Neuroendocrine proopiomelanocortin neurons are excited by hypocretin/orexin

Abstract

Hypocretin/orexin, produced by a group of neurons in the lateral hypothalamus/perifornical area, enhances cognitive arousal and also may play a crucial role in modulating the neuroendocrine system. How hypocretin modulates the endocrine system remains an open question. Hypocretin cells innervate the mediobasal hypothalamus where they can potentially influence the activity of specific cell populations within the arcuate nucleus. Here, we examine whether hypocretin modulates the median eminence-projecting proopiomelanocortin (POMC) neurons identified by selective green fluorescent protein expression and antidromic stimulation or retrograde Evans blue dye tracing in transgenic mice. We find that POMC neurons, in general, and, in addition, those that project their axons to the median eminence, were robustly activated by hypocretin in a dose-dependent manner. These excitatory actions included a threefold increase in spike frequency and direct membrane depolarization of up to 22 mV (mean, 17.9+/-7.2 mV). Direct postsynaptic depolarization was decreased at more positive membrane potentials, inhibited by the sodium-calcium exchanger antagonist KB-R7943, and reduced by lowering the bath temperature, or by buffering the postsynaptic calcium with BAPTA, suggesting that the primary mechanism for hypocretin-mediated excitation is the activation of the sodium-calcium exchanger. Hypocretin also enhanced excitatory inputs to POMC cells via a presynaptic mechanism and indirectly increased the release of GABA onto these cells in a spike-dependent manner. However, these synaptic actions were not necessary to cause postsynaptic membrane depolarization and spiking. Thus, in contrast to previous suggestions that hypocretin inhibited POMC cells, our results demonstrate robust direct excitation of POMC neurons by hypocretin.

Figure 1.

Neuroendocrine arcuate cells are activated by hypocretin. A, Left, Schematic of experimental configuration. Arcuate nucleus neurons were recorded in whole-cell mode, whereas ME axons were antidromically activated with an extracellular electrode. Right, Sodium spikes in a representative neuron triggered by ME stimulation in control conditions (top) and in the presence of cadmium (bottom) to block conventional calcium-dependent transmitter release. Arrowheads show the timing of axonal stimulation. ARC, Arcuate nucleus; NE, neuroendocrine; 3V, third ventricle; Rec, recording electrode; Stim, stimulating electrode. B, Left, A representative experiment is shown in which hypocretin increased spike frequency (per 6 s) in a physiologically identified neuroendocrine cell (top). The changes in spike frequency triggered by hypocretin were accompanied by membrane depolarization as shown for the same neuron (bottom). Right, Traces of control, hypocretin, and washout conditions from the same experiment. C, A summary is shown for averaged hypocretin actions on firing rate (top; in Hz) and membrane potential (bottom) of arcuate–neuroendocrine neurons. Error bars indicate SEM. Hcrt, Hypocretin; Ctrl, control; MP, membrane potential (in mV). Statistically signifcant (p < 0.05) compared to control condition.

Figure 2.

Hypocretin excites neuroendocrine POMC cells. A, Low-magnification DIC picture showing the mediobasal hypothalamus, including the arcuate nucleus. In the inset, a montage of a GFP-expressing POMC cell and its corresponding DIC image are shown (the recording pipette is also evident). The stimulus electrode used to determine the neuroendocrine nature of this representative POMC neuron is schematically shown in white. ARC, Arcuate nucleus; Rec, recording electrode; Stim, stimulating electrode; VMH, ventromedial nucleus. B, Excitatory actions of hypocretin on the activity of the same physiologically identified neuroendocrine POMC cell shown in A. C, Histological reconstruction of a representative experiment in which a fluorescent dye was injected intravenously 3 d before sectioning. The low-magnification picture on the top left corner (1) shows GFP expression in a hypothalamic section from a POMC transgenic mouse. Higher-magnification pictures from the region indicated by the white box (1) are shown for GFP expression (2), Evans blue fluorescence (red; 3), or both, shown by the yellow neurons (4). Some POMC cells that also contain Evans blue are highlighted by the white arrowheads (4). Scale bars: 1, 100 μm; 2, 20 μm. D, Hypocretin-mediated excitation in a representative neuroendocrine POMC cell identified by intravenous Evans blue injection. Hcrt, Hypocretin.

