Presynaptic action of neurotensin on dopamine release through inhibition of D2 receptor function
© Fawaz et al. 2009
Received: 8 October 2008
Accepted: 14 August 2009
Published: 14 August 2009
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© Fawaz et al. 2009
Received: 8 October 2008
Accepted: 14 August 2009
Published: 14 August 2009
Neurotensin (NT) is known to act on dopamine (DA) neurons at the somatodendritic level to regulate cell firing and secondarily enhance DA release. In addition, anatomical and indirect physiological data suggest the presence of NT receptors at the terminal level. However, a clear demonstration of the mechanism of action of NT on dopaminergic axon terminals is lacking. We hypothesize that NT acts to increase DA release by inhibiting the function of terminal D2 autoreceptors. To test this hypothesis, we used fast-scan cyclic voltammetry (FCV) to monitor in real time the axonal release of DA in the nucleus accumbens (NAcc).
DA release was evoked by single electrical pulses and pulse trains (10 Hz, 30 pulses). Under these two stimulation conditions, we evaluated the characteristics of DA D2 autoreceptors and the presynaptic action of NT in the NAcc shell and shell/core border region. The selective agonist of D2 autoreceptors, quinpirole (1 μM), inhibited DA overflow evoked by both single and train pulses. In sharp contrast, the selective D2 receptor antagonist, sulpiride (5 μM), strongly enhanced DA release triggered by pulse trains, without any effect on DA release elicited by single pulses, thus confirming previous observations. We then determined the effect of NT (8–13) (100 nM) and found that although it failed to increase DA release evoked by single pulses, it strongly enhanced DA release evoked by pulse trains that lead to prolonged DA release and engage D2 autoreceptors. In addition, initial blockade of D2 autoreceptors by sulpiride considerably inhibited further facilitation of DA release generated by NT (8–13).
Taken together, these data suggest that NT enhances DA release principally by inhibiting the function of terminal D2 autoreceptors and not by more direct mechanisms such as facilitation of terminal calcium influx.
Neurotensin (NT) is a peptide originally isolated from bovine hypothalamus . It is found in the CNS and gastrointestinal tract. In the CNS, NT acts as a neurotransmitter or neuromodulator and one of its better known actions is to modulate dopaminergic transmission within the mesolimbic and nigrostriatal pathways . In addition, a number of studies suggest that NT may be implicated in the pathophysiology of CNS disorders including schizophrenia, Parkinson's disease and drug abuse [2–5].
Considerable efforts have been made to characterize how NT acts to enhance dopamine (DA) release. When applied to the ventral tegmental area (VTA), NT increases the firing rate of DA neurons and DA release in terminal fields of the nucleus accumbens (NAcc) and the prefrontal cortex[6–8]. Moreover, acute microinjection of NT into the VTA enhances motor activity and facilitates DA-dependent behaviors[5, 6, 8]. At a mechanistic level, recent work has established that somatodendritic NT receptors enhance the firing rate of DA neurons through a Ca2+-dependent mechanism .
At the terminal level, NT also acts to enhance DA release. First, anatomical evidence for a presynaptic localization of NT receptors has been provided [10–12]. Second, NT facilitates K+-evoked and electrically-evoked DA release in dorsal striatal slice preparations [13, 14] as well as in dorsal striatum in vivo . These results support a presynaptic effect of NT on DA neuron axon terminals, but previous studies have not identified the mechanism involved or excluded an indirect mechanism of action. Recent work studying glutamate cotransmission in cultured DA neurons failed to provide support for a direct excitatory effect of NT on axon terminals . In addition, a previous preliminary report failed to detect an enhancement of DA release evoked by single electrical pulses in NAcc slices . Thus, the mechanism of action of NT on dopaminergic axon terminals remains unclear.
In the present study, we used fast-scan cyclic voltammetry (FCV) to better characterize the presynaptic action of NT in the NAcc. We find that although NT fails to increase DA release evoked by single pulses, it strongly enhances DA release evoked by pulse trains that lead to prolonged DA release and engage D2 autoreceptors. Our results suggest that NT acts to enhance DA release by inhibiting the function of terminal D2 autoreceptors.
Although multiple aspects of the action of NT at the cell body level in the VTA have been investigated [26–28], the mechanism of action of NT on dopaminergic axon terminals is still unclear. The present results provide new insight into the mechanism mediating the facilitation of DA release by NT at the level of axon terminals in the NAcc. We show that this mechanism implicates a decrease by NT of the effectiveness of terminal D2 autoreceptors that normally inhibit DA release. We also demonstrate that the effects of NT on DA release differ greatly depending on the electrical stimulation parameters used to elicit this release. On the one hand, NT fails to alter DA overflow triggered by single pulses in the NAcc. On the other hand, NT strongly enhances DA release evoked by pulse trains that generate prolonged DA release and strongly engage D2 autoreceptors.
