Local hypocretin-1 modulates terminal dopamine concentration in the nucleus accumbens shell

Hypocretins (hcrt), also known as orexins, play a critical role in reward-seeking behavior for natural rewards and drugs of abuse. The mesolimbic dopamine pathway that projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is critically involved in the neural mechanisms underlying reward-seeking and motivation. Hcrt immunopositive fibers densely project to the shell of the nucleus accumbens (NAcSh), suggesting that the NAcSh might be a site for the interaction between hcrt and dopaminergic modulation of reward-seeking behavior. While it is known that hcrt action in the VTA can increase dopamine in the NAc, it has not been determined if hcrt released locally at dopaminergic terminals in the NAcSh can modulate dopamine concentration. Here, we use fast scan cyclic voltammetry (FSCV) in forebrain slices containing the NAcSh to determine whether hcrt can alter evoked dopamine concentration. We found bath application of hcrt-1 increases phasically evoked dopamine release, without altering reuptake at dopamine terminals in the NAcSh. Hcrt-1-induced potentiation of dopamine concentration was inhibited by SB334867, a hcrt receptor 1 antagonist, as well as ionotropic glutamate receptor antagonists, AP-5, CNQX and DNQX. Taken together, these results suggest that local hcrt-1 can modulate dopamine in the NAcSh and may play a role in reward-seeking and appetitive behaviors.


ANIMALS
All protocols were in accordance with the ethical guidelines established by the Canadian Council for Animal Care and were approved by the University of British Columbia Animal Care Committee. C57BL/6J mice were obtained from the University of British Columbia breeding facility.

FAST-SCAN CYCLIC VOLTAMMETRY
Evoked [DA] o was measured using FSCV with carbon-fiber microelectrodes. Carbon fibers (7 μm diameter; Goodfellow) were pulled in glass electrodes and cut to a final exposed length of ∼150 μm. Triangular waveforms (holding at −0.4 V) at 10 Hz (−0.4 to 1.0 V vs. Ag/AgCl at 400 V/s scan rate) were used. Catecholamine release was evoked using electrical stimulation applied with a bipolar stimulating electrode positioned flush with the tissue for local surface stimulation. To evoke [DA] o in the NAcSh, either a single pulse was delivered or 100 Hz, 5 pulses to mimic phasic dopamine release (Rice and Cragg, 2004). The voltammetric electrode was positioned between the tips with the aid of a binocular microscope, and then lowered 50-100 μm into the tissue. Dopamine was identified by characteristic oxidation and reduction peak potentials (approx. +600 and −200 mV vs. Ag/AgCl). To determine the time course of dopamine, the current at the peak oxidation was plotted against time. Relative electrode sensitivities for dopamine were determined by obtaining voltammograms from exogenous application of dopamine (0.1-1 μM) made from stock solutions in 0.1 M HClO 4 immediately before use.

DRUGS
Hcrt-1 or AP-5 was purchased from Tocris (Ellisville), reconstituted in ddH 2 0 and stored in aliquots at −20 • C. Ten minutes prior to drug application aliquots were thawed and diluted to the working concentration in aCSF buffer. CNQX or DNQX was purchased from Sigma (Oakville) reconstituted in DMSO and stored in aliquots, protected from light, at −20 • C. Prior to the experiment, these compounds were diluted in aCSF and used at 1/1000 working concentrations of DMSO. SB334867 was purchased from Tocris (Ellisville) reconstituted in DMSO and stored in aliquots at −20 • C. Prior to the experiment, SB334867 was diluted in aCSF and used at 1/1000 working concentrations of DMSO. Our previous work has demonstrated that 1/1000 DMSO does not influence evoked [DA] o (Mebel et al., 2012).

STATISTICS
Values listed are means ± SEM. Statistical significance was assessed using a paired student t-test comparing a time-point on the baseline prior to drug application to a time-point after drug application. For multiple comparisons, One-Way ANOVA was used unless otherwise indicated. A difference of p < 0.05 was considered significant. Statistical tests were performed with GraphPad Prism v.5.

Hcrt-1 INCREASES PHASIC [DA] o AT TERMINALS
Phasic dopamine release accompanies reward-predicting stimuli (Phillips et al., 2003;Roitman et al., 2004;Day et al., 2007). To test if hcrt-1 was able to modulate terminal [DA] o under phasic conditions, we evoked dopamine release every 5 min with 100 Hz, 5 pulses, a parameter which increases dopamine concentration relative to a single pulse (Rice and Cragg, 2004  The signal decay was fit with a one phase exponential curve to determine the rate of decay 5 min before and 15 min after immediately after hcrt-1 application. The rate of decay, Tau (τ), before and after hcrt-1 application was not significantly different (P > 0.05, n = 6). Bars represent mean and SEM.

