charles River, St-Constant, Qc, Canada) weighing 280-320 g at the time of surgery were used. They were housed 1 (after surgery) or 2 per cage in a temperature (22 ± 1 • C) and humidity (40-50%) controlled room with a 12 h light/dark cycle (lights on 06:00); standard rat chow and water were available ad libitum. All testing was performed during the light phase of the light-dark cycle. All animal experimental procedures were approved by the Institutional Animal Ethics Committee (Comité de déontologie de l'expérimentation sur les animaux de l'Université de Montréal), in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (n • : 85-23, revised 1996). All efforts were made to minimize the suffering and number of animals used.

Surgery
Following 1 week habituation period to the colony room, each rat was anesthetized with isoflurane (2.5-3.5%, 0.75 L/min O 2 ); solutions of 0.1 ml of Anafen (5 mg/kg, s.c.) and 0.05 ml (i.m.) of duplocillin LA containing 15,000 I.U. of penicillin were administered to prevent inflammation and infection. The animals was then mounted on a stereotaxic apparatus, the surface of the skull was exposed and a guide cannula (Model C315G, Plastic One, VA, USA,) was implanted in each hemisphere, above the ventral tegmental area (VTA), using the following stereotaxic coordinates: 5.5 mm posterior to bregma, 1.7 mm lateral and 6.3 mm below the surface of the cranium (Paxinos and Watson, 1986); cannulae were inserted into the brain with a mediolateral angle of 8 • and were closed with an obturator of the same length. Four stainless-steel screws were threaded into the bone and the cannulae were anchored to the skull with dental acrylic. Behavioral tests started 1 week after the surgery.

Conditioned Place Preference (CPP) Paradigm
The CPP apparatus (Med Associates, St. Albans, VT, USA) consisted of a rectangular Plexiglas box divided into two large compartments (26 × 21 × 21 cm) separated by a smaller central compartment (21 × 12 × 21 cm). Two sliding doors separated the central gray compartment from the two others which have distinct wall colors (white or black) and floors (grid or bar). Locomotor activity and times spent in each chamber were measured by computer-interfaced infrared photobeams (Med Associates, St. Albans, VT, USA). The CPP experiment lasted 10 days and consisted of a habituation phase, a conditioning phase and a test phase. On the first day of the habituation phase, rats were allowed to explore the entire CPP apparatus for 20-min to reduce neophobia. On day 2, all animals received a first intra-VTA injection of 0.5 µl/side of saline and were allowed to explore freely the entire CPP apparatus for 20-min. On the third day, animals were allowed to explore the entire CPP apparatus for 20-min and time spent in each of the two large compartments was measured; rats exhibiting higher or lower time interval than 20-80% of time in a compartment were excluded (unbiased procedure). Conditioning began the next day. Conditioning trials lasted 30-min and were conducted daily for 6 days. Control and drug treatment groups were conditioned in either the black or the white compartment of the apparatus. On the first day of the conditioning phase, the drug-conditioned animals were injected in the VTA with vehicle and were immediately placed into one compartment of the apparatus for 30 min. The next day, animals were injected with [D-Tyr 11 ]NT (1.5 or 3 nmol/0.5 µl/side), SR142948 (1.2 nmol/0.5 µl/side), SR48692 (1.2 nmol/0.5 µl/side), [D-Tyr 11 ]NT (3 nmol/0.5 µl/side) + SR142948 (1.2 nmol/0.5 µl/side) or [D-Tyr 11 ]NT (3 nmol/0.5 µl/side) + SR48692 (1.2 nmol/0.5 µl/side) and were immediately placed into the other compartment of the apparatus for 30 min. This procedure was repeated three times so that rats received three vehicle (Day 4, 6, and 8) and three drug (Day 5, 7, and 9) injections. Animals in the control group were injected with the vehicle on each day and were similarly conditioned for 6 days. Twenty four hours after the last day of the conditioning phase, on day 10, animals were allowed to explore the apparatus for 20 min and the time spent in each compartment was measured. Animals were tested between 11:00 and 17:00 under an ambient light intensity of 5 lux and were habituated to the experimental room for 1-h prior to the behavioral testing.