NT is known to facilitate K+ and electrically-evoked DA release in dorsal striatal slice preparations [13, 14] as well as in vivo . Although these observations imply a presynaptic effect of NT on dopaminergic axon terminals, previous results have not excluded an indirect mechanism of action. For example, NT could act through receptors located in the striatum on other elements than dopaminergic axon terminals. These receptors could for instance be located on corticostriatal glutamatergic axon terminals. If this were the case, NT could facilitate the spontaneous release of glutamate, which would then enhance DA release by depolarizing dopaminergic axon terminals. Although we cannot formally exclude this possibility or other possible indirect mechanisms, a major role for glutamate in mediating the ability of NT to enhance train-evoked DA overflow is not easily reconcilable with our observation that this facilitatory effect of NT is blocked in the presence of the D2 antagonist sulpiride. However, we cannot exclude a possible partial implication of postsynaptic D2 receptors located on NAcc medium spiny neurons, through some retrograde signaling mechanism.
Previous experiments have explored the characteristics of DA D2 autoreceptors under different stimulation conditions varying the pulse/train duration or interpulse intervals, but didn't directly address the issue of the control of DA release by NT at the terminal level [22–25]. Nonetheless, a previous preliminary report showed that NT fails to enhance DA release evoked by single electrical pulses in NAcc slices , and suggested that DA release is not directly facilitated by NT. This previous report is consistent with our observation of an apparent lack of effect of NT on DA overflow evoked by single pulses. Although this finding could be taken as arguing against a role of terminal NT receptors in regulating DA release, our findings using train pulse-evoked release shed light on this paradox by showing that NT acts instead to enhance DA release by inhibiting the function of terminal D2 autoreceptors. Our results support a recent report in which the authors monitored glutamate co-release in cultured DA neurons and found that NT does not directly increase glutamate release, but rather attenuates the function of presynaptic D2 receptors that otherwise inhibit glutamate release .
Two of our present findings argue in favor of the hypothesis that NT mainly acts to enhance DA release by inhibiting the function of terminal D2 autoreceptors. First, we show that only pulse train-evoked DA overflow is facilitated by NT. This observation is compatible with previous reports showing that a minimum of 150–300 milliseconds is required for D2 autoreceptor activation to inhibit DA release, conditions that are satisfied during train stimulation, but are not optimal to inhibit release induced by single short pulses [22–25]. In our experiments, the lack of facilitation by sulpiride of single-pulse evoked DA overflow is in favor of this interpretation. Second, we demonstrate that the facilitating effect of NT on pulse train-evoked DA overflow is almost completely prevented by pre-blockade of D2 receptors. An alternate interpretation of this later finding is that in the presence of sulpiride, the releasable pools of DA become depleted during stimulus trains or the release probability cannot be further increased, thus masking any subsequent facilitating effect of NT. Considering our observation that elevating extracellular Ca2+ strongly enhances DA overflow after pre-application of sulpiride, this hypothesis of a ceiling effect is unlikely. Nonetheless, we cannot completely exclude that releasable pools of DA are modified in elevated extracellular Ca2+. Alternate explanations of the ability of D2 receptor blockade to prevent the facilitating effect of NT on DA release thus cannot completely be discounted.
Together, our results thus suggest that in the NAcc, NT acts mainly by inhibiting the function of terminal D2 autoreceptors, leaving room for only a minor contribution of an additional mechanism. Although this second mechanism is presently unidentified, the capacity of NT receptors to mobilize intracellular Ca2+ in DA neurons  leaves opens the possible implication of a Ca2+-dependent priming of synaptic vesicles. Although we have not directly examined the dorsal striatum in the present experiments, our results are compatible with previous biochemical and in vivo microdialysis work also suggesting that NT acts to inhibit the function of terminal D2 autoreceptors in the dorsal striatum [19–21, 29].
In the present study, we found that the preferential NTR1 antagonist SR48692 prevented the facilitatory effect of NT on train-evoked DA overflow. Although we cannot exclude a partial contribution of the type 2 NT receptor (NTR2), our finding is compatible with previous data showing that DA neurons of the VTA and substantia nigra express abundant levels of NTR1 [30, 31], but only modest amounts of NTR2 . Second, the excitatory effects of NT on DA neurons are maintained in NTR2 knockout mice but strongly decreased in NTR1 knockout mice . Compatible with this, SR48692 has been shown to block the ability of NT to increase the firing rate of DA neurons in culture . Finally, SR48692 blocks the ability of NT to reduce the effect of the D2 agonist pergolide on extracellular DA levels in the striatum in microdialysis experiments .
In summary, our results provide a better understanding of the mechanism of action of NT on dopaminergic axon terminals. We suggest that NT through its type 1 receptor enhances DA release mainly by inhibiting the function of D2-type autoreceptors, thus disinhibiting DA release. Future experiments should be oriented toward identifying the specific mechanism involved, such as heterologous desensitization of the D2 autoreceptor by NTR1 or direct receptor-receptor interactions.