DISCUSSION
While several studies have demonstrated that hcrt-1 administered into the VTA can increase dopamine concentration in the NAcSh (Vittoz et al., 2008), NAc core (España et al., 2011) or both (Narita et al., 2006), our data suggests an additional mechanism by which hcrt-1 can modulate dopamine concentrations in the NAcSh. We demonstrate that hcrt-1 promotes local dopamine release in NAcSh slices, suggesting that direct projections of hcrt neurons from the lateral hypothalamus can modulate dopamine release onto medium spiny neurons of the NAcSh. Notably hcrt-1-induced increase of [DA] o under phasic conditions reached a maximum 15 after hcrt-1 application. This delayed effect is likely due to the necessity for the 33 amino acid peptide to penetrate the tissue to reach its receptors. Interestingly, hcrt-1 modulates NMDA receptors on VTA dopamine neurons with a similar time course (Borgland et al., 2006). Hcrt-1 potentiation of local [DA] o was due to an increase in dopamine release as opposed to modulation of reuptake mechanisms as there was no effect of hcrt-1 on the decay of evoked dopamine. Taken together, in addition to hcrt-1 acting at the VTA to increase dopamine concentration in the NAc, hcrt-1 can also act at terminals to modulate release. We observed that hcrt-1 modulated evoked [DA] o under phasic conditions, but not after a single pulse repeated every 2 min. Therefore, it is likely that hcrt-1 is acting indirectly in the NAcSh to increase terminal dopamine release. One possibility is that hcrt-1 modulates GABAergic activity onto dopamine terminals in the NAcSh. While it has not been demonstrated how dopamine  (Garris et al., 1994;Gonon, 1997;Gonon et al., 2000), but may also activate presynaptic dopamine receptors on neighboring glutamatergic afferents (Wang and Pickel, 2002). Furthermore, glutamate released from excitatory inputs in the NAc can also modulate dopamine release, although its effects are complicated by the heterogeneity of expression and location of ionotropic and metabotropic glutamate receptors. There is a large body of evidence suggesting that glutamate is excitatory toward dopamine release (Cheramy et al., 1986;Clow and Jhamandas, 1988;Kalivas et al., 1989;Leviel et al., 1990;Carrozza et al., 1992;Desce et al., 1992;Jin and Fredholm, 1997;Borland and Michael, 2004). However, these studies mainly employ use of microdialysis techniques or dopamine release from isolated cells or synaptosomes. Using FSCV in NAcSh slices, we demonstrate that application of ionotropic glutamate receptor antagonists, AP-5 and CNQX or DNQX alone, significantly inhibited phasically evoked [DA] o . In contrast to the present study, phasically evoked dopamine from dorsal striatum slices was increased in the presence of the AMPA receptor antagonist, GYKI-52466 (Avshalumov et al., 2003(Avshalumov et al., , 2008, the NMDA receptor antagonist, AP-5 or the broad spectrum ionotropic glutamate receptor antagonist, kynurenate (Wu et al., 2000). Moreover, glutamate spillover can activate metabotropic glutamate receptors to inhibit evoked dopamine (Zhang and Sulzer, 2003). A possible reason for the discrepancy between our study and the other studies employing FSCV in slices is that the distribution of ionotropic receptors and glutamatergic inputs may differ between dorsal striatum and NAcSh (Pennartz et al., 1994;Sesack and Grace, 2010). Indeed, NMDA or AMPA receptors increase dopamine efflux in a study employing in vivo voltammetry in the NAc (Svensson et al., 1994) and that DNQX can inhibit quisqualate evoked dopamine concentration in a study employing microdialysis in the NAc (Imperato et al., 1990).
Hcrt-1 acts with similar efficacy at both hcrt-R1 and hcrt-R2 (Sakurai et al., 1998). Because hcrt-1 has been implicated in addiction-related behaviors and modulation of dopamine via its action in the VTA, we were interested in testing if this peptide altered local dopamine release at axon terminals. Interestingly, the hcrt-1-mediated increase in NAcSh dopamine was inhibited by SB 334867, suggesting this effect was mediated by hcrt-R1. Hcrt-R2, and to a lesser extent hcrt-R1 are expressed within the NAcSh (Trivedi et al., 1998;Marcus et al., 2001;Martin et al., 2002). Notably, cell bodies of glutamatergic neuron that project to the NAcSh, including the prefrontal cortex and basolateral amydala, express hcrt-R1 (Trivedi et al., 1998;Lu et al., 2000;Marcus et al., 2001) and it is possible that their axon terminals may also express hcrt-R1. Furthermore, hcrt-1 promotion of glutamate release has been demonstrated in other brain regions such as the VTA (Borgland et al., 2006(Borgland et al., , 2009Wang et al., 2009), amygdala (John et al., 2003) and hippocampus (Stanley and Fadel, 2011). Therefore, it is feasible that activation of hcrt-R1s on glutamatergic inputs in the NAcSh can increase glutamate release. Our results support the possibility that hcrt-R1 activation on glutamatergic inputs can promote dopamine release in the NAcSh.
Dense staining of hcrt terminals in the NAcSh has been observed Baldo et al., 2003), suggesting that hcrt-1 has direct action in the NAc. Local action of hcrt-1 at dopaminergic terminals may potentiate effects of hcrt-1-mediated dopamine release via its action in the VTA. Indeed, other studies have demonstrated that application of hcrt-1 or hcrt-2 into the NAcSh can potentiate dopaminergic activity. For example, in NAcSh slices, when hcrt-2 was co-applied with dopamine, its effect on firing rate was significantly potentiated in 2/3 of MSNs when compared to hcrt-2-induced or dopamine-induced firing alone (Mori et al., 2011). Another study demonstrated that hcrt-1 or hcrt-2 administered directly in the NAcSh potentiated the effects of dopamine receptor agonists on contraversive pivoting behavior (Kotani et al., 2008). Thus, consistent with our study, it is likely that an interaction between hcrt and dopaminergic systems for reward seeking behavior may occur in the NAcSh. Furthermore, one can speculate that local action of hcrt-1 in the NAcSh may potentiate dopaminergic responses mediated by hcrt-1 or other drug action in the VTA. In summary, hcrt-1 increased phasically evoked [DA] o in the NAcSh. This effect required activation of AMPA receptors and hcrt-R1. This study demonstrates an additional mechanism by which hcrt-1 can modulate dopamine release and potentially influence appetitive behaviors.