ATP-Sensitive Potassium Channel-Mediated Lactate Effect on Orexin Neurons: Implications for Brain Energetics during Arousal

Active neurons have a high demand for energy substrate, which is thought to be mainly supplied as lactate by astrocytes. Heavy lactate dependence of neuronal activity suggests that there may be a mechanism that detects and controls lactate levels and/or gates brain activation accordingly. Here, we demonstrate that orexin neurons can behave as such lactate sensors. Using acute brain slice preparations and patch-clamp techniques, we show that the monocarboxylate transporter blocker α-cyano-4-hydroxycinnamate (4-CIN) inhibits the spontaneous activity of orexin neurons despite the presence of extracellular glucose. Furthermore, fluoroacetate, a glial toxin, inhibits orexin neurons in the presence of glucose but not lactate. Thus, orexin neurons specifically use astrocyte-derived lactate. The effect of lactate on firing activity is concentration dependent, an essential characteristic of lactate sensors. Furthermore, lactate disinhibits and sensitizes these neurons for subsequent excitation. 4-CIN has no effect on the activity of some arcuate neurons, indicating that lactate dependency is not universal. Orexin neurons show an indirect concentration-dependent sensitivity to glucose below 1mM, responding by hyperpolarization, which is mediated by ATP-sensitive potassium channels composed of Kir6.1 and SUR1 subunits. In conclusion, our study suggests that lactate is a critical energy substrate and a regulator of the orexin system. Together with the known effects of orexins in inducing arousal, food intake, and hepatic glucose production, as well as lactate release from astrocytes in response to neuronal activity, our study suggests that orexin neurons play an integral part in balancing brain activity and energy supply

Electrophysiological Properties of Melanin-Concentrating Hormone and Orexin Neurons in Adolescent Rats

Orexin and melanin-concentrating hormone (MCH) neurons have complementary roles in various physiological functions including energy balance and the sleep/wake cycle. in vitro electrophysiological studies investigating these cells typically use post-weaning rodents, corresponding to adolescence. However, it is unclear whether these neurons are functionally mature at this period and whether these studies can be generalized to adult cells. Therefore, we examined the electrophysiological properties of orexin and MCH neurons in brain slices from post-weaning rats and found that MCH neurons undergo an age-dependent reduction in excitability, but not orexin neurons. Specifically, MCH neurons displayed an age-dependent hyperpolarization of the resting membrane potential (RMP), depolarizing shift of the threshold, and decrease in excitatory transmission, which reach the adult level by 7 weeks of age. In contrast, basic properties of orexin neurons were stable from 4 weeks to 14 weeks of age. Furthermore, a robust short-term facilitation of excitatory synapses was found in MCH neurons, which showed age-dependent changes during the post-weaning period. On the other hand, a strong short-term depression was observed in orexin neurons, which was similar throughout the same period. These differences in synaptic responses and age dependence likely differentially affect the network activity within the lateral hypothalamus where these cells co-exist. In summary, our study suggests that orexin neurons are electrophysiologically mature before adolescence whereas MCH neurons continue to develop until late adolescence. These changes in MCH neurons may contribute to growth spurts or consolidation of adult sleep patterns associated with adolescence. Furthermore, these results highlight the importance of considering the age of animals in studies involving MCH neurons

Dopamine Acts as a Partial Agonist for a2A Adrenoceptor in Melanin-Concentrating Hormone Neurons

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that promotes positive energy balance and anxiety. Since dopamine (DA) is also closely implicated in these functions, the present study investigated the effect of DA on MCH neurons. Using whole-cell patch-clamp recordings in rat brain slices, we found that DA hyperpolarizes MCH neurons by activating G-protein-activated inwardly rectifying K+ (GIRK) channels. Pharmacological study indicated that the effect was mediated by α2A adrenoceptors, not DA receptors. DA-induced outward current was also observed in the presence of tetrodotoxin or the dopamine β-hydroxylase inhibitor fusaric acid, suggesting thatDAdirectly binds toα2Areceptors onMCHneurons, rather than acting presynaptically or being transformed into norepinephrine (NE) in the slice preparation. The effects of NE and DA were concentration-dependent with EC50 of 5.9 and 23.7μM, respectively, and a maximal effect of 106.6 and 57.2 pA, respectively, suggesting that DA functions as a partial agonist. Prolonged (5 min) activation ofα2A receptors by eitherDAor NE attenuated the subsequent response toDAor NE, while 5 s applications were not sufficient to induce desensitization. Therefore, a history of α2A receptor activation by DA or NE can have a lasting inhibitory effect on the catecholaminergic transmission to MCH neurons. Our study suggests that α2A receptors expressed by MCH neurons may be one of the pathways by which DA and NE can interact and modulate mood and energy homeostasis, and this cross talk may have functional implications in mood disorders and obesity