Figure 3.

Both cell-attached and whole-cell recordings failed to detect hypocretin-mediated inhibition of POMC firing activity. A, Extracellular recordings in control (left) and hypocretin (right) conditions for doses of 0.1 μm (top) and 1 μm (bottom), respectively, in two typical cells. B, Averaged effects of low (left) and high (right) doses of hypocretin on POMC spiking studied in cell-attached configuration. C, Whole-cell recording from two representative POMC neurons in control conditions (left) and in the presence of low (top right) or high (bottom right) doses of hypocretin. D, Summary displaying the dose-dependent actions of hypocretin on POMC firing detected by whole-cell recording. Hcrt, Hypocretin; Ctrl, control. *p < 0.05.

Figure 4.

Presynaptic modulation of excitatory synaptic input to POMC neurons. A, B, Hypocretin effects on the frequency of EPSCs are shown for a representative experiment (A) and summarized for 21 cells (B). C, D, mEPSCs detected in TTX. The mEPSC waveform (C) or cumulative distribution (D) is shown before and after hypocretin application. No apparent changes in mean EPSC waveform (in black), mean amplitude, or cumulative distribution of peak EPSC amplitude were observed. E, F, Bar graphs summarizing the actions of hypocretin on mean mEPSC amplitude (E) and frequency (F). G, Population bar graph depicting the reversible increase in EPSC frequency that follows hypocretin application in eight POMC cells loaded with 20 mm BAPTA. Hcrt, Hypocretin; Ctrl, control. *p < 0.05.

Figure 5.

Spike-dependent regulation of GABA release onto POMC neurons by hypocretin. A, B, Traces from a representative experiment (A) and summary of nine cells (B) showing IPSC frequency before and during hypocretin application. C, IPSC waveforms detected in the same experiment presented in A. Individual events are shown in gray, and the mean is shown in black. Note the increased IPSC amplitude in the presence of hypocretin. D, Averaged effect of hypocretin on IPSC amplitude (n = 9). E, F, Lack of hypocretin effect is shown either for mIPSC frequency (E) or amplitude (F) in six POMC neurons. Hcrt, Hypocretin; Ctrl, control. *p < 0.05.

Figure 6.

Hypocretin excitatory actions are independent of synaptic input. A, Cell-attached recordings in three representative POMC cells in control (left) and hypocretin (right) conditions after pharmacological blockade of their glutamatergic (1), GABAergic (2), or both glutamatergic/GABAergic (3) synaptic inputs. B, The summary of the population data for all three conditions is displayed. Hcrt, Hypocretin; Ctrl, control; AP, action potential. *p < 0.05.

Figure 7.

Mechanisms of postsynaptic hypocretin depolarization. A, Whole-cell voltage-clamp recording from a representative POMC neuron showing the effect of hypocretin on the holding potential and channel activity in the presence of TTX and blockers of glutamate and GABA transmission. B, Top, Effect of hypocretin on current response recorded in voltage clamp after a slow voltage ramp from −110 to −20 mV. Bottom, Net hypocretin-induced current obtained by subtracting control from hypocretin condition. C, Summary of current-clamp recordings displaying the actions of hypocretin on POMC membrane potential in control (Ctrl) conditions, or in the presence of BAPTA in the pipette solution, or KB-R7943 in the bath. *p < 0.05. D, The impact of initial membrane potential on hypocretin-evoked POMC depolarization in 22 cells. These neurons were grouped (10 mV bins) according to their initial membrane potential. Net depolarization (ΔV) was plotted against initial membrane potential (init MP), and a linear function was fitted (thin line; Y = 17.2 + 0.4X). E, Effect of hypocretin on membrane potential in a representative POMC cell maintained either near −40 mV (top) or around −60 mV (see Materials and Methods). Hcrt, Hypocretin; MP, membrane potential.