Microinjection Procedure
Bilateral microinjections were made by inserting into each guide cannula an injection cannula (model C315I) that extended 2 mm beyond the tip of the guide. Each cannula was connected with polyethylene tubing to a 2-µl microsyringe and a volume of 0.5 µl of solution was injected into each hemisphere simultaneously with a micro-infusion pump over a period of 60 s; cannulae were left in place for an additional 60 s to allow diffusion into the surrounding brain tissue.

Histology
At the end of the experiment, animals were deeply anesthetized with urethane (2 g/kg, i.p.) and transcardially perfused with 0.9% saline followed by 10% formalin. Brains were removed, stored in 10% formalin and subsequently sliced in serial 40-µm sections that were stained with formal-thionin solution. Locations of the injection sites were determined under light microscopic examination. Only animals that had both injection sites within the VTA, including the rostral and caudal linear nuclei, the paranigral, parabrachial, and the interfascicular nuclei between 5.0 and 6.0 mm behind bregma (Paxinos and Watson, 1986) were included in the analyses.

Drugs
[D-Tyr 11 ]neurotensin-(1-13) was purchased from Bachem (Sunnydale, CA, USA) and dissolved in sterile 0.9% saline at a concentration of 3 or 6 nmol/µl. The neurotensin antagonist, SR-142948 and SR-48692 were purchased from Tocris Bioscience (Burlington, ON, Canada) and were dissolved at a concentration of 2.4 nmol/µl in a sterile 0.9% sodium chloride solution that contained 20% dimethylsufoxyde (DMSO). All solutions were stored at −20 • C in 50 µl aliquots in silicone-coated tubes; they were thawed just before testing and were used only once.

Statistical Analysis
Preference score was determined by subtracting the time spent in the drug-paired compartment measured before the conditioning phase (Pre) to the time spent in the same compartment measured on the conditioning test day (Post). Preference score and locomotor activity (horizontal and stereotypic-like movements) measured during the conditioning test day were analyzed with a One-way analysis of variance (ANOVA). The Duncan's multiple range posthoc tests was used for individual group comparisons. The accepted value for significance was set at 0.05 (Statistica V5.0, StatSoft).

Animals and Slice Preparation
Fourteen to 21-day-old (P14-P21) Long Evans pups of either sex obtained from Charles River (St-Constant, QC) were used. Pups were anesthetized by methoxyflurane vapor inhalation in a closed chamber, decapitated and their brain quickly removed and transferred to chilled, oxygenated artificial cerebrospinal fluid (ACSF) in which NaCl had been replaced by equivalent osmolarity of sucrose and containing (in mM) sucrose 252 (NaCl 126 in standard ACSF); KCl, 3; NaH 2 PO 4 , 1.25; MgSO 4 7 H 2 O, 1.3; CaCl 2 , 2.5; NaHCO 3 , 26; and glucose, 10, and saturated with a gas mixture of 95% O 2 and 5% CO 2 . Two hundred and fifty micrometer thick horizontal slices preserving the VTA afferents (Margolis et al., 2006) were cut using a vibrating microtome (DSK Microslicer). Slices were transferred to a submerged recording chamber maintained between 32 and 34 • C and superfused with standard ACSF at a rate of 2 ml/min; slices were incubated for at least 1 h before recording began.
Whole-cell recordings were made with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA) in continuous single-electrode voltage-clamp mode. The output of the amplifier was fed into a LPF 200A DC amplifier/filter (Warner Instruments Corp., Hamden, CT, USA) and digitized at 5-10 kHz with a real-time acquisition system (CED 1401 Power). Data acquisition was achieved using the Signal 4.0 software (Cambridge Electronic Design, Cambridge, England). Recording pipette's capacitance was optimally adjusted before whole-cell configuration was achieved. The resting membrane potential was measured just after rupturing the cell membrane and the offset potential, measured upon withdrawal of the electrode from the cell, was accounted for assuming that it drifted in a linear fashion with time from the start of the recording session. We did not correct for liquid junction potential which for a pipette containing 140 mM potassium gluconate amounts for an additional potential shift of around −10 mV (Spigelman et al., 1992).