Nociceptin Induces Hypophagia in the Perifornical and Lateral Hypothalamic Area

Nociceptin/orphanin FQ (N/OFQ) is known to induce food intake when administered into the lateral ventricle or certain brain areas. This is somewhat contradictory to its reward-suppressing role, as food is a strong rewarding stimulus. This discrepancy may be due to the functional diversity of N/OFQ’s target brain areas. N/OFQ has been shown to inhibit orexin and melanin-concentrating hormone (MCH) neurons, both of which are appetite-inducing cells. As the expression of these neurons is largely confined to the lateral hypothalamus/perifornical area (LH/PFA), we hypothesized that N/OFQ inhibits food intake by acting in this area. To test this hypothesis, we examined the effect of local N/OFQ infusion within the LH/PFA on food intake in the rat and found that N/OFQ decreased sugar pellet as well as chow intake. This effect was not seen when the injection site was outside of the LH/PFA, suggesting a site-specific effect. Next, to determine a possible cellular mechanism of N/OFQ action on food intake, whole cell patch clamp recordings were performed on rat orexin neurons. As previously reported in mice, N/OFQ induced a strong and long lasting hyperpolarization. Pharmacological study indicated that N/OFQ directly inhibited orexin neurons by activating ATP-sensitive potassium (KATP) channels. This effect was partially but significantly attenuated by the inhibitors of PI3K, PKC and PKA, suggesting that the N/OFQ signaling is mediated by these protein kinases. In summary, our results demonstrate a KATP channel-dependent N/OFQ signaling and that N/OFQ is a site-specific anorexic peptide

Activity-dependent potentiation of mEPSC amplitude and frequency are dissociated.

<div><p>A1-3) A representative magnocellular neuron that shows an extended mEPSC amplitude potentiation that outlasts the frequency response. A1) Voltage clamp traces taken at time points as indicated. A2) Cumulative plots showing mEPSC amplitude (left panel) and interevent interval (right panel). Late post HFS is after the frequency has recovered. A3) Time-effect plots of mEPSC amplitude (top) and frequency (bottom). Solid vertical line indicates delivery of HFS, dotted vertical line with arrows denotes time when frequency is fully recovered.</p><p>B1-3) A representative magnocellular neuron that displays an extended potentiation of mEPSC frequency that outlasts the amplitude response. B1) Voltage clamp traces taken at time points as indicated. B2) Cumulative plots of mEPSC amplitude (left panel) and interevent interval (right panel) at time points as indicated. Late post HFS is after the amplitude has recovered. B3) Time-effect plots of mEPSC amplitude (top) and frequency (bottom). Solid vertical line indicates delivery of HFS, dotted vertical line with arrows denotes time when the amplitude is fully recovered.</p><p>C) Time-effect plot of mEPSC amplitude change in response to 50 Hz, 1 sec (filled circles) or 100 Hz, 1 sec x 2 stimulation (open circles). HFS was applied at time 0.</p><p>D) Relationship between the initial degree of potentiation for amplitude and frequency (1^st min post HFS). Each symbol denotes a cell. There is no significant correlation between the two parameters.</p></div

Relationships between basal mEPSC amplitude, peak change and duration of response to HFS.

<div><p>In all graphs in this figure and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077402#pone-0077402-g003" target="_blank">Figure 3</a>, each circle denotes a single neuron. p values are indicated within graphs if they reached a significance.</p><p>A-C) Linear regression analysis showing that the basal mEPSC amplitude is not related to the absolute amplitude change during the 1^st min post-HFS (A) or duration of the change (B). In contrast, a negative correlation exists between the amplitude in baseline control and 1^st min post-HFS conditions upon normalizing the amplitude response to control values (C).</p><p>D and E) Linear regression analysis showing that both absolute (D) and normalized (relative; E) responses during the 1^st min post-HFS are positively related to how long the response persists.</p></div

mEPSCs before and after HFS have similar kinetics.

<div><p>A) Superimposed mEPSCs (grey lines) from control (left panel) and late post-HFS (middle panel) with averaged traces shown in black. Right panel: averaged traces, scaled and superimposed (solid line: control; dotted line: late post-HFS).</p><p>B) Characteristics of mEPSCs recorded from a representative cell during baseline condition. 10-90 percentage points rise (B1) or decay times (B2) are plotted against their amplitudes. Each circle indicates individual mEPSC. Linear regression analysis indicates no relationship between the factors (p>0.05).</p><p>C) Scatter plot of 10-90% rise (C1) or decay times (C2) vs. amplitudes of mESPCs recorded from the same cell as (B) at late post-HFS after frequency has recovered to baseline. Linear regression analysis indicates no relationship between the factors (p>0.05).</p><p>D and E) Summary graphs showing 10-90% rise (D) and decay (E) of mEPSCs in baseline and late post-HFS. Post-HFS mEPSCs are grouped according to the amplitude as small (< 20 pA) or large (> 45 pA). D and E show data from the same group of cells.</p></div

Relationships between basal mEPSC frequency, peak change and duration of response to HFS.

<div><p>A-C) Linear regression analysis showing that the basal mEPSC frequency is not related to the absolute frequency change during the 1^st min post-HFS (A) or duration of the change (B). In contrast, there is a negative correlation between the control baseline values and the changes normalized to control (C).</p><p>C and D and E) Linear regression analysis showing that both absolute (D) and normalized responses (E) during the 1^st min are positively related to how long the response persists.</p><p>E) Linear regression analysis showing no relationship between the basal mEPSC frequency and the duration of response.</p></div