Excitation of ventral tegmental area dopaminergic and nondopaminergic neurons by orexins/hypocretins

Abstract

Orexins/hypocretins are involved in mechanisms of emotional arousal and short-term regulation of feeding. The dense projection of orexin neurons from the lateral hypothalamus to mesocorticolimbic dopaminergic neurons in the ventral tegmental area (VTA) is likely to be important in both of these processes. We used single-unit extracellular and whole-cell patch-clamp recordings to examine the effects of orexins (A and B) and melanin-concentrating hormone (MCH) on neurons in this region. Orexins caused an increase in firing frequency (EC(50) 78 nm), burst firing, or no change in firing in different groups of A10 dopamine neurons. Neurons showing oscillatory firing in response to orexins had smaller afterhyperpolarizations than the other groups of dopamine neurons. Orexins (100 nm) also increased the firing frequency of nondopaminergic neurons in the VTA. In the presence of tetrodotoxin (0.5 microm), orexins depolarized both dopaminergic and nondopaminergic neurons, indicating a direct postsynaptic effect. Unlike the orexins, MCH did not affect the firing of either group of neurons. Single-cell PCR experiments showed that orexin receptors were expressed in both dopaminergic and nondopaminergic neurons and that the calcium binding protein calbindin was only expressed in neurons, which also expressed orexin receptors. In narcolepsy, in which the orexin system is disrupted, dysfunction of the orexin modulation of VTA neurons may be important in triggering attacks of cataplexy.

Fig. 1.

Electrophysiological properties of dopaminergic neurons and their response to orexins. A, Double stainings of biocytin-filled neuron (red) and TH-immunoreactive neurons (green).Arrows indicate the position of the neuron in the tissue. This biocytin-filled neuron is TH positive. Scale bar, 50 μm.B, Voltage responses to current pulses (−0.4, 0, +0.1 pA). C, Chart recording of membrane potential and spontaneous action potentials before and after application of orexin A (100 nm). Orexin A was applied for 2 min. D, Orexin B (100 nm) caused depolarization in the presence of tetrodotoxin (0.5 μm). E, Example of a TH-positive neuron in which application of orexin B (100 nm) caused burst firing. F, A typical orexin-mediated burst.

Fig. 2.

Presumed GABAergic neurons with high spontaneous firing rate are excited by orexins. A, Double stainings of biocytin-filled neuron (red) and TH-immunoreactive neurons (green). This neuron is TH negative.Arrows indicate the position of the neuron.B, Voltage responses to current pulses (−0.3, 0, +0.1 pA). C, Chart recording of membrane potential and spontaneous action potentials of the neuron before and after application of orexin B (100 nm). D, Chart recording of the membrane potential of the neuron after application of 0.5 μm TTX. Orexin A causes depolarization of the cell.

Fig. 3.

Presumed GABAergic neurons with low spontaneous firing rate are excited by orexins. A, Double stainings of biocytin-filled neuron (red) and TH-immunoreactive neurons (green). This neuron is TH negative.B, Voltage responses to current pulses (−0.2, 0,+0.1 pA). C, Chart recording of membrane potential and spontaneous action potentials before and after application of orexin A (100 nm).