Synaptic Activation and Drug Application
The presence of I h current was determined by voltage clamping cells at −60 mV and stepping to −40, −50, −70,−80, −90, −100, −110, and −120 mV. Input resistance was monitored with hyperpolarizing pulses in current clamp mode. A monopolar tungsten stimulating microelectrode was placed rostral to the recording site in the medial VTA, on the slice superficial layer, 0.5-1.0 mm from the recording electrode. Excitatory postsynaptic currents were evoked by 0.1 ms, 3-6 V cathodal pulses delivered at 15 sec intervals. In order to isolate glutamate receptor-mediated EPSCs, all experiments were performed in the presence of (−) bicuculline methiodide (BMI, 10 µM) in bath solution to block GABA A receptor-mediated synaptic currents. BMI was applied 30 min before obtaining whole-cell configuration to ensure a complete diffusion in the slice tissue. In all experiments the EPSCs were recorded from an online voltage-clamped potential of −70 mV. The effects of D-Tyr [11] NT on glutamatergic EPSCs were assessed at a holding membrane potential of −70 mV. Three concentrations of the peptide (0.01, 0.1, and 0.5 µM) were tested, one concentration per cell. Upon agonist application, the change in amplitude of the glutamatergic EPSC was measured. Five minutes of baseline EPSC activity was recorded before superfusion with the peptide. The EPSC amplitudes were recorded during 7 min after the onset of the peptide application and averaged over the last 5 min. A washout period of 15 min was allowed before the amplitude of the recovered EPSC was measured. In some experiments, the control EPSC amplitude was measured for 4 min before a NT antagonist was added to the superfusion medium. SR142948 nor SR48692 produced any change in EPSC amplitude (n = 12; data not shown); therefore in further experiments where NTS receptor antagonists were used, SR142948 or SR48692 was added to the superfusing medium for 7 min and a control response was measured in the presence of the antagonist.

Drugs and Peptides
The following pharmacological agents were applied through the superfusing ACSF: (−) bicuculline methiodide obtained from Sigma Aldrich (Oakville, Ontario, Canada); D-Tyr [11] NT from Bachem (Sunnyvale, CA, USA); SR-48692 and SR1429482 obtained from Tocris Biosciences (Burlington, ON, Canada). All drugs were made up as 10 mM stock solutions in distilled water and diluted with ACSF solution to final concentration just before addition to the perfusion medium with the exception of SR48692 which was dissolved in DMSO (final concentration 0.1%) and distilled water.

Data analysis
Data analysis was performed using Signal software (Cambridge Electronic Design, Cambridge, England). The magnitude of EPSC recorded after application of the peptide was expressed as percent of baseline and group means were calculated for drug condition. A One-way ANOVA was performed and Duncan post-hoc test used to determine significant differences between concentration or drug and peptide condition when justified; level of significance was set at 0.05 (Statistica V5.0, StatSoft).

