Bidirectional dopaminergic modulation of excitatory synaptic transmission in orexin neurons

Abstract

Orexin neurons in the lateral hypothalamus (LH)/perifornical area (PFA) are known to promote food intake as well as provide excitatory influence on the dopaminergic reward pathway. Dopamine (DA), in turn, inhibits the reward pathway and food intake through its action in the LH/PFA. However, the cellular mechanism by which DA modulates orexin neurons remains largely unknown. Therefore, we examined the effect of DA on the excitatory neurotransmission to orexin neurons. Whole-cell patch-clamp recordings were performed using acute rat hypothalamic slices, and orexin neurons were identified by their electrophysiological and immunohistochemical characteristics. Pharmacologically isolated action potential-independent miniature EPSCs (mEPSCs) were monitored. Bath application of DA induced a bidirectional effect on the excitatory synaptic transmission dose dependently. A low dose of DA (1 microM) increased mEPSC frequency, which was blocked by the D1-like receptor antagonist SCH 23390, and mimicked by the D1-like receptor agonist SKF 81297. In contrast, higher doses of DA (10-100 microM) decreased mEPSC frequency, which could be blocked with the D2-like receptor antagonist, sulpiride. Quinpirole, the D2-like receptor agonist, also reduced mEPSC frequency. None of these compounds affected the mEPSCs amplitude, suggesting the locus of action was presynaptic. Furthermore, DA (1 microM) induced an increase in the action potential firing, whereas DA (100 microM) hyperpolarized and ceased the firing of orexin neurons, indicating the effect of DA on excitatory synaptic transmission may influence the activity of the postsynaptic cell. In conclusion, our results suggest that D1- and D2-like receptors have opposing effects on the excitatory presynaptic terminals impinging onto orexin neurons.

Figure 1.

Identification of orexin neurons. A, Electrophysiological characteristics of an orexin neuron. In a typical orexin neuron, hyperpolarization induced a sag characteristic of an Ih current (arrow head) and rebound depolarization at the current offset (arrow). B, Immunohistochemical identification of recorded orexin neurons. Left, An example of a cell filled with biocytin during recording. Middle, Orexin A immunoreactivity is shown in red. Right, Overlay showing the biocytin labeled cell is orexin A-immunopositive. C, Electrophysiological characteristic of an MCH neuron. A typical MCH neuron displays neither Ih current nor rebound depolarization, but shows a strong spike adaptation. D, Immunohistochemical identification of recorded MCH cell. Left, An example of a cell filled with biocytin during recording. Middle, MCH immunoreactivity is shown in green. Right, Overlay showing the biocytin labeled cell is MCH-immunopositive. Calibration: A, C, 50 ms, 25 mV (same bar applies to A and C). Scale bars: B, D, 20 μm.

Figure 2.

Dopamine induces a bidirectional change in the spontaneous excitatory transmission. A1, Sample traces showing mEPSCs in control condition, in the presence of DA (1 μm) and after wash as indicated. A2, Time-effect plot of the frequency of mEPSCs from a representative cell in the presence of DA (1 μm). A3, A4, Cumulative plot of interevent interval (A3) or amplitude (A4) of mEPSCs from the same cell as A2. B1, Sample traces showing mEPSCs in control condition, in the presence of DA (100 μm) and after wash as indicated. B2, A representative time-effect plot of the effect of DA (100 μm) on the frequency of mEPSCs. B3, B4, Cumulative plot of interevent interval (B3) or amplitude (B4) of mEPSCs recorded from the same cell as shown in B2. C, Summary of the effect of DA on mEPSC frequency at different concentrations. Calibration: A1, B1, 100 ms, 50 pA. *p < 0.05; ***p < 0.005. Error bars indicate SE.

Figure 3.

D₁-like receptor activation increases the frequency of mEPSCs. A, Sample traces showing basal mEPSCs (control), in the presence of SKF 81297 (10 μm) and after wash as indicated. Calibration: 200 ms, 20 pA. B1, A representative time-effect plot showing the frequency of mEPSCs in presence of SKF 81297 (10 μm). B2, B3, Cumulative plot of interevent interval (B2) or amplitude (B3) of mEPSCs from the same cell as shown in B1. C, Time-effect plot of the frequency of mEPSCs depicting the response to DA (1 μm) in a cell pretreated with SCH 23390 (10 μm). D, Summary of the effect of DA 1 μm, SCH 23390 plus DA 1 μm, and SKF 81297 on mEPSC frequency. *p < 0.05; **p < 0.01. Error bars indicate SE.

Figure 4.

D₂-like receptor activation decreases the frequency of mEPSCs. A, Sample traces showing basal mEPSCs (control), in the presence of quinpirole (10 μm) and after wash as indicated. Calibration: 200 ms, 20 pA. B1, Time-effect plot of a representative cell showing the effect of quinpirole (10 μm) on the frequency of mEPSCs. B2, B3, Cumulative plot of interevent interval (B2) or amplitude (B3) of mEPSCs from the same cell as shown in B1. C, Time-effect plot depicting the response of the frequency of mEPSCs to DA (100 μm) in a cell pretreated with sulpiride (10 μm). D, Summary of the effect of 100 μm DA, sulpiride plus 100 μm DA, and quinpirole (10–50 μm) on mEPSC frequency. *p < 0.05; ***p < 0.005. Error bars indicate SE.

Figure 5.

Bidirectional modulation of the firing activity by low and high dose of dopamine. A, DA (1 μm) increases the rate of action potentials in orexin neurons. A1, Sample recording showing the effect of 1 μm DA. A2, Expanded traces from another cell recorded before, during, and after DA application. A3, Time-effect plot of the same experiment as shown in A2. B, DA (100 μm) hyperpolarizes and diminishes the firing activity. B1, Typical recording depicting the effect of 100 μm DA. B2, Traces from another orexin neuron recorded before, during, and after DA application, shown at an expanded time scale. B3, Time-effect plot of the same experiment as in B2. Calibration: A1, B1, 50 s, 20 mV; A2, B2, 5 s, 20 mV.

Figure 6.

D₁- and D₂-like receptors modulate synaptic inputs to the same neuron. A, A time-effect plot of a representative cell showing the frequency of mEPSCs in response to sequential applications of DA (100 μm) and SKF 81297 (10 μm). B, An example of the frequency of mEPSCs modulated in opposite direction by consecutive applications of quinpirole (10 μm) and SKF 81297 (10 μm). C, A typical cell showing the rate of action potential firing being bidirectionally modulated by low and high dose of DA.