Experiments were performed in accordance with the Université de Montréal animal ethics committee guidelines. Sprague Dawley rats, aged 4 to 6 weeks, were lightly anaesthetized with halothane and decapitated. Their brains were rapidly removed and transferred into ice-cold artificial cerebrospinal fluid (aCSF) containing: 125.2 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 0.3 mM anhydrous KH2PO4, 2.4 mM anhydrous CaCl2, 1.3 mM anhydrous MgSO4 and 10 mM D-glucose. NAcc brain slices were then cut on a VT 1000s vibratome (Leica Microsystems, Nussloch, Germany) at a thickness of 400 μm. The brain slices were separated into right and left sections at the mid-sagittal line after transfer to a holding chamber. Slices were allowed to recover for 1 hour at room temperature in the holding chamber in oxygenated (95% O2, 5% CO2) aCSF and then transferred to a custom-built recording chamber and perfused (1 ml/min) with aCSF at 35°C. For experiments performed in the absence of extracellular calcium, the composition of the zero calcium saline was NaCl 140 mM, KCl 5 mM, MgCl2 4 mM, EGTA 1 mM, Hepes 10 mM, Sucrose 4 mM, Glucose 10 mM.
Freshly-cut, disk carbon fibre electrodes of 5 μm diameter were fabricated according to procedures described by Kawagoe et al.  and by Kuhr and Wightman . Electrodes were backfilled with a 4.0 M potassium acetate solution. The background current ranged from 50 to 180 nA. Fast-scan cyclic voltammetry was used to monitor DA release . A triangular voltage waveform (-400 to +1000 mV at a rate of 300 V/s) was applied to the electrode every 100 ms and was computer-controlled using Clampex 9 software and a Digidata 1200B analog to digital converter (Axon Instruments, Union City, CA). This voltage ramp was applied to the electrode via an Axopatch 200B amplifier (Axon Instruments, Union City, CA) in voltage-clamp mode. The background-subtracted voltammograms were used to identify the released substance. Prior work has also established that the primary catecholamine released in the rat NAcc slice is DA . Calibration was performed in vitro for each working electrode before and after the experiment in a 1 μM DA solution, quantifying the peak of the oxidation current after adding DA.
Single-pulse or train-pulse electrical stimulations were generated by an S-900 stimulator (Dagan, Minneapolis, MN) and computer-triggered. Stimulations were applied to the NAcc brain slice through a twisted bipolar tungsten stimulation electrode (Plastics One, Roanoke, VA). The 2 stimulating electrode tips were separated by 100 to 150 μm and were gently placed on the surface of the NAcc brain slice using a micromanipulator (Newport, Fountain Valley, CA). Single-pulse (1 ms) and train-pulse (10 Hz, 30 pulses, 0.1 ms/pulse) stimuli were then generated to evoke DA release. Pulse amplitude was 400 μA for both single and train pulses.
The recording chamber was connected to a temperature-controlled perfusion and aspiration system. The carbon fibre electrode was then inserted ~75 μm into the slice by a piezoelectric micromanipulator (Burleigh Instruments, Victor, NY) and placed at approximately 100 μm in front of the central position of the stimulating electrode tips. After insertion of the carbon fibre electrode, the slice was allowed to recuperate for at least 5 minutes before starting recordings. Electrical stimulations were applied at intervals of 2 minutes. The first 10 minutes served as a control period. The peak of each response was measured and plotted over time.
Chemicals were purchased from Sigma (St. Louis, MO) except for tetrodotoxin (TTX) (Alomone laboratories, Jerusalem, Israel). The 8–13 fragment of NT was used as a NT receptor agonist. It was used at a concentration of 100 nM, shown to produce maximal effects in previous studies . The D2 receptor agonist quinpirole was used at a saturating concentration of 1 μM, while the antagonist sulpiride was used at a saturating dose of 5 μM, since numerous previous physiological studies use these ligands in the micromolar dose range [38–40]. Most drugs were stored in aliquots at -20°C as stock solutions and dissolved into aCSF immediately before use. Calcium, magnesium and glucose were freshly added to the aCSF, which was pre-oxygenated before use.
Data are provided as mean ± SEM, with n representing the number of slices. Simple two group comparisons were performed using Student's t-test. Multiple group means were compared by analyses of variance (ANOVA), followed by a Tukey post hoc test. The minimal significance level for tests was set at p < 0.05. Graphs are expressed in percentage of control, where the control for each slice represents the average of [DA] recorded in normal saline before the entrance of the drug (T = 0 to T = 8). The response after a drug application was then analysed by comparing the maximal effect of the drug with the corresponding time of the control graph.
fast-scan cyclic voltammetry
ventral tegmental area
artificial cerebrospinal fluid
analysis of variance
type 1 neurotensin receptor.
This work was supported by grants (MOP-49591) from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC) to L.-É. Trudeau and by an infrastructure grant from the FRSQ to the Groupe de Recherche sur le Système Nerveux Central. L.-É. Trudeau is supported by a senior scholar award from the Fonds de la Recherche en Santé du Québec (FRSQ). Charbel Simon Fawaz received support from the CIHR, delivered by way of the Summer Scholarship Program of the Université de Montréal. Damiana Leo was supported by a postdoctoral fellowship from the Department of Foreign Affairs and International Trade of Canada. We wish to thank Marie-Josée Bourque for her help with drug preparation and animal maintenance, Shouwei Yang for his helpful suggestions for preparing brain slices and Drs. Pierre-Paul Rompré and Laurent Descarries for their comments on an earlier version of this manuscript.
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