Behavioral Experiment
From the 86 rats that completed the experiment, 10 were excluded from the analysis because the injection sites were dorsal or anterior to the VTA, or because the sites (left and right hemisphere) overlapped on the midline; an additional rat was excluded because the injection sites could not be located. Figure 1 illustrates the preference score (top panel) and locomotor activity (middle and bottom panels) measured during the conditioning test in different groups of rats that were conditioned with the vehicle and one of two doses of [D-Tyr 11 ]NT. As can be seen, animals that were conditioned with VTA [D-Tyr 11 ]NT microinjections spent more time in the peptide associated compartment than the animal conditioned with VTA microinjections of the vehicle. The ANOVA yielded a significant effect of treatment [F (2, 31) = 13.1, p < 0.001] and post-hoc test showed that preference score of each [D-Tyr 11 ]NT group was significantly different than vehicle; although the preference score for the group treated with the highest dose was superior to that of the lower dose there was no significant difference between the two doses (p > 0.05). In order to determine whether the preference for the [D-Tyr 11 ]NT-paired compartment was in part related to a conditioned aversion to the unpaired compartment, we compared the preference score for this compartment and the neutral compartment among the three groups. Animals that were injected with [D-Tyr 11 ]NT spent less time in the unpaired compartment on the conditioned test day than those injected with the vehicle (Figure 2, top panel) but the ANOVA yielded no significant effect of treatment [F (2, 31) = 2.4, p > 0.05]. Moreover, the animals conditioned with the high dose of [D-Tyr 11 ]NT spent slightly less time in the neutral compartment (Figure 2, bottom panel) compared to the other groups but the difference was not significant [F (2, 31) = 2.33, p > 0.05]. Altogether, these results suggest that the reduction in the time spent in the compartment non-associated with VTA [D-Tyr 11 ]NT in the conditioned groups was due to an increase in the amount of time spent in the conditioned but not the neutral compartment, hence confirming the occurrence of a conditioned preference effect.

Ventral Midbrain Microinjection of [D-Tyr 11 ]NT Induced a Conditioned Place Preference
The overall locomotor activity (in the entire apparatus) measured during the conditioned test did not differ between groups suggesting that repeated exposure to VTA [D-Tyr 11 ]NT did not induce conditioned locomotor activity (Figure 1, middle and bottom panels). The ANOVA performed on each measure of activity, horizontal and stereotypy-like, yielded no significant effect of treatment [horizontal activity, F (2, 31) = 0.63 p > 0.05; stereotypy-like activity, F (2, 31) = 0.14, p > 0.05].

[D-Tyr 11 ]NT-Induced a Conditioned Place Preference: Role of NTS1 Receptors
To determine which NT receptor is involved in the induction of a CPP by VTA [D-Tyr 11 ]NT, we compared the preference score obtained from animals that were conditioned with vehicle and [D-Tyr 11 ]NT alone to that of animals conditioned with either SR142948 with [D-Tyr 11 ]NT or SR48692 with [D-Tyr 11 ]NT, or each NT antagonist alone. Results presented in Figure 3 (top panel) shows that SR48692 blocked the induction of a CPP. The ANOVA yielded a significant effect of treatments [F (5, 61) = 5.74, p < 0.001] and post-hoc test showed that the preference score of the group injected with preferred NTS1 antagonist, SR48692, with [D-Tyr 11 ]NT is not significantly different than that of the vehicle injected animals but is significantly different than that of the [D-Tyr 11 ]NT alone injected animals. The NTS1/NTS2 antagonist, SR142948, attenuated the induction of a CPP. When administered alone during the conditioning phase, the antagonists induced no conditioned  Measures of locomotor activity recorded during the preference test for the animals in each treatment group are presented in the middle panel (horizontal) and bottom panel (stereotypy-like). The asterisks and the cross indicate a statistical significant difference with VEH (*p < 0.01; ***p < 0.001) and NT ( + p < 0.05) respectively.
effect. Altogether, these results show that the conditioned preference is due to activation of VTA NTS1 receptors. Locomotor activity measured in the entire apparatus during the conditioned test did not differ between groups [ Figure 3, middle and bottom panels; horizontal activity, F (5, 61) = 1.14, p > 0.05; stereotypy-like activity, F (5, 61) = 0.93. p > 0.05].

ELECTROPHYSIOLOGICAL RESULTS
Whole-cell voltage-clamp recording was carried out on 96 physiologically identified VTA neurons. Neurons were designated as I + h (n = 54) or I − h (n = 42) based on the presence or absence of the hyperpolarization activated cationic current (I h ). The amplitude of I h in I + h positive neurons ranged from 67 pA to 419 pA with an average of 192.3 pA (n = 54; data not shown).

