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

Abstract

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.

Keywords: dopamine; glutamate; hypocretin; nucleus accumbens shell; orexin; voltammetry.

Figure 1

Hcrt-1 does not modify single pulse dopamine in the NAcSh. [DA]_o was electrically evoked using a single pulse in slices containing the NAcSh. (A) An example voltammogram from a single experiment for electrically evoked [DA]_o before (black line) and immediately after a 5 min hcrt-1 (100 nM, grey line) application. (B) Bath application of hcrt-1 (100 nM, 5 min, filled circles) did not modify evoked [DA]_o in NAcSh slices (n = 6) compared to control slices (n = 5, open circles) (P < 0.05). (C) A representative current-time plot from a single experiment showing [DA]_o evoked before (open black line) and 15 min after (grey line) application of hcrt-1 (100 nM). (D) 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.

Figure 2

Hcrt-1 increases phasic dopamine in the NAcSh. Phasic [DA]_o was electrically evoked using 100 Hz, 5 pulses in slices containing the NAcSh. (A) An example voltammogram from a single experiment for electrically evoked [DA]_o before (black line) and immediately after a 5 min hcrt-1 (100 nM, grey line) application. (B) Bath application of hcrt-1 (100 nM, 5 min, filled circles) significantly increased evoked [DA]_o in NAcSh slices (n = 11) compared to control slices (n = 6, open circles) (^*P < 0.05). (C) A representative current-time plot from a single experiment showing [DA]_o evoked before (open black line) and 25 min after (grey line) application of hcrt-1 (100 nM). (D) The signal decay was fit with a one phase exponential curve to determine the rate of decay 5 min before and 25 min after immediately after hcrt-1 application. Tau before and after hcrt-1 application was not significantly different (P > 0.05, n = 11). Bars represent mean and SEM.

Figure 3

Hcrt-1 modulation of phasic dopamine in the NAcSh is mediated by hcrt-R1. Phasic [DA]_o was electrically evoked using 100 Hz, 5 pulses in slices containing the NAcSh. Slices were preincubated with SB 334967 (1 μM) for 30 min prior to application of hcrt-1 and throughout the experiment. (A) An example voltammogram from a single experiment for electrically evoked [DA]_o before (black line) and 25 min after a 5 min hcrt-1 (100 nM, grey line) application. (B) In the presence of SB 334867, bath application of hcrt-1 (100 nM, 5 min) did not significantly alter evoked [DA]_o in NAcSh slices (n = 4; P > 0.05).

Figure 4

Hcrt-1 modulation of phasic dopamine in the NAcSh requires glutamatergic signaling. Phasic [DA]_o was electrically evoked using 100 Hz, 5 pulses in slices containing the NAcSh. (A) CNQX (10 μM) and AP5 (50 μM) were bath applied to slices for 20 min prior to application of hcrt-1 and throughout the experiment. An example voltammogram from a single experiment for electrically evoked [DA]_o before (hatched line), during CNQX and AP5 treatment (black line) and 25 min after a 5 min hcrt-1 (100 nM, grey line) application. (B) CNQX and AP5 significantly reduced [DA]_o in NAcSh slices (P < 0.05). In the presence of CNQX and AP5, bath application of hcrt-1 (100 nM, 5 min) did not significantly alter evoked [DA]_o in NAcSh slices (n = 7; P > 0.05). (C) A bargraph demonstrating maximal effects relative to baseline (open bar) of CNQX and AP5 treatment with (filled bar) or without hcrt-1 (shaded bar). CNQX and AP5 with or without hcrt-1 was significantly different from baseline (repeated measures ANOVA with a tukey's post hoc test (n = 7; P < 0.05). (D) An example voltammogram from a single experiment for electrically evoked [DA]_o before (hatch line), during DNQX treatment (black line) and 25 min after a 5 min hcrt-1 (100 nM, grey line) application. (E) DNQX significantly reduced [DA]_o in NAcSh slices (P < 0.05). In the presence of DNQX, bath application of hcrt-1 (100 nM, 5 min) did not significantly alter evoked [DA]_o in NAcSh slices (n = 5; P > 0.05). (F) A bar graph demonstrating maximal effects relative to baseline (open bar) of DNQX treatment with (filled bar) or without hcrt-1 (shaded bar). DNQX with or without hcrt-1 was significantly different from baseline (repeated measures ANOVA with a tukey's post hoc test (n = 5; P < 0.05). Bars represent means and SEM. ^*p < 0.05.