Effects of D-Tyr [11] NT on Glutamatergic EPSCs in I + h and I − h Neurons
The  Figure 4. It can be seen that [D-Tyr 11 ]NT attenuated the EPSC and that this effect was completely reversible. As shown in Figure 5 [D-Tyr 11 ]NT produced a dose dependent reduction in the amplitude of the glutamatergic EPSCs in I + h cells. At concentrations of 0.01, 0.1, and 0.5 µM, the mean decrease in EPSC amplitude was 20 ± 1.5% (n = 6), 29 ± 3% (n = 6), and 47 ± 4% (n = 7) respectively. A Oneway ANOVA yielded significant effect [F (2, 16) = 20.95, p < 0.001] and post-hoc test confirmed that the highest concentration produced a decrease that was significantly different than that produced by the lower concentration (p < 0.001).
In To identify the neurotensin receptor subtype(s) involved in the enhancement and attenuation effects of [D-Tyr 11 ]NT on the evoked EPSCs in each cell population, we measured the EPSCs in the presence of SR142948 or SR48692. We found that in I + h cells, SR48692 (0.5 µM) and SR142948 (0.5 µM) were both effective at blocking the decrease in EPSC amplitude produced by  ]NT (Figure 7, top panel). It can be noted that in the in the presence of this high concentration of the antagonists, [D-Tyr 11 ]NT produced an enhancement of the EPSC amplitude. A One way ANOVA yielded a significant effect [F (2, 11, 89.3) = p < 0.001] and post-hoc test confirmed that the EPSCs measured in the presence of the antagonist were significantly different than the EPSCs measured in the presence of [D-Tyr 11 ]NT alone; there was also a significant difference between the effect of SR142948 and SR48692. Interestingly, different results were obtained when the concentration of the antagonist was reduced to 0.1 µM. At this concentration, SR142948 blocked the attenuation effect of [D-Tyr 11 ]NT while SR48692 had no effect (Figures 4, 7). The ANOVA yielded significant effect [F (2, 11) = 37.7, p < 0.001] and post-hoc test confirmed that the mean EPSC measured in the presence of SR142948 was significantly different than that measured in the presence of [D-Tyr 11 ]NT alone or SR48692 + [D-Tyr 11 ]NT.
In I − h cells, at the highest concentration (0.5 µM), SR48692 and SR142948 blocked the enhancement effect of [D-Tyr 11 ]NT on the EPSC (Figure 7, bottom panel); in the presence of SR142948, [D-Tyr 11 ]NT produced a large attenuation of the EPSC. The ANOVA yielded significant effect [F (2, 14) = 40.6, p < 0.001] and post-hoc test showed that the EPSC measured in the presence of SR142948 and SR48692 was significantly different than that measured in the presence of [D-Tyr 11 ]NT alone. There was also a significant difference in EPSCs measured in the presence of SR142948 and SR48692, confirming that SR142948 led to a significant attenuation. Similarly to what we observed in I + h cells, different results were obtained when the concentration of the antagonists was reduced to 0.1 µM. At this concentration, SR142948 and SR48692 similarly blocked the enhancement effect of [D-Tyr 11 ]NT (Figures 6, 7). The ANOVA yielded significant effect [F (2, 13) = 18.5, p < 0.001] and post-hoc test confirmed that the mean EPSC measured in the presence of SR142948 and SR48692 were not different but were both different than that measured in the presence of [D-Tyr 11 ]NT alone.

DISCUSSION
The main finding of this study is that activation of ventral midbrain NTS1 receptor induces a CPP and that this effect can be mediated, at least in part, through an enhancement of glutamatergic synaptic input in nondopamine neurons in the VTA. Our results also show that [D-Tyr 11 ]NT activates NTS2 receptors to reduce glutamatergic synaptic input to VTA dopamine and nondopamine neurons. To our knowledge these findings constitute the first evidence that NT acts on two different NT receptor sub-types to modulate in an opposite manner glutamatergic neurotransmission in different populations of VTA neurons.
Previous studies have shown that ventral midbrain NT microinjection produces a rewarding effect as it sustains self-administration (Glimcher et al., 1987), enhances brain stimulation reward (Rompré et al., 1992) and induces a CPP (Glimcher et al., 1984). Consistently, we found that ventral midbrain microinjections of [D-Tyr 11 ]NT dose-dependently induced a preference for the environment associated with the peptide. The induction of a CPP by  ]NT is consistent with many other results showing that this NT analog mimics several behavioral, physiological and neurochemical effects of NT (Jolicoeur et al., 1984;Donoso et al., 1986;al-Rodhan et al., 1991;Steinberg et al., 1995;Rompré, 1997). According to Glimcher et al. (1984), however, the induction of CPP by repeated ventral midbrain NT is not reproduced by an equimolar concentration of NT-(8-13); such a result was unexpected because NT-(8-13) displays a high affinity for the NTS1 (Kitabgi et al., 1980) and has been shown to be as effective as NT at inducing locomotor activity (Kalivas and Taylor, 1985;Steinberg et al., 1995), enhancing brain stimulation reward (Rompré and Gratton, 1993), increasing dopamine cell firing (Seutin et al., 1989;Shi and Bunney, 1991) and inducing polydipsia (Hawkins et al., 1989). One possible explanation is that Glimcher et al. (1984) compared the effectiveness of bilateral VTA microinjections of NT to equimolar unilateral microinjections of NT-(8-13); it could be that at the concentration used unilateral microinjections were insufficient to induce a CPP.
The induction of a CPP by [D-Tyr 11 ] was blocked by SR48692 hence suggesting that it is mediated by NTS1 receptors. It has been shown that activation of NTS1 receptors expressed on dopamine neurons stimulates cell firing and dopamine release in brain regions known to play a key role in reward (Woulfe and Beaudet, 1989;Leonetti et al., 2004;St-Gelais et al., 2006;Thibault et al., 2011). Kempadoo et al. (2013) also showed that activation of VTA NTS1 receptors by local NT release reinforces operant responding. Altogether, this suggests that the induction of a CPP by [D-Tyr 11 ]NT could be due to activation of NTS1 receptors expressed on VTA dopamine neurons. A role for NTS1 is in contradiction, however, with Glimcher et al's results showing a CPP is induced by repeated injections of NT-(1-11), a Nterminal fragment that fails to interact with the NTS1 receptor (Kitabgi et al., 1980). To our knowledge, no other studies have reported NT-like effects following central injection of . That suggests that the conditioned rewarding effect of NT-(1-11) is NT-independent. This hypothesis is reinforced by an in vitro study reporting that NT-(1-11) dose-dependently inhibits cortisol production in cultured adrenocortical cells, an effect that is not reproduced by NT-(1-13) and not mediated by any of the known NT receptors (Sicard et al., 2006).
Conditioned place-preference is a learning process that involves neural plasticity. Drugs that induce a CPP, such as cocaine and morphine, induce lasting changes in VTA glutamatergic neurotransmission (Zweifel et al., 2008) and blockade of VTA glutamatergic receptors prevents cocaine- (Harris and Aston-Jones, 2003) and morphine-induced CPP (Harris et al., 2004). Kempadoo et al. (2013) have also shown that the rewarding effect of VTA NT release is associated with an enhancement of glutamatergic input to dopamine neurons. In order to determine whether the induction of a conditioned reward by NT was related to a modulation of glutamatergic inputs to VTA neurons, we investigated the effect of [D-Tyr 11 ]NT on glutamatergic EPSCs in different population of neurons distinguished with the presence or absence of an I h current. Nearly, all dopaminergic neurons express an I h current while I + h neurons represent a subset of non-dopaminergic neurons; some GABA and glutamatergic neurons also express an I h current (Lacey et al., 1989;Johnson and North, 1992;Margolis et al., 2006Margolis et al., , 2012Hnasko et al., 2012).
In the present study, we observed that bath application of varying concentrations of [D-Tyr 11 ]NT generated a dosedependent enhancement in the amplitude of glutamatergic EPSCs in I − h neurons (non-dopamine neurons). This enhancement was most likely mediated by activation of NTS1 receptors as it was blocked by SR142948 and SR48692. These findings are in parallel with the behavioral results and suggest the action of NT on glutamatergic inputs to non-dopamine neurons may also play a key role in conditioned reward. Since the VTA contains a high density of NT terminals, it is thus possible that the effect of NT is not limited to glutamatergic inputs to dopamine neurons in this region (Jennes et al., 1982;Geisler and Zahm, 2006). Luo et al. (2010), for instance, showed that cocaine still induces a CPP in animals that had selective deletion of NMDA receptors onto dopamine neuron, and that this conditioned rewarding effect was NMDA-dependent.
In several limbic regions such as in the entorhinal cortex and the dentate gyrus of the hippocampus, activation of NTS1 receptors induces an excitatory effect and an increase in glutamate release (Rostène and Alexander, 1997;Antonelli et al., 2007Antonelli et al., , 2008; these effects are dependent on coupling to PLC, phosphokinase C (PKC) and Ca 2+ influx through Ltype Ca 2+ channels and activation of myosin light chain kinases respectively (Xiao et al., 2014;Zhang et al., 2015). Additionally, evidence of a facilitatory NTS1-NMDA receptor interaction at cortico-striatal glutamate terminals strengthens the role of NT in modulating glutamate release (Antonelli et al., 2004). Although, within the scope of our study, we were not able to identify the exact effector molecules mediating this action, association of NTS1 receptors to such downstream excitatory signaling cascades might have come into play. When tested over the same range of concentrations, [D-Tyr 11 ]NT generated a dose-dependent attenuation in the amplitude of glutamatergic EPSCs in I + h neurons. This attenuation was most likely mediated by activation of NTS2 receptors as it was blocked by a low concentration of SR142948 but not SR48692. As mentioned previously, all VTA dopamine neurons are I + h and that strongly suggests that [D-Tyr 11 ]NT reduces glutamatergic inputs to at least a population of VTA dopamine neurons. In view of the evidence of a role for VTA dopamine in reward, and of the enhancement effect of NT on VTA dopamine impulse flow, these results were unexpected. They suggest that the action of [D-Tyr 11 ]NT on glutamatergic inputs to VTA dopamine neurons through activation of NTS2 receptors is unlikely involved in the induction of a conditioned reward. In fact, the action of [D-Tyr 11 ]NT on NTS2 should oppose its action on NTS1 and contribute to reduce its effectiveness at inducing a conditioned reward. This may explain why SR142948 which displays a similar affinity for NTS1 and NTS2 (Gully et al., 1997) was less effective than SR48692, a preferred NTS1 antagonist (Gully et al., 1995), at attenuating the induction of CPP.
There is also a large proportion of VTA GABA neurons that express an I h current and it has been shown that GABA provides an inhibitory drive to dopamine neurons that is under the control of glutamate (Grace et al., 2007). An attenuation of glutamatergic input to these neurones is likely to enhance dopamine impulse flow resulting in reward and/or reward enhancement. If [D-Tyr 11 ] NT is acting on NTS2 receptors to reduce glutamatergic EPSCs to these neurons, SR142948 would have been more effective than SR48692 at attenuating the induction of CPP; but as mentioned above, we observed the opposite.
The attenuation effect of [D-Tyr 11 ]NT on glutamatergic EPSCs in I + h neurons contrasts with the enhancement effect of NT and NT-(8-13) reported in previous studies (Kempadoo et al., 2013;Bose et al., 2015). Indeed both NT and NT-(8-13) enhance glutamatergic EPSC in I + h neurons by activating NTS1 receptors. Kempadoo et al. (2013), however, observed a biphasic effect with NT-(8-13), an enhancement of NMDA EPSCs at a low concentration and an attenuation at a high concentration; the former but not the latter was blocked by SR48692 suggesting that the attenuation is not mediated by the NTS1 receptor. It thus appears that both NTS1 and NTS2 modulate glutamatergic inputs to I + h neurons and that [D-Tyr 11 ]NT has a predominant effect on the NTS2 receptor subtype. [D-Tyr 11 ]NT has a higher affinity for NTS2 than NTS1 (Kitabgi et al., 1980;Labbé-Jullié et al., 1994) and activation of NTS2 receptors do not induce excitatory effects. For instance, activation of human NTS2 receptors expressed on CHO cell lines lacks the potential to elevate intracellular Ca 2+ levels by mobilizing internal calcium reserves or accumulation IP3; it was rather associated with activation mitogen activated protein kinases (MAPK) that led to inhibition (Sarret et al., 2002). It could be that activation of NTS2 receptors on putative dopamine neurons enhances MAPK signaling and produces a reduction in glutamatergic signaling. Although there is no evidence reported in the literature that [D-Tyr 11 ]NT can generate a NTS1-or NTS2-mediated biased signalization, it has been recently reported that modified analogs of NT-(8-13) at the 11 amino acid position displayed NTS1-mediated biased activation of G αq and β-arrestin signaling pathways (Fanelli et al., 2015).
The 11th position substitution in [D-Tyr 11 ]NT by a Dtyrosine residue makes it more resistant to cleavage by endopeptidases (Checler et al., 1983). In fact, after an intracerebroventricular injection of NT, 98% of the NT was cleared and degraded in brain tissues during a 30 min period after the injection. Under the same conditions, 33% of [D-Tyr 11 ]NT was retained, suggesting a half-life 1.5 times greater than that of NT (Checler et al., 1983). Owing to the relatively stable metabolic profile of [D-Tyr 11 ]NT it is possible that the reduction in EPSC observed by Kempadoo et al. (2013) with higher concentrations of NT8-13 reflects that produced by the lower concentrations of  ]NT used in the present study.
Interestingly, in the presence of a high concentration (0.5 µM) of SR142948, but not SR48692, [D-Tyr 11 ]NT produced an opposite, significant inhibition, of glutamatergic EPSCs in I − h neurons. This could possibly arise because at this concentration, SR142948 interacts with an NT receptor subtype other than NTS1 and NTS2 (possibly NTS3). The NTS3 protein and its mRNA are present in VTA and are mainly expressed on cell bodies and dendrites. According to Mazella et al. (1998), the NTS3 receptor is nearly insensitive to SR48692. Others, however, reported that SR142948 is effective at blocking the NTS3-mediated growth response to NT in cancer cells (Dal Farra et al., 2001); that would rather exclude a role for this receptor in the opposite effect of [D-Tyr 11 ]NT on EPSC in the presence of SR142948. In I + h neurons, the presence of a high concentration of SR142948 and SR48692 had the same impact; [D-Tyr 11 ]NT enhanced the EPSC, an effect opposite to what was observed when it was infused alone. As mentioned previously, it remains unclear why the antagonists produce such a reverse effect.

AUTHOR CONTRIBUTIONS
PR and KR designed the behavioral experiments; KR carried out the behavioral experiments and analyzed the data with PR. RW and PB designed the electrophysiological experiments; PB carried out the electrophysiological experiments and analyzed the data with RW and PR. All authors contributed and approved the final version of the manuscript.

FUNDING
Funding for this study was provided by Canadian Institutes of Health Research (Grant #102572 to PR and RW), Natural Sciences and Engineering Research Council of Canada (Grant # 184095-2009 to RW) and Fonds de Recherche Santé Québec.